Targeted metabolomics in human and animal biofluids and tissues using liquid chromatography coupled with tandem mass spectrometry

Summary

Here, we present a targeted polar metabolomics protocol for the analysis of biofluids and frozen tissue biopsies using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We describe steps for sample pretreatment, liquid-liquid extraction, and isolation of polar metabolites. We then detail procedures for target LC-MS/MS analysis. In this protocol, we focus on the analysis of plasma and serum samples. We also provide brief instructions on how to process other biological matrices as supplemental information.

For complete details on the use and execution of this protocol, please refer to Coskun et al. (2022).¹

Graphical abstract

Highlights

• •Targeted protocol for polar metabolomics using protein precipitation and LC-MS/MS
• •Semiquantitative analysis of 260 polar metabolites in biological samples
• •Guidance on peak integration, quantitation strategy, and data processing

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Here, we present a targeted polar metabolomics protocol for the analysis of biofluids and frozen tissue biopsies using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We describe steps for sample pretreatment, liquid-liquid extraction, and isolation of polar metabolites. We then detail procedures for target LC-MS/MS analysis. In this protocol, we focus on the analysis of plasma and serum samples. We also provide brief instructions on how to process other biological matrices as supplemental information.

Before you begin

Metabolomics aims to comprehensively measure small metabolites (usually within a mass range of 50–1500 Da) in biological systems and map metabolism under both physiological and pathological conditions.² Unlike upstream genomics and genetic risk scores, metabolite profiling provides a functional readout and gives a snapshot of biochemical processes occurring in the biological system at the time of sample collection.

Metabolomics is extensively used in drug discovery and development, from early preclinical stages to late clinical monitoring. Metabolomics focuses on the measurement of metabolites that result from internal biological regulations as well as external environmental influences that can be used for phenotypic characterization of patient populations, patient stratification, better understanding of disease progression and etiology; thus, advancing the field of precision medicine.

Metabolomics can help elucidate mechanisms of action of drug candidates, explore metabolic responses associated with drug administration, thus finding applications in toxicology and drug monitoring, identify targets and drug repurposing strategies, and measure markers of target engagement.^3,4,5,6,7,8

Our protocol describes the metabolite extraction process for plasma and serum samples, the two chromatographic assays used to collect metabolite data, the settings of multiple reaction monitoring (MRM) for data acquisition using a triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source, and the quantification strategy. The description of our statistical analysis strategy is outside the scope of this protocol. We redirect the readers to other publications for further details.^{9,10,11,12,13,14,15,16,17}

Note that the protocol has also been used to analyze urine, liver, muscle, kidney, brown and white adipose tissue samples, as well as cultured cells and organoids using minor adjustments.

Note that one chromatographic assay relies on hydrophilic interaction liquid chromatography (HILIC), and the other on reversed phase (RP) chromatography. The assays are referred as BEH AMIDE (HILIC assay) and HSS T3 (RP assay) throughout the protocol. Separate assays have been developed for targeted measure of lipid species, the description of which is considered outside the scope of this protocol. We state so to emphasize that the list of metabolites targeted in this protocol is narrow by design and limited to polar metabolites below 800 Da.

Institutional permissions

Data generated using this protocol have been published in several clinical and preclinical studies. Published results can be found in Pirro et al., 2022,¹⁸ Samms et al., 2021,¹⁹ Coskun et al., 2022,¹ and Samms et al., 2022.²⁰ Information on the samples used in these studies can be found in the original publications. Unpublished data used in figures and tables are labeled as such.

Plasma sample preparation

Aliquoting the samples

Timing: 1 day+

This step describes the preparation of plasma and serum samples. The protocol has been successfully used on K2 and K3 ethylenediamine tetraacetic acid (EDTA) plasma, citrate plasma, and serum. However, for simplicity, we will refer generically to plasma samples throughout the protocol.

Note: Depending on the nature of the study, the number of experimental samples can vary from a few tens (e.g., preclinical studies) to thousands (e.g., for late phase clinical trials); thus, the time needed to aliquot the experimental samples varies from a couple of hours to several days.

Note: In our institution, plasma samples are aliquoted from personnel outside the metabolomics laboratory as numerous analytical measures are planned besides exploratory -omics analyses. This limits the volume of plasma that is generally available to no more than 50 μL. This also precludes the addition of stable-labeled internal standards (ISs) directly into the experimental samples, a limitation that is discussed later in the protocol.

• 1.Thaw frozen plasma samples on wet ice.
• 2.Pipette two 25-μL plasma aliquots in two polymerase chain reaction (PCR) 96-well clear plates with V-shaped bottom: one aliquot for each assay (BEH AMIDE and HSS T3).
• 3.Seal the 96-well plates with an adhesive sealing foil and place them at –80°C if liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis is not executed immediately.

Note: When sorting and aliquoting the experimental samples, make sure proper randomization strategy is followed according to study design and objectives of analysis.

CRITICAL: Highly accurate and precise pipetting is necessary. This is particularly critical if ISs cannot be added to the experimental samples prior to aliquoting into the PCR 96-well plates. This step is usually performed manually by skilled operators using single-channel pipettes.

CRITICAL: Plasma samples are to be considered biohazardous. Use of appropriate personal protective equipment (PPE) and compliance to institutional biosafety protocols are always required. This warning applies to the entire protocol when handling biological material.

Preparing the extraction solutions with labeled ISs

Timing: 2–3 h

This step described the preparation of the stable-labeled IS stocks and working solutions, as well as the preparation of the extraction solutions, containing the ISs, used for protein precipitation.

Note: Internal standards allow us to correct for analytical variations during sample preparation and data acquisition (e.g., matrix effects, ion suppression, chromatographic shifts, instrument drifts) and normalize data across batches. We use IS normalization to calculate relative concentration of targeted metabolites against pooled calibrators as well as calculate absolute concentrations of matching endogenous metabolites in solution. The list of ISs is presented in Table 1. The list covers a diversified range of metabolite classes, with good coverage of retention times (RTs) and chemical diversity for both polarities of molecular ions. Internal standards have also been added to help detect and quantify targeted metabolites that are more challenging to measure in the assays due to significant matrix effects and ion suppression, and poor chromatographic behavior. The present list counts 46 ISs but it is constantly revised as endogenous metabolites are added to the assays and additional assays are developed. We recommend a subset of key ISs (see Table S3), if adopting the full list is cost prohibitive.

Note: The labeled ISs are kept in solution at –20°C at 1 mg/mL unless otherwise noted (Table 1) and are prepared from powders two times a year, on average. It is not recommended to store the 1-mg/mL stock solutions for more than 1 year. Solvent mixtures used to prepare the stock solutions are detailed in Table 1. Exact volumes of ISs spiked in 500 mL of extraction solution are also reported in Table 1 as example.

• 4.
  For the BEH AMIDE assay:

  • a.
    Mix acetonitrile and methanol in the ratio of 50:50% (v/v) in a clean glass bottle.
  • b.
    While keeping the extraction solvent on wet ice, add the appropriate volume of each IS solution to reach the final concentration reported in Table 1.
  • c.
    Mix thoroughly.
• 5.
  For the HSS T3 assay:

  • a.
    Mix methanol and water in 80:20% (v/v) in a clean glass bottle.
  • b.
    While keeping the extraction solvent on wet ice, add the appropriate volume of each IS solution to reach the final concentration reported in Table 1.
  • c.
    Mix thoroughly.

Note: Make fresh extraction solutions for each study. Store the extraction solutions at –20°C for a maximum of 8 months.

Note: Consistent use of the same solvent brands and grades (MS grades) is preferred whenever possible.

Table 1.

Stable labeled internal standards

Internal standards	Assay	Source (preferred)	Catalog number	Internal ID	Stock solution concentration (mg/mL)	Solvent for stock solution % v/v	Extraction solution concentration (μg/mL)	Volume (μL) to prepare 500 mL of extraction solution
1-methylhistidine, methyl-D3	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-2949	LABELED_1-methylhistidine	1.0	Methanol-Water 50:50	0.20	100
1-methylnicotinamide-D7 iodide	BEH AMIDE	BDG Synthesis	140138–25	LABELED_1-MeNAM	1.0	Methanol-Water 50:50	0.10	50
2-Hydroxyglutaric acid13C5 disodium salt	HSS T3	Cambridge Isotope Laboratories (isotope.com)	CLM-10351	LABELED_2KG	1.0	Methanol-Water 10:90	1.00	500
3-Hydroxybutyric acid13C4 sodium salt	HSS T3	Cambridge Isotope Laboratories (isotope.com)	CLM-3853	LABELED_3HB	1.0	Methanol-Water 50:50	1.00	500
alpha-ketoisocaproic acid13C6 sodium salt	HSS T3	Cambridge Isotope Laboratories (isotope.com)	CLM-4785	LABELED_ketoleucine	1.0	Methanol-Water 50:50	1.00	500
alpha-ketoisovaleric acid, 3-methyl13C,D4 sodium salt	HSS T3	Cambridge Isotope Laboratories (isotope.com)	CDLM-7317	LABELED_ketovaline	1.0	Methanol-Water 50:50	0.50	250
Cholesteryl-D7 sulfate sodium salt	HSS T3	Sigma Aldrich 903752	903752	LABELED_CS	1.0	Methanol-Chloroform 50:50	0.20	100
Cholic acid-D5	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-9549	LABELED_cholic acid	0.5	Acetonitrile-Methanol 50:50	1.00	1000
Citric acid-D4	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-3487	LABELED_citric acid	1.0	Methanol-Water 10:90	2.00	1000
Creatinine, N-methyl-D3	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-3653	LABELED_creatinine	1.0	Methanol-Water 50:50	0.20	100
Cyclic adenosine monophosphate13C5	HSS T3	Toronto Research Chemicals (trc-canada.com)	A280457	LABELED_cAMP	1.0	Water	1.00	500
Cytosine13C,15N2	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CNLM-4424	LABELED_cytosine	1.0	Methanol-Water 50:50	0.20	100
Deoxycholic acid-D6	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-9546	LABELED_deoxycholic acid	0.5	Acetonitrile-Methanol 50:50	1.00	1000
DL-phenylalanine, ring-D5	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-2986	LABELED_phenylalanine	1.0	Methanol-Water 50:50	0.20	100
DL-valine-D8	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-311	LABELED_valine	1.0	Methanol-Water 50:50	0.20	100
Glycine-D2	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-1674	LABELED_glycine	1.0	Methanol-Water 10:90	2.50	1250
Histamine:2HCL-D4	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-2911	LABELED_histamine	1.0	Methanol-Water 50:50	0.20	100
Lactic acid-D3 sodium salt 20% W/W in H2O	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-9071	LABELED_lactic acid	200	Water	10.00	25
L-aspartic acid-D3	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-546	LABELED_aspartic acid	1.0	Water	2.50	1250
L-carnitine C0, trimethyl-D9, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_carnitine	25.9 (μg/mL)∗	Methanol	0.13	2500∗∗
L-citrulline-D4	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-6039	LABELED_citrulline	1.0	Methanol-Water 50:50	0.20	100
L-glutamine13C5,15N2	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CNLM-1275-H	LABELED_glutamine	1.0	Methanol-Water 10:90	0.50	250
L-histidine:HCL:H2O13C6	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CLM-2264	LABELED_histidine	1.0	Methanol-Water 10:90	0.20	100
L-isoleucine-D10	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-141	LABELED_isoleucine	1.0	Methanol-Water 80:20	0.20	100
L-leucine13C6	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CLM-2262-H	LABELED_leucine	1.0	Methanol-Water 50:50	0.20	100
L-methionine13C5	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CLM-893-H	LABELED_methionine	1.0	Methanol-Water 50:50	0.20	100
L-ornithine13C5	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CLM-4724-H	LABELED_rnithine	1.0	Methanol-Water 10:90	0.20	100
L-serine-D3	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-582	LABELED_serine	1.0	Methanol-Water 50:50	2.50	1250
N-acetyl-DL-aspartic-D3 acid	BEH AMIDE	CDN Isotopes D-5980	D-5980	LABELED_NAA	1.0	Methanol-Water 50:50	0.10	50
N-acetyl-L-leucine-D10	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-476	LABELED_N-acetylleucine	1.0	Methanol-Water 50:50	1.00	500
N-acetyl-L-serine-D3	BEH AMIDE	CDN Isotopes D-6999	D-6999	LABELED_N-acetylserine	1.0	Methanol-Water 50:50	0.20	100
Nicotinamide13C6	HSS T3	Cambridge Isotope Laboratories (isotope.com)	CLM-9925	LABELED_nicotinamide	1.0	Methanol	1.00	500
Nicotinic acid-D4	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-4578	LABELED_nicotinic acid	1.0	Methanol-Water 50:50	1.00	500
O-acetyl-L-carnitine HCl (C2), N-methyl-D3, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C2	9.2 (μg/mL)∗	Methanol	0.046	N/A
O-butyryl-L-carnitine HCl (C4), N-methyl-D3, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C4	3.17 (μg/mL)∗	Methanol	0.016	N/A
O-isovaleryl-L-carnitine HCl (C5), N,N,N-trimethyl-D9, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C5	2.48 (μg/mL)∗	Methanol	0.012	N/A
O-myrystoyl-L-carnitine HCl (C14), N,N,N-trimethyl-D9, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C14	2.06 (μg/mL)∗	Methanol	0.010	N/A
O-octanoyl-L-carnitine HCl (C8), N-methyl-D3, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C8	2.21 (μg/mL)∗	Methanol	0.011	N/A
O-palmitoyl-L-carnitine HCl (C16), N-methyl-D3, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C16	1.95 (μg/mL)∗	Methanol	0.010	N/A
O-propionyl-L-carnitine HCl (C3), N-methyl-D3, 98%	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	NSK-B (mix)	LABELED_C3	6.67 (μg/mL)∗	Methanol	0.033	N/A
S-adenosyl-L-homocysteine, adenosine13C10	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CLM-8906	LABELED_SAH	1.0	Methanol-Water 50:50	0.05	25
Succinic acid-D4 disodium salt	HSS T3	Cambridge Isotope Laboratories (isotope.com)	DLM-2307	LABELED_succinic acid	1.0	Methanol-Water 10:90	2.00	1000
Taurine-D4	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-8057	LABELED_taurine	1.0	Methanol-Water 10:90	0.20	100
trans-4-hydroxy-L-proline-D3	BEH AMIDE	CDN Isotopes D-7186	D-7186	LABELED_4-hydroxyproline	1.0	Methanol-Water 10:90	0.20	100
Trimethylamine N-oxide-D9	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	DLM-4779	LABELED_TMAO	1.0	Methanol-Water 10:90	0.20	100
Urea13C,15N2	BEH AMIDE	Cambridge Isotope Laboratories (isotope.com)	CNLM-234	LABELED_urea	1.0	Methanol-Water 10:90	0.20	100

N/A, not applicable.

^∗Reconstitute vial in 1 mL of methanol.

^∗∗Volume of NSK-B mix to add only once to the extraction solution.

Key resources table provides a summary of the preferred reagents and resources.

Note: It is possible to leave out certain ISs or add others depending on the study objectives, supply chain issues, and the evolution of the metabolomics protocol. However, test additional ISs for interferences with endogenous metabolites and existing ISs. When leaving out an IS, make sure there is still adequate coverage of the entire RT range and chemical diversity of the targeted metabolites for both polarities of molecular ions.

CRITICAL: Prepare sufficient volume of extraction solutions to extract all experimental samples, prepare all calibrators and quality controls to avoid biases between batches of extraction solutions. Tables S2 and S3 provide an example of how to calculate the volume of extraction solution required for the analysis of 500 experimental samples (for one assay). The description of how to extract plasma samples and how to prepare calibrators and quality controls follows this paragraph and is key to understand the rationale behind the calculations presented in Tables S2 and S3.

CRITICAL: The extraction solution is added to all the experimental samples. Be sure to use clean disposable plastic and glass consumables to prepare the extraction solutions and all the IS stock solutions. Any contamination may interfere with the analysis.

CRITICAL: Always use proper PPE in accordance with safety regulations of the laboratory. Organic solvent vapors can be harmful. This warning applies to the entire protocol when handling organic solvents.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Control plasma	Ab Sciex (sciex.com)	4386703
Metabolites in frozen human plasma	NIST (-s.nist.gov)	SRM 1950
Chemicals, peptides, and recombinant proteins
Acetonitrile, Optima LC/MS grade	Fisher Chemical (fishersci.com)	172421
Water, Optima LC/MS grade	Fisher Chemical (fishersci.com)	221441
Acetonitrile 0.1% formic acid Optima LC/MS grade	Thermo Scientific (fishersci.com)	LS120-4
Water 0.1% formic acid HPLC grade	Fisher Chemical (fishersci.com)	HB523-4
Ammonium formate 99%	Thermo Scientific (fishersci.com)	401150050
Methanol, Optima LC/MS grade	Fisher Chemical (fishersci.com)	218262
Formic acid 98%–100% for LC-MS/MS LiChropur	Sigma-Aldrich (sigmaaldrich.com)	5.33002
Isopropanol HPLC ≥99.8%	Sigma-Aldrich (sigmaaldrich.com)	59307
PPGs chemical standards kit (MS calibration)	Ab Sciex (us-store.sciex.com)	4406127
Software and algorithms
Analyst 1.7.2	Ab Sciex	https://sciex.com/support/software-support/software-downloads (license required)
MultiQuant 3.0.3	Ab Sciex	https://sciex.com/support/software-support/software-downloads (license required)
RefMet (Metabolomics Workbench: Databases)	https://metabolomicsworkbench.org/	https://metabolomicsworkbench.org/databases/refmet/index.php
Human Metabolome Database (HMBD)	https://hmdb.ca/	https://hmdb.ca/metabolites
Kyoto Encyclopedia of Genes and Genomes (KEGG)	https://www.genome.jp/kegg/	https://www.genome.jp/kegg/compound/
Other
Waters ACQUITY UPLC BEH Amide 2.1 × 100 mm; 1.7 μm particle size	Waters Corp. (waters.com)	186004801
Waters ACQUITY UPLC BEH Amide VanGuard pre-column 2.1 × 5 mm; 1.7 μm particle size	Waters Corp. (waters.com)	186004799
Waters ACQUITY UPLC HSS T3 2.1 × 100 mm; 1.8 μm particle size	Waters Corp. (waters.com)	186003539
Waters ACQUITY UPLC HSS T3 VanGuard pre-column 2.1 × 5 mm; 1.8 μm particle size	Waters Corp. (waters.com)	186009637
Mixer for 35 μL mixing volume	Thermo Scientific (fishersci.com)	6040.5000
Red PEEK tubing 0.005 ID	Thermo Scientific (fishersci.com)	CH105834
SuperPlate PCR plate, 96-well, clear, low profile, skirted	Thermo Scientific (fishersci.com)	AB2800
Aluminum sealing film 50 μm	Axygen (fishersci.com)	PCR-AS-600
Thermo-Seal heat sealing foil	Thermo Scientific (fishersci.com)	AB0559
Thermo sealer ALPS 50 V-manual heat sealer	Thermo Scientific (fishersci.com)	AB1443A
Falcon 15 mL conical centrifuge tubes	Corning (fishersci.com)	352099
Falcon 50 mL conical centrifuge tubes	Corning (fishersci.com)	352098
Snap-cap microcentrifuge Safe-Lock tubes (Biopur preferred) 1.5 or 2.0 mL	Eppendorf (fishersci.com)	22600044
LTS pipette tips (variable volumes)	Rainin (shoprainin.com)	Not specified
Combitips advanced PCR pipet tips (variable volumes)	Eppendorf (fishersci.com)	Not specified
Single- and multichannel Rainin LTS pipettes (variable volumes)	Rainin (shoprainin.com)	Not specified
Repeater E3	Eppendorf (fishersci.com)	4987000118
5810R centrifuge and rotor packages	Eppendorf (fishersci.com)	022627133
Microcentrifuge 5430 R	Eppendorf (fishersci.com)	022620663
ThermoMixer F1.5	Eppendorf (fishersci.com)	5384000020
Analytical balance	Mettler Toledo (fishersci.com)	30029076
Spectrum Bessman tissue pulverizer	Thomas Scientific (thomassci.com)	189470
LCGC certified clear glass 12 × 32 mm screw neck vial, with cap and preslit PTFE/silicone septum, 2 mL volume	Waters Corp. (waters.com)	186000307C
Fisherbrand 300 μL PolySpring autosampler vial inserts	Fisherband (fishersci.com)	13-622-217
Nexera UHPLC 30 series	Shimadzu (ssi.shimadzu.com)	N/A
Triple quadrupole 6500+ system	Ab Sciex (sciex.com)	N/A
Turbo spray ion drive ESI source	Ab Sciex (sciex.com)	N/A

Step-by-step method details

Extracting plasma samples

Timing: 2 days+

This section describes metabolite extraction and protein precipitation with organic solvents ahead of the LC/MS analysis. A solution of acetonitrile-methanol 50:50%(v/v) is used for metabolite extraction with the BEH AMIDE assay. A solution of methanol-water 80:20%(v/v) is used for metabolite extraction with the HSS T3 assay.

Note: The optimization of peak shapes for each chromatography was the main driver for the adoption of two extraction solutions rather than significant differences in recovery of the targeted metabolites from plasma.

Note: When the number of samples exceed 5000, approximately 4–5 days are needed for each assay to complete this task (counting two operators).

• 1.Thaw the sample 96-well plate containing 25-μL aliquots of plasma on wet ice.
• 2.Unseal the sample 96-well plate on a steady surface when plasma samples are thawed.
• 3.Using a repeater pipette, add 150 μL of ice-cold extraction solution containing the ISs to the 25-μL plasma aliquots. Skip wells that do not contain any plasma aliquot.
• 4.Use multi-channel pipettes to mix the samples thoroughly.
• 5.Seal the sample 96-well plate with an adhesive sealing foil and place the 96-well plate at –20°C for 8–16 h extraction.
• 6.Repeat steps 1–5 until all the study sample 96-well plates are prepared.

Note: Use cold extraction solution kept at –20°C to maximize quenching of metabolism and protein precipitation. Keep the extraction solution on wet ice during the sample preparation. When finished, store the extraction solution back at –20°C.

CRITICAL: As the extraction solution contains the labeled ISs, highly accurate and precise pipetting is necessary. We recommend that highly skilled operators perform step 3 using repeater pipettes to be faster, more precise, and more accurate than using single-channel pipettes. Adjust the speed at which solvent aspiration and dispensing occurs based on the viscosity of the extraction solution. It is necessary to avoid solvent and sample splashing outside the well when dispensing the extraction solution. Also, avoid that the tip of the pipette touches the plasma inside the well while dispensing the extraction solution to prevent sample cross-contamination.

CRITICAL: Be aware of solvent evaporation. Prepare one plate at a time to minimize the time plates sit uncovered on wet ice. This warning applies to the entire protocol when handling organic solvents.

• 7.After the 8–16 h extraction, chill the centrifuge to +4°C.
• 8.Centrifuge all sample 96-well plates for 15 min at 4000 rpm (3200 × g) at +4°C.
• 9.Store all centrifuged plates at –20°C until ready to proceed with the next steps.

CRITICAL: Ensure that all the wells have a compact protein pellet at the bottom of the well and the supernatant is clean. If necessary, centrifuge the sample 96-well plate twice. We recommend the use of clear 96-well plates with a V-shaped bottom to be able to observe the protein precipitate in the wells and to maximize the recovery of the supernatant (Figure 1).

Figure 1.

Plasma protein precipitatation

Photograph of a PCR 96-well plate (clear, low profile, skirted, V-bottom) showing human plasma (25 μL) protein precipitate and organic solvent supernatant (acetonitrile-methanol 50:50%[v/v], 150 μL) after 8–16 h extraction at −20°C and centrifugation for 15 min at 4000 rpm (3200 × g), at +4°C.

Preparing the pool calibrators

Timing: 2 days+

This step describes how to prepare calibrators by pooling aliquots of all extracted experimental samples as well as plasma quality controls and blanks.

Note: The number and level of the calibrators can change based on project needs and study-specific considerations. The most common scheme we employ is detailed below.

Note: Our protocol targets 260 endogenous small polar metabolites. We aim to provide relative quantitation of the metabolites against pooled calibrators. We pool an aliquot of every experimental sample to create a reference standard for the study (referred to as ‘100% pool calibrator’). This is done independently for the two assays (viz., one ‘100% pool calibrator’ is prepared using the BEH AMIDE extracts in acetonitrile-methanol 50:50%[v/v], and another is prepared using the HSS T3 extracts in methanol-water 80:20%[v/v]). For each assay, the ‘100% pool calibrator’ is diluted to create additional calibrators that are used to linearly model metabolite area counts (normalized by IS area counts) to the dilution factor of the ‘100% pool calibrator’.

Note: The volume of supernatant to pool from each experimental sample to prepare the ‘100% pool calibrator’ is calculated based on the total number of calibration curves to run throughout the LC-MS/MS analysis and the number of calibrators in each calibration curve. We provide an example of how we calculate volumes in Table S4 where we simulate a study of 500 experimental samples. As general guidance, studies with over 500 experimental samples will require volumes of supernatant ≤10 μL to be pooled from each sample. Smaller preclinical cohorts might require pooling volumes up to 20–30 μL of supernatant from each experimental sample. We acknowledge this is a step where the decision-making strategy can be tailored to specific needs and could be different from what is presented here as an example.

• 10.Unseal the centrifuged sample 96-well plate on a steady surface.
• 11.Place the sample 96-well plate on wet ice.
• 12.Take an aliquot of each experimental sample and pool it into a Biopur 2.0 mL Eppendorf tube or a Falcon tube (if the total volume pooled is > 2.0 mL) to obtain the 100% pool calibrator.
• 13.Reseal the centrifuged sample 96-well plate with an unused adhesive sealing foil and store the plate back at –20°C.
• 14.Repeat steps 10–13 for all the sample 96-well plates.

CRITICAL: Avoid disturbing the protein precipitate at the bottom of the well when using a pipette to transfer the supernatant. Make sure that the transferred supernatant is free from precipitate and debris.

CRITICAL: Be aware of solvent evaporation. Prepare one sample 96-well plate at a time to minimize the time plates are left unsealed. Always keep the plates on wet ice when unsealed. Keep the Eppendorf or Falcon tube on wet ice and capped as much as possible while pooling the supernatant from all experimental samples.

• 15.Label clean Biopur 2.0 mL Eppendorf tubes or Falcon tubes (if total volume of each calibrator is larger than 2.0 mL).
• 16.Prepare calibrators 5%, 10%, 15%, 30%, 50%, and 75% by mixing the 100% pool calibrator and the extraction solution (see example in Table S5).
• 17.Cap the tubes and vortex for 20 s.
• 18.Store the calibrators at –20°C until ready for LC-MS/MS analysis.

Note: While preparing the calibration curve, we recommend using one pipette tip to dispense all the volumes of ‘100% pool calibrator’ into the clean Eppendorf or Falcon tubes of calibrators 5%–75% and then use clean pipette tips to add subsequently the extraction solution in each tube, rather than the opposite. This avoids the need to condition multiple pipette tips with the ‘100% pool calibrator’, which introduces air into the sample tube and can cause additional solvent evaporation.

Preparing the plasma quality controls

Timing: 1 day+

This step describes the preparation of additional quality controls we run together with pooled calibrators (e.g., NIST SRM 1950) to track instrument performance over time.

Note: We prepare independent sets of quality controls for each assay (BEH AMIDE and HSS T3).

The number of quality control plasma aliquots to prepare is chosen based on the number of experimental samples, total run time, and other study-specific considerations. Our default strategy is to analyze one quality control plasma aliquot for every sample 96-well plate. Preferred sources for the quality control plasma are reported in key resources table.

Note: To avoid many freeze-and-thaw cycles of the quality control plasma over time, we recommend thawing frozen material or reconstituting lyophilized material upon arrival in the laboratory, transfer as many 25 μL aliquots in Biopur 2.0 mL Eppendorf tubes as possible, as described in steps 19–20, then thaw on wet ice as many tubes as required for a study and proceed with the extraction procedure (thus starting the quality control sample preparation from step 21).

• 19.Label a clean Biopur 2.0 mL Eppendorf tube for each aliquot of quality control plasma.
• 20.Aliquot 25 μL of quality control plasma in each tube using a single-channel pipette.
• 21.Add 150 μL of ice-cold extraction solution containing the labeled ISs to each tube using a repeater pipette.
• 22.Cap the tubes and vortex for 20 s.
• 23.Store the tubes at –20°C for 8–16 h.
• 24.After the 8–16 h extraction, chill the centrifuge to +4°C.
• 25.Centrifuge the tubes for 15 min at 4000 rpm (3200 × g) at +4°C.
• 26.Store all centrifuged tubes at –20°C until ready to proceed with the next steps.

Note: Goal is to store the quality control plasma extracts in the same conditions as the extracted experimental samples.

Transferring experimental samples into the final LC-MS 96-well plates for LC-MS/MS analysis

Timing: 1 day+

This step describes the transfer of the extracted experimental samples into the final 96-well plates ahead of the LC-MS/MS analysis.

Note: We recommend working on one plate at a time to minimize the time each plate is left unsealed.

• 27.Label a new clear PCR 96-well plate (LC-MS 96-well plate).
• 28.Unseal one centrifuged sample 96-well plate on a steady surface.
• 29.Place the two 96-well plates (the sample 96-well plate and the LC-MS 96-well plate) on wet ice alongside each other.
• 30.Using a repeater pipette, add 25 μL of extraction solution in the wells of the LC-MS 96-well plate (only in the same well coordinates where the experimental samples are located into the sample 96-well plate).
• 31.Using an 8-channel pipette, transfer 25 μL of the supernatant from the sample 96-well plate to the LC-MS 96-well plate and mix thoroughly. Transfer the experimental samples without altering the well coordinates.
• 32.Reseal the sample 96-well plate with an unused adhesive sealing foil and store the plate back at –20°C in case reruns are necessary.

Note: Our quantitation strategy justifies the need for diluting the supernatant 1:1 with the extraction solution (steps 30 and 31), as explained later in the protocol.

Note: The minimum volume of supernatant to transfer in the LC-MS 96-well plates depends on the minimum amount of solvent needed for accurate aspiration with the LC needle. Our LC setup requires a minimum of 40 μL of dead volume, implying that no less than 20 μL should be transferred in steps 30 and 31. In general, we recommend using V-shaped shirted PCR 96-well plates with 200 μL capacity (key resources table) as LC-MS 96-well plates to minimize dead volumes.

CRITICAL: High accuracy and precision in pipetting is necessary. We recommend using the repeater pipette to dispense the extraction solution into a clean LC-MS 96-well plate (step 30).

Transferring blank ISs and pool calibrators into the final LC-MS 96-well plates for LC-MS/MS analysis

Timing: 1–3 h

This step describes how to add blank IS and pool calibrators to the LC-MS 96-well plates already containing the extracted experimental samples.

• 33.Place the tubes containing the blank IS and the calibrators on wet ice.
• 34.Transfer 40 μL of blank IS and each calibrator into empty wells of the LC-MS 96-well plate. Transfer two sets of one blank IS and calibrators per plate.

Note: When aliquoting plasma into the sample 96-well plates, we are accustomed to leave two empty columns, hence never aliquoting more than 80 experimental samples per plate. By doing so, two sets of one blank IS and 7 calibrators can be transferred to the LC-MS 96-well plate without altering the well coordinates of the experimental samples from the sample 96-well plate (see example in Figure 2).

Note: The blank IS is an aliquot of the extraction solution containing the ISs.

• 35.Heat seal the LC-MS 96-well plate using aluminum sealing foils at 170°C for 3 s.
• 36.Store the LC-MS 96-well plate at –20°C until ready for LC-MS/MS analysis.

Note: At step 35, the use of aluminum heat sealing foil is preferred over the use of adhesive seals as the glue could be a source of significant contamination when in contact with organic solvents and could also cause pieces of the foil to adhere to the LC needle, possibly clogging the LC autosampler. It is convenient, however, to use the adhesive aluminum foil to seal the sample 96-well plates (step 5) as the sample preparation requires the operator to unseal and reseal the sample 96-well plate multiple times.

CRITICAL: Inspect the plate to make sure homogenous and strong sealing around each well has occurred. Press the plate again in the thermo-sealer if necessary. A plate roller can also be used to manually press the foil after heat-sealing.

CRITICAL: Be careful. Hot surfaces can cause burns.

Figure 2.

Plate map

Graphical representation of a LC-MS 96-well plate map showing position of experimental samples in blue; blank IS and calibrators (5%, 10%, 15%, 30%, 50%, 75%, and 100%) in red (from top to bottom in darker shade of red); empty wells in gray.

Transferring quality controls and double blanks into autosampler vials for LC-MS/MS analysis

Timing: 1–3 h

This step describes how to prepare the final dilutions of the extracted quality controls ahead of LC-MS/MS analysis.

• 37.Place the Biopur 2.0 mL Eppendorf tubes of extracted quality control plasma on wet ice.
• 38.Label clean 1.5-mL autosampler vials with conical clear inserts.
• 39.Transfer 25 μL of supernatant from each Eppendorf tube into an autosampler vial.
• 40.Add 25 μL of extraction solution into each autosampler vial.
• 41.Cap the vials with pre-slit septa screw caps and vortex for 20 s.
• 42.Store the vials at –20°C until ready for LC-MS/MS analysis. Goal is to store the autosampler vials in the same conditions as the extracted experimental samples.

CRITICAL: Be aware of solvent evaporation. Keep the tubes on wet ice while preparing the quality control plasma. Keep the tubes capped as much as possible.

This is done independently for the BEH AMIDE and the HSS T3 assay.

• 43.
  For the BEH AMIDE assay:

  • a.
    Label a clean 1.5-mL autosampler vial.
  • b.
    Transfer 1 mL of acetonitrile-methanol 50:50%(v/v) in an autosampler vial.
  • c.
    Cap the vial using pre-slit septa screw caps.
  • d.
    Store at –20°C until ready for LC-MS/MS analysis.
• 44.
  For the HSS T3 assay:

  • a.
    Label a clean 1.5-mL autosampler vial.
  • b.
    Transfer 1 mL of methanol-water 80:20%(v/v) in a vial for the HSS T3 assay.
  • c.
    Cap the vial using pre-slit septa screw caps.
  • d.
    Store at –20°C until ready for LC-MS/MS analysis.

Preparing LC mobile phases

Timing: 1 h

This step described the preparation of the solvent used as mobile phases for both the BEH AMIDE and the HSS T3 assay.

Note: The same solvents are used as mobile phases for both the BEH AMIDE and HSS T3 assays, although the solvent gradient is reversed (starting with a high percentage of organic mobile phase for the HILIC assay and with a high percentage of aqueous mobile phase for the RP assay). Different needle wash solvents are used for the two assays.

• 45.
  Prepare solvent A: Water with 0.1% formic acid and 10 mM ammonium formate:

  • a.
    Add 2.52 g of ammonium formate to 4 L of water.
  • b.
    Add 4 mL of formic acid to 4 L of water.
  • c.
    Cap the bottle and swirl until fully dissolved.
  • d.
    Store at 20°C–25°C for no longer than 10 days.

Solvent A

Reagent	Final concentration	Amount
Ammonium formate	10 mM	2.52 g
Formic acid	0.1%	4 mL
Water	N/A	4 L
Total	N/A	4 L

Storage conditions: 20°C–25°C, maximum storage time is 10 days.

• 46.
  Prepare solvent B: Acetonitrile 0.1% formic acid:

  • a.
    Add 4 mL of formic acid to 4 L of acetonitrile.
  • b.
    Cap the bottle and mix thoroughly.
  • c.
    Store at 20°C–25°C for no longer than 10 days in flammable solvent cabinets.

Note: We typically prepare two 4-L bottles of both solvent A and B every 10 days. Each 4-L bottle lasts about 5 days of interrupted instrument use. The total volume of solvent (sum of A and B) per injection approximates 8 mL for the BEH AMIDE assay and 9 mL for the HSS T3 assay. Twenty-four hours of uninterrupted run roughly equates to 1.1 L of total solvent consumed for the BEH AMIDE assay, and 0.9 L of total solvent consumed for the HSS T3 assay.

• 47.
  Prepare the needle wash solution for the BEH AMIDE assay:

  • a.
    Mix acetonitrile and water in the ratio 50:50%(v/v). Store at 20°C–25°C.
• 48.
  Prepare the needle wash solution for the HSS T3 assay:

  • a.
    Mix methanol and water in the ratio 80:20%(v/v). Store at 20°C–25°C.

CRITICAL: Do not use detergents to wash solvent bottles. Avoid using bottles that have been used to prepare salty solutions or to transfer hydrophobic solvents, especially if they are not of mass spectrometry grade.

CRITICAL: Make sure all glassware is clean to avoid possible contaminations. Periodically inspect, replace the solvent, and wash bottles to avoid algal and bacterial growth (especially for the aqueous mobile phase). Usage of amber colored bottles to avoid light exposure is recommended.

CRITICAL: Use only MS-grade solvents.

Conditioning the LC-MS system for analysis

Timing: 1–2 h

This step provides guidance on how to set up the LC system ahead of analysis.

Note: The methodology requires use of LC and ESI-triple quadrupole MS instrumentation. Common issues, such as leaking pumps or aging column or any hardware and firmware issues of sorts, are something operators are accustomed to coming across and are experienced in troubleshooting, thus they are not discussed here. Troubleshooting steps discussed in the quality control section will not be mentioned again here.

• 49.
  Connect the UPLC column to the LC system.

  • a.
    The BEH AMIDE assay requires a Waters Corp ACQUITY UPLC BEH Amide column with 130 Å, 1.7 μm, 2.1 mm × 100 mm as specifications.
  • b.
    The HSS T3 assay requires a Waters Corp ACQUITY UPLC HSS T3 column, 130 Å, 1.8 μm, 2.1 mm × 100 mm with a 35-μL pre-column mixer to achieve better solvent mixing.
• 50.Equilibrate the column following vendor recommendations.^21,22

Note: Use of guard columns is recommended (key resources table).

Note: The total time needed to complete the data acquisition is highly variable and depends on the total number of samples. Usually, large clinical trials require 4–6 months of uninterrupted instrument run to complete using one LC-MS system. Proper maintenance of the LC-MS/MS system is key for the successful execution of the protocol. It is not uncommon to clean the first optics of the mass spectrometer, replace the ESI electrode, peek tubings, and the pump seals before we start collecting the data from a large clinical trial. Every time the ESI electrode is replaced, we optimize the outer position of the electrode to the probe and the relative position of the ESI probe to the MS inlet, as well as ESI source parameters.

Performing LC-MS/MS analysis

Timing: 15 min+

This step describes the operations needed to set up and start the LC-MS/MS analysis.

Note:Table 2 summarizes the LC conditions and MS settings for both BEH AMIDE and HSS T3 assay. Table 3 lists the targeted metabolites and provides the chemical formulas, exact masses, human metabolome database (HMDB) identifiers, and chemical taxonomy. CAS numbers, Pubchem and KEGG identifiers, and the standardized RefMet nomenclature are detailed in Table S6. The MRM instrument settings used for data acquisition are listed in Table 4.

Note: Timing needed for data acquisition is 15 min per chromatographic run for the BEH AMIDE assay; 20 min per chromatographic run for the HSS T3 assay (including column re-equilibration)

• 51.When ready for LC-MS/MS analysis, centrifuge the LC-MS 96-well plates at +4°C for 5 min at 4000 rpm (3200 × g).
• 52.Place the LC-MS 96-well plates into the LC autosampler or rack changer (if available).
• 53.
  Create the sequence file for each LC-MS 96-well plate and submit in the ‘instrument queue’:

  • a.
    Randomize the experimental sample injection order in the batch file to avoid analytical biases, if needed.
  • b.
    Add a double blank injection at the beginning and at the end of each sequence.
  • c.
    At the beginning of every sequence, include an injection of a quality control plasma.
  • d.
    Add an injection for the blank IS and each calibrator every 40 experimental samples (thus acquiring two sets of blank IS and calibrators data for each LC-MS 96-well plate).
  • e.
    Add a double blank injection after every ‘100% pool calibrator’ run to be able to assess carry-over.
  • f.
    Start data acquisition.
  • g.
    Keep placing the LC-MS 96-well plates and quality control plasma autosampler vials in the system as data acquisition progresses. The number of plates and vials that can be accommodated into the LC system is dependent on the specific instrumentation used.
  • h.
    Refill and/or replace the mobile phase and wash solvent bottles as sample run continues.

Note: Apply proper randomization strategies based on study design and sample characteristics (e.g., randomize by gender, treatment group, patient country of origin).

Note: Solvent B is consumed in larger percentage than solvent A in the BEH AMIDE assay. The opposite occurs for the HSS T3 assay. Monitor the volume of each solvent pumped as the data acquisition progresses to schedule preventive maintenance of the LC system as needed.

Note: A temperature of +7°C is set for the LC rack changer and autosampler to increase stability of metabolites (see Table 2). However, we have not observed significant differences between +7°C-15°C. We recommend not keeping the samples at temperatures ≤+4°C to avoid formation of condensation in the LC system and on top of the aluminum seal foils.

Note: One quantifier ion and one or two qualifier ions are monitored for all targeted metabolites forming multiple reliable fragments though collision-induced dissociation (CID). The most selective and specific fragments are picked as quantifiers, providing better signal-to-noise, showing less interferences from unknown chemical and biological background in different biological matrices, and less interferences from other targeted metabolites and internal standards (see for example the MRM traces of symmetric dimethyl arginine and asymmetric dimethyl arginine in Figure S1). Whenever possible, exclude water-loss fragment as either qualifier or quantifier ions because they provide low-specificity and carry higher background noise. Only one quantifier ion is monitored for each IS to reduce MRM cycle time.

Note: For all targeted metabolites and ISs, synthetic standards were infused during method development to optimize the ion optics in the quadrupoles for maximal ion transmission, fragmentation, and select the quantifier ions. We remind the readers these steps are key to ensure optimal instrument performances but the settings from one instrument cannot necessarily be directly transferred to other instruments. Alternative protocols described in commercial application notes may list source conditions and MRM settings and provide a starting point for users to set up similar methodologies on different systems.^{23,24,25,26,27}

Table 2.

LC-MS/MS instrument settings

Parameter	BEH AMIDE		HSS T3
LC settings
Injection volume	4 μL		3 μL
Total flow	0.8 mL/min		0.6 mL/min
Cooler temperature	7°C		7°C
Column temperature	40°C		40°C
Mobile phase A	Water 0.1% formic acid 10 mM ammonium formate		Water 0.1% formic acid 10 mM ammonium formate
Mobile phase B	Acetonitrile 0.1% formic acid		Acetonitrile 0.1% formic acid
Gradient	Time (min)	Percentage of B	Time (min)	B%
	0	99	0	0.5
	0.67	99	0.89	0.5
	6.33	55	6.7	30
	6.5	40	8.89	98
	7.2	40	10.5	98
	7.4	99	11	0.5
	9.9	99	14	0.5
Valve diverter
Position B switch	0.1 min		0.1 min
Position A switch	7.2 min		10.5 min
Electrospray settings
Curtain gas (CUR)	20		20
Collision gas (CAD)	8		8
IonSpray voltage	±4500 V		±4500 V
Source temperature	550°C		550°C
Ion source gas 1 (GS1)	50		60
Ion source gas 2 (GS2)	50		60
Entrance potential	±10 V		±10 V
Advanced scheduled MRM settings
Target cycle time	0.25 s		0.2 s
Ionization start time	0 min		0 min
Ionization stop time	7.2 min		10.5 min
Resolution Q1	Unit		Unit
Resolution Q3	Unit		Unit
Min. dwell	3 ms		3 ms
Max. dwell	250 ms		250 ms
Primary/secondary	1		1
Trigger threshold	0		0

Table 3.

List of targeted metabolites

Metabolite group ID	Formula	Exact mass	Pubchem ID	CAS #	KEGG ID	HMDB ID	HMDB super class	HMDB class	HMDB sub class
1-methyladenosine	C11H15N5O4	281.1124	27476	15763-06-1	C02494	HMDB0003331	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
1-methylhistamine	C6H11N3	125.0953	3614	501-75-7	C05127	HMDB0000898	Organic nitrogen compounds	Organonitrogen compounds	Amines
1-methyl-L-histidine	C7H11N3O2	169.0851	92105	332-80-9	C01152	HMDB0000001	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
1-methylnicotinamide	C7H9N2O+	137.0715	457	3106-60-3	C02918	HMDB0000699	Organoheterocyclic compounds	Pyridines and derivatives	Pyridinecarboxylic acids and derivatives
2,3-dihydroxybenzoic acid	C7H6O4	154.0266	19	303-38-8	C00196	HMDB0000397	Benzenoids	Benzene and substituted derivatives	Benzoic acids and derivatives
2,3-pyridinedicarboxylic acid	C7H5NO4	167.0219	1066	89-00-9	C03722	HMDB0000232	Organoheterocyclic compounds	Pyridines and derivatives	Pyridinecarboxylic acids and derivatives
2-aminoadipic_acid	C6H11NO4	161.0688	469	542-32-5	C00956	HMDB0000510	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
2-aminobutyric_acid	C4H9NO2	103.0633	80283	1492-24-6	C02356	HMDB0000452	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
2-aminoisobutyric_acid	C4H9NO2	103.0633	6119	62-57-7	C03665	HMDB0001906	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
2-aminooctanoic_acid	C8H17NO2	159.1259	69522	644-90-6	-	HMDB0000991	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
2-deoxyadenosine	C10H13N5O3	251.1018	13730	958-09-8	C00559	HMDB0000101	Nucleosides, nucleotides, and analogs	Purine nucleosides	Purine 2′-deoxyribonucleosides
2-deoxyguanosine	C10H13N5O4	267.0968	135398592	961-07-9	C00330	HMDB0000085	Nucleosides, nucleotides, and analogs	Purine nucleosides	Purine 2′-deoxyribonucleosides
2-hydroxy-3-methylbutyric acid	C5H10O3	118.0630	99823	4026-18-0	-	HMDB0000407	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
2-hydroxybutyric acid	C4H8O3	104.0473	11266	600-15-7	C05984	HMDB0000008	Organic acids and derivatives	Hydroxy acids and derivatives	Alpha hydroxy acids and derivatives
2-hydroxyglutaric acid	C5H8O5	148.0372	43	2889-31-8	C03196	HMDB0000694	Organic acids and derivatives	Hydroxy acids and derivatives	Short-chain hydroxy acids and derivatives
2-hydroxyisocaproic acid	C6H12O3	132.0786	92779	498-36-2	-	HMDB0000665	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
2-isopropylmalic acid	C7H12O5	176.0685	77	3237-44-3	C02504	HMDB0000402	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
2-oxo-4-methylthiobutyric acid	C5H8O3S	148.0194	473	583-92-6	C01180	HMDB0001553	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
3-aminobutyric_acid	C4H9NO2	103.0633	10932	541-48-0	-	HMDB0031654	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
3-aminoisobutyric_acid	C4H9NO2	103.0633	64956	144-90-1	C05145	HMDB0003911	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
3-guanidinopropionic_acid	C4H9N3O2	131.0695	67701	353-09-3	C03065	HMDB0013222	Organic nitrogen compounds	Organonitrogen compounds	Guanidines
3-hydroxybenzoic acid	C7H6O3	138.0317	7420	99-06-9	C00587	HMDB0002466	Benzenoids	Benzene and substituted derivatives	Benzoic acids and derivatives
3-hydroxybutyric acid	C4H8O3	104.0473	441	300-85-6	C01089	HMDB0000357	Organic acids and derivatives	Hydroxy acids and derivatives	Beta hydroxy acids and derivatives
3-hydroxy-DL-kynurenine	C10H12N2O4	224.0797	11811	606-14-4	C03227	HMDB0011631	Organic oxygen compounds	Organooxygen compounds	Carbonyl compounds
3-hydroxyhippuric acid	C9H9NO4	195.0532	450268	1637-75-8	-	HMDB0006116	Benzenoids	Benzene and substituted derivatives	Benzoic acids and derivatives
3-hydroxyisobutyric acid	C4H8O3	104.0473	87	2068-83-9	C06001	HMDB0000023	Organic acids and derivatives	Hydroxy acids and derivatives	Beta hydroxy acids and derivatives
3-hydroxyphenylacetic acid	C8H8O3	152.0473	12122	621-37-4	C05593	HMDB0000440	Benzenoids	Phenols	1-hydroxy-4-unsubstituted benzenoids
3-methyl-2-oxovaleric acid	C6H10O3	130.0630	47	1460-34-0	C00671	HMDB0000491	Organic acids and derivatives	Keto acids and derivatives	Short-chain keto acids and derivatives
3-methylbutyrylglycine	C7H13NO3	159.0895	546304	16284-60-9	-	HMDB0000678	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
3-methylhistamine	C6H11N3	125.0953	69520	644-42-8	-	HMDB0001861	Organic nitrogen compounds	Organonitrogen compounds	Amines
3-methyl-L-histidine	C7H11N3O2	169.0851	64969	368-16-1	C01152	HMDB0000479	Organic acids and derivatives	Organic acids and derivatives	Amino acids, peptides, and analogs
3-methylphenylacetic acid	C9H10O2	150.0681	12121	621-36-3	-	HMDB0002222	Benzenoids	Benzene and substituted derivatives	Toluenes
3-methylthiopropionic acid	C4H8O2S	120.0245	563	646-01-5	C08276	HMDB0001527	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
3-phenyllactic acid	C9H10O3	166.0630	3848	828-01-3	-	HMDB0000779	Phenylpropanoids and polyketides	Phenylpropanoic acids	NA
4-aminobutyric_acid	C5H11NO2	117.0790	119	56-12-2	C00334	HMDB0000112	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
4-hydroxyphenylpyruvic acid	C9H8O4	180.0423	979	156-39-8	C01179	HMDB0000707	Benzenoids	Benzene and substituted derivatives	Phenylpyruvic acid derivatives
4-oxo-L-proline	C5H7NO3	129.0426	107541	4347-18-6	C01877	-	-	-	-
4-pyridoxic acid	C8H9NO4	183.0532	6723	82-82-6	C00847	HMDB0000017	Organoheterocyclic compounds	Pyridines and derivatives	Pyridinecarboxylic acids and derivatives
5-hydroxyindoleacetic acid	C10H9NO3	191.0582	1826	54-16-0	C05635	HMDB0000763	Organoheterocyclic compounds	Indoles and derivatives	Indolyl carboxylic acids and derivatives
5-methoxy-DL-tryptophan	C12H14N2O3	234.1004	151018	2504-22-5	-	HMDB0002339	Organoheterocyclic compounds	Indoles and derivatives	Tryptamines and derivatives
7-methylguanosine	C11H15N5O5	297.1073	135445750	20244-86-4	C20674	HMDB0001107	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
Acadesine	C9H14N4O5	258.0964	17513	2627-69-2	C04663	HMDB0062179	Nucleosides, nucleotides, and analogs	Imidazole ribonucleosides and ribonucleotides	NA
Acetylcarnitine	C9H17NO4	203.1158	7045767	3040-38-8	C02571	HMDB0000201	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Adenine	C5H5N5	135.0545	190	73-24-5	C00147	HMDB0000034	Organoheterocyclic compounds	Imidazopyrimidines	Purines and purine derivatives
Adenosine	C10H13N5O4	267.0968	60961	58-61-7	C00212	HMDB0000050	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
Asymmetric dimethyl arginine	C8H18N4O2	202.1430	123831	30315-93-6	C03626	HMDB0001539	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Agmatine	C5H14N4	130.1218	199	306-60-5	C00179	HMDB0001432	Organic nitrogen compounds	Organonitrogen compounds	Guanidines
Alanine	C3H7NO2	89.0477	5950	56-41-7	C00041	HMDB0000161	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Allantoic acid	C4H8N4O4	176.0546	203	99-16-1	C00499	HMDB0001209	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Allantoin	C4H6N4O3	158.0440	204	97-59-6	C01551	HMDB0000462	Organoheterocyclic compounds	Azoles	Imidazoles
Alpha-ketoglutaric acid	C5H6O5	146.0215	51	328-50-7	C00026	HMDB0000208	Organic acids and derivatives	Keto acids and derivatives	Gamma-keto acids and derivatives
Alpha-ketoisovaleric acid	C5H8O3	116.0473	49	759-05-7	C00141	HMDB0000019	Organic acids and derivatives	Keto acids and derivatives	Short-chain keto acids and derivatives
Arachidonoylcarnitine	C31H54NO4	504.4053	137628528	36816-11-2	-	HMDB0006455	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Arachidoylcarnitine	C27H53NO4	455.3975	53477833	-	-	-	-	-	-
Arginine	C6H14N4O2	174.1117	6322	74-79-3	C00062	HMDB0000517	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Argininosuccinic acid	C10H18N4O6	290.1226	16950	2387-71-5	C03406	HMDB0000052	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Asparagine	C4H8N2O3	132.0535	6267	70-47-3	C00152	HMDB0000168	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Aspartic acid	C4H7NO4	133.0375	5960	56-84-8	C00049	HMDB0000191	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Atrolactic acid	C9H10O3	166.0630	1303	515-30-0	-	HMDB0142137	Benzenoids	Benzene and substituted derivatives	NA
Beta_alanine	C3H7NO2	89.0477	239	107-95-9	C00099	HMDB0000056	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Beta-hydroxyisovaleric acid	C5H10O3	118.0630	69362	625-08-1	-	HMDB0000754	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Betaine	C5H11NO2	117.0790	247	107-43-7	C00719	HMDB0000043	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Beta-muricholic acid	C24H40O5	408.2876	119473	39016-49-4	-	HMDB0000865	Lipids and lipid-like molecules	Steroids and steroid derivatives	Bile acids, alcohols and derivatives
Butenoylcarnitine	C11H19NO4	229.1314	134822127	-	-	-	-	-	-
Butyrylcarnitine	C11H21NO4	231.1471	213144	25576-40-3	C02862	HMDB0002013	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
cAMP	C10H12N5O6P	329.0525	6076	60-92-4	C00575	HMDB0000058	Nucleosides, nucleotides, and analogs	Purine nucleotides	Cyclic purine nucleotides
Carnitine	C7H15NO3	161.1052	10917	541-15-1	C00318	HMDB0000062	Organic nitrogen compounds	Organonitrogen compounds	Quaternary ammonium salts
Carnosine	C9H14N4O3	226.1066	439224	305-84-0	C00386	HMDB0000033	Organic acids and derivatives	Peptidomimetics	Hybrid peptides
CDP-choline	C14H26N4O11P2	488.1073	13804	987-78-0	C00307	HMDB0001413	Nucleosides, nucleotides, and analogs	Pyrimidine nucleotides	Pyrimidine ribonucleotides
CDP-ethanolamine	C11H20N4O11P2	446.0604	123727	3036-18-8	C00570	HMDB0001564	Nucleosides, nucleotides, and analogs	Pyrimidine nucleotides	Pyrimidine ribonucleotides
cGMP	C10H12N5O7P	345.0474	135398570	7665-99-8	C00942	HMDB0001314	Nucleosides, nucleotides, and analogs	Purine nucleotides	Cyclic purine nucleotides
Chenodeoxycholic acid	C24H40O4	392.2927	10133	474-25-9	C02528	HMDB0000518	Lipids and lipid-like molecules	Steroids and steroid derivatives	Bile acids, alcohols and derivatives
Cholesterol sulfate	C27H46O4S	466.3117	65076	1256-86-6	C18043	HMDB0000653	Lipids and lipid-like molecules	Steroids and steroid derivatives	Cholestane steroids
Cholic acid	C24H40O5	408.2876	221493	81-25-4	C00695	HMDB0000619	Lipids and lipid-like molecules	Steroids and steroid derivatives	Bile acids, alcohols and derivatives
Choline	C5H14NO	104.1075	305	62-49-7	C00114	HMDB0000097	Organic nitrogen compounds	Organonitrogen compounds	Quaternary ammonium salts
Citraconic acid	C5H6O4	130.0266	643798	498-23-7	C02226	HMDB0000634	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Citramalic acid	C5H8O5	148.0372	1081	597-44-4	C00815	HMDB0000426	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Citric acid	C6H8O7	192.0270	311	77-92-9	C00158	HMDB0000094	Organic acids and derivatives	Carboxylic acids and derivatives	Tricarboxylic acids and derivatives
Citrulline	C6H13N3O3	175.0957	9750	372-75-8	C00327	HMDB0000904	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
CMPF	C12H16O5	240.0998	123979	86879-39-2	-	HMDB0061112	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Creatine	C4H9N3O2	131.0695	586	57-00-1	C00300	HMDB0000064	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Creatinine	C4H7N3O	113.0589	588	60-27-5	C00791	HMDB0000562	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Cyclic-di-GMP	C20H24N10O14P2	690.0949	135440063	61093-23-0	C16463	-	-	-	-
Cysteine sulfinic acid	C3H7NO4S	153.0096	1549098	1115-65-7	C00606	HMDB0000996	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Cystine	C6H12N2O4S2	240.0239	67678	56-89-3	C00491	HMDB0000192	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Cytidine	C9H13N3O5	243.0855	6175	65-46-3	C00475	HMDB0000089	Nucleosides, nucleotides, and analogs	Pyrimidine nucleosides	NA
Cytosine	C4H5N3O	111.0433	597	71-30-7	C00380	HMDB0000630	Organoheterocyclic compounds	Diazines	Pyrimidines and pyrimidine derivatives
Decadienoylcarnitine	C17H29NO4	311.2097	71464495	128305-29-3	-	HMDB0013325	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Decanoylcarnitine	C17H33NO4	315.2410	10245190	1492-27-9	C03299	HMDB0000651	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
decatrienoylcarnitine	C17H27NO4	309.1940	71464493	-	-	-	-	-	-
Decenoylcarnitine	C17H31NO4	313.2253	129628702	-	-	HMDB0013205	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Deoxycholic acid	C24H40O4	392.2927	222528	83-44-3	C04483	HMDB0000626	Lipids and lipid-like molecules	Steroids and steroid derivatives	Bile acids, alcohols and derivatives
Dihydroorotic acid	C5H6N2O4	158.0328	439216	5988-19-2	C00337	HMDB0003349	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Dodecanoylcarnitine	C19H37NO4	343.2723	168381	25518-54-1	-	HMDB0002250	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Dodecenoylcarnitine	C19H35NO4	341.2566	129664620	-	-	HMDB0013326	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Eicoseneoylcarnitine	C27H51NO4	453.3818	71464507	-	-	-	-	-	-
Ethanolamine	C2H7NO	61.0528	700	141-43-5	C00189	HMDB0000149	Organic nitrogen compounds	Organonitrogen compounds	Amines
Ethylmalonic acid	C5H8O4	132.0423	11756	601-75-2	-	HMDB0000622	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
FAD	C27H33N9O15P2	785.1571	643975	146-14-5	C00016	HMDB0001248	Nucleosides, nucleotides, and analogs	Flavin nucleotides	NA
Fumaric acid	C4H4O4	116.0110	444972	110-17-8	C00122	HMDB0000134	Organic acids and derivatives	Carboxylic acids and derivatives	Dicarboxylic acids and derivatives
Gamma-glutamylvaline	C10H18N2O5	246.1216	7015683	2746-34-1	-	HMDB0011172	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Glucosamine	C6H13NO5	179.0794	439213	3416-24-8	C00329	HMDB0001514	Organic oxygen compounds	Organooxygen compounds	Carbohydrates and carbohydrate conjugates
Glutamic_acid	C5H9NO4	147.0532	33032	56-86-0	C00025	HMDB0000148	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Glutamine	C5H10N2O3	146.0691	5961	56-85-9	C00064	HMDB0000641	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Glutaric_acid	C5H8O4	132.0423	743	110-94-1	C00489	HMDB0000661	Organic acids and derivatives	Carboxylic acids and derivatives	Dicarboxylic acids and derivatives
Glutarylcarnitine	C12H21NO6	275.1369	53481699	102636-82-8	-	HMDB0013130	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Glutathione_disulfide	C20H32N6O12S2	612.1520	65359	27025-41-8	C00127	HMDB0003337	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Glyceric acid	C3H6O4	106.0266	752	473-81-4	C00258	HMDB0000139	Organic oxygen compounds	Organooxygen compounds	Carbohydrates and carbohydrate conjugates
Glycerophosphocholine	C8H20NO6P	257.1028	657272	28319-77-9	C00670	HMDB0000086	Lipids and lipid-like molecules	Glycerophospholipids	Glycerophosphocholines
Glycine	C2H5NO2	75.0320	750	56-40-6	C00037	HMDB0000123	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Guanidinoacetic acid	C3H7N3O2	117.0538	763	352-97-6	C00581	HMDB0000128	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Guanine	C5H5N5O	151.0494	135398634	73-40-5	C00242	HMDB0000132	Organoheterocyclic compounds	Imidazopyrimidines	Purines and purine derivatives
Guanosine	C10H13N5O5	283.0917	135398635	118-00-3	C00387	HMDB0000133	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
Heptanoylcarnitine_glutaconylcarnitine	C12H19NO6	273.1212	91825718	-	-	HMDB0013129	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hexadecadienoylcarnitine	C23H41NO4	395.3036	53481687	1911579-97-9	-	HMDB0013334	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hexanoylcarnitine	C13H25NO4	259.1784	3246938	22671-29-0	-	HMDB0000756	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hexenoylcarnitine	C13H23NO4	257.1627	129846791	-	-	HMDB0013161	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Histamine	C5H9N3	111.0796	774	51-45-6	C00388	HMDB0000870	Organic nitrogen compounds	Organonitrogen compounds	Amines
Histidine	C6H9N3O2	155.0695	6274	71-00-1	C00135	HMDB0000177	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Histidinol	C6H11N3O	141.0902	776	501-28-0	C00860	HMDB0003431	Organic nitrogen compounds	Organonitrogen compounds	Amines
Homoarginine	C7H16N4O2	188.1273	9085	156-86-5	C01924	HMDB0000670	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Homocitrulline	C7H15N3O3	189.1113	65072	1190-49-4	C02427	HMDB0000679	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Homocysteic_acid	C4H9NO5S	183.0201	177491	14857-77-3	C16511	HMDB0002205	Organic acids and derivatives	Carboxylic acids and derivatives	Direct Parent
Homoserine	C4H9NO3	119.0582	12647	672-15-1	C00263	HMDB0000719	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Hydroxybutyrylcarnitine	C11H21NO5	247.1420	-	1469900-92-2	-	HMDB0013127	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hydroxydecanoylcarnitine	C17H33NO5	331.2359	129691748	-	-	-	-	-	-
Hydroxydodecenoylcarnitine	C19H35NO5	357.2515	71464579	-	-	-	-	-	-
Hydroxyisovalerylcarnitine	C12H23NO5	261.1576	53915061	99159-87-2	-	HMDB0061189	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hydroxylysine	C6H14N2O3	162.1004	3032849	1190-94-9	C16741	HMDB0000450	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Hydroxyoctenoylcarnitine	C15H27NO5	301.1889	-	-	-	-	-	-	-
Hydroxypalmitoylcarnitine	C23H45NO5	415.3298	126456228	195207-76-2	-	HMDB0013336	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hydroxyproline	C5H9NO3	131.0582	5810	51-35-4	C01157	HMDB0000725	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Hydroxytetradecenoylcarnitine	C21H39NO5	385.2828	129849063	-	-	HMDB0013330	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Hydroxytryptophan	C11H12N2O3	220.0848	439280	08-09-4350	C00643	HMDB0000472	Organoheterocyclic compounds	Indoles and derivatives	Tryptamines and derivatives
Hypotaurine	C2H7NO2S	109.0198	107812	300-84-5	C00519	HMDB0000965	Organic acids and derivatives	Sulfinic acids and derivatives	Sulfinic acids
Hypoxanthine	C5H4N4O	136.0385	135398638	68-94-0	C00262	HMDB0000157	Organoheterocyclic compounds	Imidazopyrimidines	Purines and purine derivatives
Imidazole	C3H4N2	68.0374	795	288-32-4	C01589	HMDB0001525	Organoheterocyclic compounds	Azoles	Imidazoles
Imidazole-4-acetic acid	C5H6N2O2	126.0429	96215	645-65-8	C02835	HMDB0002024	Organoheterocyclic compounds	Azoles	Imidazoles
Indole-3-carboxylic acid	C9H7NO2	161.0477	96215	645-65-8	C02835	HMDB0002024	Organoheterocyclic compounds	Azoles	Imidazoles
Inosine	C10H12N4O5	268.0808	135398641	58-63-9	C00294	HMDB0000195	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
Isocitric acid	C6H8O7	192.0270	1198	320-77-4	C00311	HMDB0000193	Organic acids and derivatives	Carboxylic acids and derivatives	Tricarboxylic acids and derivatives
Isoleucine	C6H13NO2	131.0946	6306	73-32-5	C00407	HMDB0000172	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Isovalerylcarnitine	C12H23NO4	245.1627	6426851	31023-24-2	C20826	HMDB0000688	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Itaconic acid	C5H6O4	130.0266	811	97-65-4	C00490	HMDB0002092	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Ketoleucine	C6H10O3	130.0630	70	816-66-0	C00233	HMDB0000695	Organic acids and derivatives	Keto acids and derivatives	Short-chain keto acids and derivatives
Kynurenic acid	C10H7NO3	189.0426	3845	492-27-3	C01717	HMDB0000715	Organoheterocyclic compounds	Quinolines and derivatives	Quinoline carboxylic acids
Kynurenine	C10H12N2O3	208.0848	161166	2922-83-0	C00328	HMDB0000684	Organic oxygen compounds	Organooxygen compounds	Carbonyl compounds
Lactic acid	C3H6O3	90.0317	612	79-33-4	C00186	HMDB0000190	Organic acids and derivatives	Hydroxy acids and derivatives	Alpha hydroxy acids and derivatives
Leucine	C6H13NO2	131.0946	6106	61-90-5	C00123	HMDB0000687	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Linoleoylcarnitine	C25H45NO4	423.3349	6450015	36816-10-1	-	HMDB0006469	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Lipoic acid	C8H14O2S2	206.0435	6112	1200-22-2	C16241	HMDB0001451	Organoheterocyclic compounds	Dithiolanes	Lipoic acids and derivatives
Lithocholic acid	C24H40O3	376.2977	9903	434-13-9	C03990	HMDB0000761	Lipids and lipid-like molecules	Steroids and steroid derivatives	Bile acids, alcohols and derivatives
Lysine	C6H14N2O2	146.1055	5962	56-87-1	C00047	HMDB0000182	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Maleic acid	C4H4O4	116.0110	444266	110-16-7	C01384	HMDB0000176	Organic acids and derivatives	Carboxylic acids and derivatives	Dicarboxylic acids and derivatives
Malic acid	C4H6O5	134.0215	222656	97-67-6	C00149	HMDB0000744	Organic acids and derivatives	Hydroxy acids and derivatives	Beta hydroxy acids and derivatives
Malonylcarnitine	C10H17NO6	247.1056	91825606	910825-21-7	-	HMDB0002095	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Methionine	C5H11NO2S	149.0511	6137	63-68-3	C00073	HMDB0000696	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Methionine_sulfoxide	C5H11NO3S	165.0460	158980	3226-65-1	C02989	HMDB0002005	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Methylimidazole acetic acid	C6H8N2O2	140.0586	75810	2625-49-2	C05828	HMDB0002820	Organoheterocyclic compounds	Azoles	Imidazoles
Methylmalonic acid	C4H6O4	118.0266	487	516-05-2	C02170	HMDB0000202	Organic acids and derivatives	Carboxylic acids and derivatives	Dicarboxylic acids and derivatives
Methylmalonylcarnitine	C11H19NO6	261.1212	-	256928-70-8	-	HMDB0013133	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Methylsuccinic acid	C5H8O4	132.0423	10349	498-21-5	-	HMDB0001844	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Mevalonic_acid	C6H12O4	148.0736	449	150-97-0	C00418	HMDB0000227	Lipids and lipid-like molecules	Fatty Acyls	Fatty acids and conjugates
Myristoylcarnitine	C21H41NO4	371.3036	53477791	25597-07-3	-	HMDB0005066	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
N-acetylalanine	C5H9NO3	131.0582	88064	97-69-8	-	HMDB0000766	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylaspartic_acid	C6H9NO5	175.0481	65065	997-55-7	C01042	HMDB0000812	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylaspartylglutamic_acid	C11H16N2O8	304.0907	-	3106-85-2	C12270	HMDB0001067	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylglucosamine	C8H15NO6	221.0899	24139	14131-68-1	C03878	HMDB0000803	Organic oxygen compounds	Organooxygen compounds	Carbohydrates and carbohydrate conjugates
N-acetylglutamic_acid	C7H11NO5	189.0637	70914	1188-37-0	C00624	HMDB0001138	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylglycine	C4H7NO3	117.0426	10972	543-24-8	-	HMDB0000532	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylisoleucine	C8H15NO3	173.1052	7036275	3077-46-1	-	HMDB0061684	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyl-L-citrulline	C8H15N3O4	217.1063	656979	33965-42-3	C15532	HMDB0000856	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyl-L-cysteine	C5H9NO3S	163.0303	12035	616-91-1	C06809	HMDB0001890	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylleucine	C8H15NO3	173.1052	70912	1188-21-2	C02710	HMDB0011756	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyl-L-glutamine	C7H12N2O4	188.0797	182230	2490-97-3	-	HMDB0006029	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyl-L-histidine	C8H11N3O3	197.0800	75619	01-02-2497	C02997	HMDB0032055	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyl-L-ornithine	C7H14N2O3	174.1004	439232	6205-08-9	C00437	HMDB0003357	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylmethionine	C7H13NO3S	191.0616	448580	65-82-7	C02712	HMDB0011745	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylphenylalanine	C11H13NO3	207.0895	74839	2018-61-3	C03519	HMDB0000512	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylproline	C7H11NO3	157.0739	66141	68-95-1	-	HMDB0094701	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylputrescine	C6H14N2O	130.1106	122356	5699-41-2	C02714	HMDB0002064	Organic nitrogen compounds	Organonitrogen compounds	Amines
N-acetylserine	C5H9NO4	147.0532	65249	16354-58-8	-	HMDB0002931	Organic acids and derivatives	-	-
N-acetylthreonine	C6H11NO4	161.0688	152204	17093-74-2	-	HMDB0062557	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyltryptophan	C13H14N2O3	246.1004	700653	1218-34-4	-	HMDB0013713	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetyltyrosine	C11H13NO4	223.0845	68310	537-55-3	-	HMDB0000866	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-acetylvaline	C7H13NO3	159.0895	66789	96-81-1	-	HMDB0011757	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Nalpha-acetyl-L-arginine	C8H16N4O3	216.1222	67427	155-84-0	-	HMDB0004620	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Nalpha-acetyllysine	C8H16N2O3	188.1161	92907	1946-82-3	C12989	HMDB0000446	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-butyrylglycine	C6H11NO3	145.0739	88412	20208-73-5	-	HMDB0000808	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-caprylylglycine	C10H19NO3	201.1365	84290	14246-53-8	-	HMDB0000832	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Nepsilon-acetyllysine	C8H16N2O3	188.1161	92832	692-04-6	C02727	HMDB0000206	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-formylkynurenine	C11H12N2O4	236.0797	439788	08-11-3978	C02700	HMDB0001200	Organic oxygen compounds	Organooxygen compounds	Carbonyl compounds
N-furoylglycine	C7H7NO4	169.0375	21863	5657-19-2	-	HMDB0000439	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Nicotinamide	C6H6N2O	122.0480	936	98-92-0	C00153	HMDB0001406	Organoheterocyclic compounds	Pyridines and derivatives	Pyridinecarboxylic acids and derivatives
Nicotinic acid	C6H5NO2	123.0320	938	59-67-6	C00253	HMDB0001488	Organoheterocyclic compounds	Pyridines and derivatives	Pyridinecarboxylic acids and derivatives
N-methylaspartic acid	C5H9NO4	147.0531	22880	6384-92-5	C12269	HMDB0002393	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-myristoylglycine	C16H31NO3	285.2304	72348	14246-55-0	-	HMDB0013250	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
NN-dimethylglycine	C4H9NO2	103.0633	673	1118-68-9	C01026	HMDB0000092	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-oleoylglycine	C20H37NO3	339.2773	6436908	2601-90-3	-	HMDB0013631	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-palmitoylglycine	C18H35NO3	313.2617	151008	158305-64-7	-	HMDB0013034	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
N-propionylglycine	C5H9NO3	131.0582	98681	21709-90-0	-	HMDB0000783	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Octadecanoylcarnitine	C25H49NO4	427.3662	52922056	25597-09-5	-	HMDB0000848	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Octanoylcarnitine	C15H29NO4	287.2097	11953814	25243-95-2	C02838	HMDB0000791	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Octenoylcarnitine	C15H27NO4	285.1940	129692230	-	-	HMDB0013324	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Oleoyl L-carnitine	C25H47NO4	425.3505	46907933	38677-66-6	-	HMDB0005065	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
O-phosphotyrosine	C9H12NO6P	261.0402	30819	21820-51-9	C06501	HMDB0006049	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Ornithine	C5H12N2O2	132.0899	6262	70-26-8	C00077	HMDB0000214	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Palmitoleoylcarnitine	C23H43NO4	397.3192	71464547	329321-94-0	-	HMDB0013207	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Palmityol-L-carnitine	C23H45NO4	399.3349	11953816	2364-67-2	C02990	HMDB0000222	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Pantothenic acid	C9H17NO5	219.1107	6613	79-83-4	C00864	HMDB0000210	Organic oxygen compounds	Organooxygen compounds	Alcohols and polyols
Phenylacetylglutamine	C13H16N2O4	264.1110	92258	28047-15-6	C04148	HMDB0006344	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Phenylalanine	C9H11NO2	165.0790	6140	63-91-2	C00079	HMDB0000159	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Phenylpyruvic acid	C9H8O3	164.0473	997	156-06-9	C00166	HMDB0000205	Benzenoids	Benzene and substituted derivatives	Phenylpyruvic acid derivatives
Phosphoenolpyruvic acid	C3H5O6P	167.9824	1005	138-08-9	C00074	HMDB0000263	Organic acids and derivatives	Organic phosphoric acids and derivatives	Phosphate esters
Picolinic acid	C6H5NO2	123.0320	1018	98-98-6	C10164	HMDB0002243	Organoheterocyclic compounds	Pyridines and derivatives	Pyridines and derivatives
Pipecolic_acid	C6H11NO2	129.0790	849	535-75-1	C00408	HMDB0000070	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Pivaloylglycine	C7H13NO3	159.0895	2608784	23891-96-5	-	-	-	-	-
Proline	C5H9NO2	115.0633	145742	147-85-3	C00148	HMDB0000162	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Propionylcarnitine	C10H19NO4	217.1314	188824	20064-19-1	C03017	HMDB0000824	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Purine	C5H4N4	120.0436	1044	120-73-0	C15587	HMDB0001366	Organoheterocyclic compounds	Imidazopyrimidines	Purines and purine derivatives
Putrescine	C4H12N2	88.1000	1045	111-60-1	C00134	HMDB0001414	Organic nitrogen compounds	Organonitrogen compounds	Amines
Pyridoxamine	C8H12N2O2	168.0899	1052	85-87-0	C00534	HMDB0001431	Organoheterocyclic compounds	Pyridines and derivatives	Pyridoxamines
Pyridoxine	C8H11NO3	169.0739	1054	65-23-6	C00314	HMDB0000239	Organoheterocyclic compounds	Pyridines and derivatives	Pyridoxines
Pyroglutamic_acid	C5H7NO3	129.0426	7405	98-79-3	C01879	HMDB0000267	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Pyruvic acid	C3H4O3	88.0160	1060	127-17-3	C00022	HMDB0000243	Organic acids and derivatives	Keto acids and derivatives	Alpha-keto acids and derivatives
Riboflavin	C17H20N4O6	376.1383	493570	83-88-5	C00255	HMDB0000244	Organoheterocyclic compounds	Pteridines and derivatives	Alloxazines and isoalloxazines
S-adenosyl-L-homocysteine	C14H20N6O5S	384.1216	439155	979-92-0	C00021	HMDB0000939	Nucleosides, nucleotides, and analogs	5′-deoxyribonucleosides	5′-deoxy-5′-thionucleosides
S-adenosyl-L-methionine	C15H22N6O5S	398.1451	34755	485-80-3	C00019	HMDB0001185	Nucleosides, nucleotides, and analogs	5′-deoxyribonucleosides	5′-deoxy-5′-thionucleosides
Sarcosine	C3H7NO2	89.0477	1088	107-97-1	C00213	HMDB0000271	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Symmetric dimethyl arginine	C8H18N4O2	202.1430	169148	30344-00-4	-	HMDB0003334	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Serine	C3H7NO3	105.0426	5951	56-45-1	C00065	HMDB0000187	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Shikimic_acid	C7H10O5	174.0528	8742	138-59-0	C00493	HMDB0003070	Organic oxygen compounds	Organooxygen compounds	Alcohols and polyols
S-methylcysteine	C4H9NO2S	135.0354	24417	1187-84-4	C22040	HMDB0002108	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
S-methylcysteine_sulfoxide	C4H9NO3S	151.0.0303	99483	6853-87-8	-	HMDB0029432	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
S-methylglutathione	C11H19N3O6S	321.0995	115260	2922-56-7	C11347	-	-	-	-
S-methylthioadenosine	C11H15N5O3S	297.0896	439176	2457-80-9	C00170	HMDB0001173	Nucleosides, nucleotides, and analogs	5′-deoxyribonucleosides	5′-deoxy-5′-thionucleosides
Succinic acid	C4H6O4	118.0266	1110	110-15-6	C00042	HMDB0000254	Organic acids and derivatives	Carboxylic acids and derivatives	Dicarboxylic acids and derivatives
Succinoadenosine	C14H17N5O8	383.1077	20849086	4542-23-8	-	HMDB0000912	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
Succinylcarnitine	C11H19NO6	261.1212	131802075	256928-74-2	-	HMDB0061717	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Taurine	C2H7NO3S	125.0147	1123	107-35-7	C00245	HMDB0000251	Organic acids and derivatives	Organic sulfonic acids and derivatives	Organosulfonic acids and derivatives
Tetradecadienoylcarnitine	C21H37NO4	367.2723	129691756	-	-	HMDB0013331	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Tetradecenoylcarnitine	C21H39NO4	369.2879	129631631	835598-21-5	-	HMDB0002014	Lipids and lipid-like molecules	Fatty Acyls	Fatty acid esters
Thiamine	C12H17N4OS+	265.1123	1130	70-16-6	C00378	HMDB0000235	Organoheterocyclic compounds	Diazines	Pyrimidines and pyrimidine derivatives
Threonine	C4H9NO3	119.0582	6288	72-19-5	C00188	HMDB0000167	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Thymidine	C10H14N2O5	242.0903	5789	50-89-5	C00214	HMDB0000273	Nucleosides, nucleotides, and analogs	Pyrimidine nucleosides	Pyrimidine 2′-deoxyribonucleosides
Thymine	C5H6N2O2	126.0429	1135	65-71-4	C00178	HMDB0000262	Organoheterocyclic compounds	Diazines	Pyrimidines and pyrimidine derivatives
Trans-aconitic acid	C6H6O6	174.0164	444212	4023-65-8	C02341	HMDB0000958	Organic acids and derivatives	Carboxylic acids and derivatives	Tricarboxylic acids and derivatives
Trimethylamine_N-oxide	C3H9NO	75.0684	1145	1184-78-7	C01104	HMDB0000925	Organic nitrogen compounds	Organonitrogen compounds	Aminoxides
Tryptophan	C11H12N2O2	204.0899	6305	73-22-3	C00078	HMDB0000929	Organoheterocyclic compounds	Indoles and derivatives	Indoles and derivatives
Tyrosine	C9H11NO3	181.0739	6057	60-18-4	C00082	HMDB0000158	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
UDP-GlcNac	C17H27N3O17P2	607.0816	10705	528-04-1	C00043	HMDB0000290	Nucleosides, nucleotides, and analogs	Pyrimidine nucleotides	Pyrimidine nucleotide sugars
Uracil	C4H4N2O2	112.0273	1174	66-22-8	C00106	HMDB0000300	Organoheterocyclic compounds	Diazines	Pyrimidines and pyrimidine derivatives
Urea	CH4N2O	60.0324	1176	57-13-6	C00086	HMDB0000294	Organic acids and derivatives	Organic carbonic acids and derivatives	Ureas
Uric acid	C5H4N4O3	168.0283	1175	69-93-2	C00366	HMDB0000289	Organoheterocyclic compounds	Imidazopyrimidines	Purines and purine derivatives
Uridine	C9H12N2O6	244.0695	6029	58-96-8	C00299	HMDB0000296	Nucleosides, nucleotides, and analogs	Pyrimidine nucleosides	NA
Ursodeoxycholic acid	C24H40O4	392.2927	31401	128-13-2	C07880	HMDB0000946	Lipids and lipid-like molecules	Steroids and steroid derivatives	Bile acids, alcohols and derivatives
Valine	C5H11NO2	117.0790	6287	72-18-4	C00183	HMDB0000883	Organic acids and derivatives	Carboxylic acids and derivatives	Amino acids, peptides, and analogs
Xanthine	C5H4N4O2	152.0334	1188	69-89-6	C00385	HMDB0000292	Organoheterocyclic compounds	Imidazopyrimidines	Purines and purine derivatives
Xanthosine	C10H12N4O6	284.0757	64959	146-80-5	C01762	HMDB0000299	Nucleosides, nucleotides, and analogs	Purine nucleosides	NA
Xanthurenic acid	C10H7NO4	205.0375	5699	59-00-7	C02470	HMDB0000881	Organoheterocyclic compounds	Quinolines and derivatives	Quinoline carboxylic acids

Table 4.

Targeted metabolite MRM settings and retention times

Assay	Polarity	Metabolite ID	RT (min)	MRM window(s)	Molecular ion	Q1 (m/z)	Q3 (m/z)	DP (V)	CE (V)	CXP (V)
BEH AMIDE	(+)	1-methyladenosine	3.7	25	[M + H]+	282	150.1 (Q)	60	29	14
BEH AMIDE	(+)	1-methyladenosine	3.7	25	[M + H]+	282	133.1	60	59	12
BEH AMIDE	(+)	1-methylhistamine	4	55	[M + H]+	126.1	109.1 (Q)	36	19	10
BEH AMIDE	(+)	1-methylhistamine	4	55	[M + H]+	126.1	67.9	36	27	10
BEH AMIDE	(+)	1-methyl-L-histidine	4.8	100	[M + H]+	170.1	124.1 (Q)	21	21	18
BEH AMIDE	(+)	1-methyl-L-histidine	4.8	100	[M + H]+	170.1	81	21	55	12
BEH AMIDE	(+)	1-methylnicotinamide	3	25	[M]+	137	78.0 (Q)	56	35	10
HSS T3	(−)	2,3-dihydroxybenzoic acid	4.1	30	[M−H]−	152.9	109.0 (Q)	−20	−22	−9
HSS T3	(−)	2,3-dihydroxybenzoic acid	4.1	30	[M−H]−	152.9	107.9	−20	−34	−13
HSS T3	(−)	2,3-pyridinedicarboxylic acid	0.6	60	[M−H]−	165.9	121.9 (Q)	−15	−14	−13
HSS T3	(−)	2,3-pyridinedicarboxylic acid	0.6	60	[M−H]−	165.9	77.9	−15	−20	−11
BEH AMIDE	(+)	2-aminoadipic_acid	4.2	25	[M + H]+	162	98.1 (Q)	36	21	14
BEH AMIDE	(+)	2-aminoadipic_acid	4.2	25	[M + H]+	162	144.1	36	15	10
BEH AMIDE	(+)	2-aminobutyric acid	3.9	25	[M + H]+	103.9	58.1	40	15	10
BEH AMIDE	(+)	2-aminobutyric acid	3.9	25	[M + H]+	103.9	41.1 (Q)	40	31	18
BEH AMIDE	(+)	2-aminoisobutyric acid	3.9	25	[M + H]+	104	59.0 (Q)	40	27	10
BEH AMIDE	(+)	2-aminooctanoic acid	3.1	25	[M + H]+	160.1	114.2 (Q)	31	15	10
BEH AMIDE	(+)	2-aminooctanoic acid	3.1	25	[M + H]+	160.1	55.1	31	29	10
BEH AMIDE	(+)	2-deoxyadenosine	2.4	25	[M + H]+	252	136.1 (Q)	61	21	18
BEH AMIDE	(+)	2-deoxyadenosine	2.4	25	[M + H]+	252	119	61	59	16
BEH AMIDE	(+)	2-deoxyguanosine	3.3	25	[M + H]+	268.1	152.1 (Q)	81	15	18
BEH AMIDE	(+)	2-deoxyguanosine	3.3	25	[M + H]+	268.1	135.1	81	47	10
HSS T3	(−)	2-hydroxy-3-methylbutyric acid	3.7	30	[M−H]−	117.1	71.0 (Q)	−20	−16	−9
HSS T3	(−)	2-hydroxy-3-methylbutyric acid	3.7	30	[M−H]−	117.1	45	−20	−16	−7
HSS T3	(−)	2-hydroxybutyric acid	1.6	60	[M−H]−	103	56.9 (Q)	−15	−16	−7
HSS T3	(−)	2-hydroxybutyric acid	1.6	60	[M−H]−	103	44.9	−15	−16	−7
HSS T3	(−)	2-hydroxyglutaric acid	0.9	60	[M−H]−	146.9	129.0 (Q)	−20	−14	−9
HSS T3	(−)	2-hydroxyglutaric acid	0.9	60	[M−H]−	146.9	85	−20	−20	−9
HSS T3	(−)	2-hydroxyisocaproic acid	5.1	30	[M−H]−	130.9	85.0 (Q)	−25	−18	−11
HSS T3	(−)	2-hydroxyisocaproic acid	5.1	30	[M−H]−	130.9	68.9	−25	−28	−9
HSS T3	(−)	2-isopropylmalic acid	4.2	30	[M−H]−	174.9	115.0 (Q)	−25	−20	−13
HSS T3	(−)	2-isopropylmalic acid	4.2	30	[M−H]−	174.9	113	−25	−20	−7
HSS T3	(−)	2-oxo-4-methylthiobutyric acid	5.1	40	[M−H]−	147.1	99.0 (Q)	−10	−12	−11
HSS T3	(−)	2-oxo-4-methylthiobutyric acid	5.1	40	[M−H]−	147.1	47	−10	−28	−7
BEH AMIDE	(+)	3-aminobutyric acid	3.7	25	[M + H]+	104	44.1 (Q)	40	15	8
BEH AMIDE	(+)	3-aminobutyric acid	3.7	25	[M + H]+	104	86	40	11	14
BEH AMIDE	(+)	3-aminoisobutyric acid	3.7	25	[M + H]+	104	86	40	11	12
BEH AMIDE	(+)	3-aminoisobutyric acid	3.7	25	[M + H]+	104	57.1 (Q)	40	19	10
BEH AMIDE	(+)	3-guanidinopropionic acid	3.6	25	[M + H]+	132	72.0 (Q)	21	21	12
BEH AMIDE	(+)	3-guanidinopropionic acid	3.6	25	[M + H]+	132	90	21	17	14
HSS T3	(−)	3-hydroxybenzoic acid	4.8	30	[M−H]−	136.9	93.0 (Q)	−15	−16	−11
HSS T3	(−)	3-hydroxybutyric acid	1.5	60	[M−H]−	103.1	59.0 (Q)	−15	−14	−9
HSS T3	(−)	3-hydroxybutyric acid	1.5	60	[M−H]−	103.1	41	−15	−32	−19
BEH AMIDE	(+)	3-hydroxy-DL-kynurenine	3.8	35	[M + H]+	225	208.0 (Q)	31	13	14
BEH AMIDE	(+)	3-hydroxy-DL-kynurenine	3.8	35	[M + H]+	225	162.2	31	27	12
HSS T3	(+)	3-hydroxyhyppuric acid	3.9	40	[M + H]+	196	121.0 (Q)	71	19	14
HSS T3	(+)	3-hydroxyhyppuric acid	3.9	40	[M + H]+	196	93	71	37	12
HSS T3	(+)	3-hydroxyhyppuric acid	3.9	40	[M + H]+	196	65	71	49	10
HSS T3	(−)	3-hydroxyisobutyric acid	1.7	60	[M−H]−	103.1	73.0 (Q)	−10	−16	−11
HSS T3	(−)	3-hydroxyphenylacetic acid	5	30	[M−H]−	150.9	107.0 (Q)	−10	−12	−7
HSS T3	(−)	3-hydroxyphenylacetic acid	5	30	[M−H]−	150.9	65	−10	−32	−9
HSS T3	(−)	3-methyl-2-oxovaleric acid	3.7	40	[M−H]−	129	129.0 (Q)	−10	−5	−5
HSS T3	(+)	3-methylbutyrylglycine	3.9	40	[M + H]+	160.1	57.0 (Q)	71	21	12
HSS T3	(+)	3-methylbutyrylglycine	3.9	40	[M + H]+	160.1	76	71	11	10
HSS T3	(+)	3-methylbutyrylglycine	3.9	40	[M + H]+	160.1	85	71	13	12
BEH AMIDE	(+)	3-methylhistamine	4.7	90	[M + H]+	126.1	109.0 (Q)	46	21	14
BEH AMIDE	(+)	3-methylhistamine	4.7	90	[M + H]+	126.1	96.1	46	27	14
BEH AMIDE	(+)	3-methyl-L-histidine	5.2	100	[M + H]+	170.1	96	21	27	14
BEH AMIDE	(+)	3-methyl-L-histidine	5.2	100	[M + H]+	170.1	95 (Q)	21	41	14
HSS T3	(−)	3-methylphenylacetic acid	7.7	40	[M−H]−	148.9	105.0 (Q)	−10	−12	−9
HSS T3	(−)	3-methylthiopropionic acid	4.3	30	[M−H]−	118.9	47.0 (Q)	−5	−18	−7
HSS T3	(−)	3-phenyllactic acid	5.6	30	[M−H]−	164.9	147.0 (Q)	−20	−16	−7
HSS T3	(−)	3-phenyllactic acid	5.6	30	[M−H]−	164.9	103	−20	−22	−11
BEH AMIDE	(+)	4-aminobutyric acid	3.7	25	[M + H]+	104	87.0 (Q)	40	15	14
BEH AMIDE	(+)	4-aminobutyric acid	3.7	25	[M + H]+	104	69	40	21	10
HSS T3	(−)	4-hydroxyphenylpyruvic acid	3.1	30	[M−H]−	178.9	106.9 (Q)	−10	−12	−13
BEH AMIDE	(+)	4-oxo-L-proline_Pipecolic acid	3.8	25	[M + H]+	130	84.1 (Q)	41	15	12
BEH AMIDE	(+)	4-oxo-L-proline_Pipecolic acid	3.8	25	[M + H]+	130	56	41	25	10
HSS T3	(−)	4-pyridoxic acid	3.2	30	[M−H]−	182	138.0 (Q)	−15	−18	−9
HSS T3	(−)	4-pyridoxic acid	3.2	30	[M−H]−	182	108	−15	−28	−7
HSS T3	(+)	5-hydroxyindoleacetic acid	4.6	30	[M + H]+	192.1	146.1 (Q)	36	23	12
HSS T3	(+)	5-hydroxyindoleacetic acid	4.6	30	[M + H]+	192.1	91.1	36	47	12
BEH AMIDE	(+)	5-methoxy-DL-tryptophan	3.4	25	[M + H]+	235.1	218.2 (Q)	26	15	16
BEH AMIDE	(+)	5-methoxy-DL-tryptophan	3.4	25	[M + H]+	235.1	176.2	26	25	12
BEH AMIDE	(+)	7-methylguanosine	3.8	25	[M + H]+	298	166.1 (Q)	11	21	12
BEH AMIDE	(+)	7-methylguanosine	3.8	25	[M + H]+	298	149.1	11	53	14
BEH AMIDE	(+)	Acadesine	2.9	25	[M + H]+	259	127.1 (Q)	56	15	10
BEH AMIDE	(+)	Acadesine	2.9	25	[M + H]+	259	110.1	56	33	18
BEH AMIDE	(+)	Acetylcarnitine	2.9	40	[M + H]+	204	85.0 (Q)	51	25	12
BEH AMIDE	(+)	Acetylcarnitine	2.9	40	[M + H]+	204	145	51	17	14
BEH AMIDE	(+)	Adenine	2.6	25	[M + H]+	136.1	119.0 (Q)	81	31	14
BEH AMIDE	(+)	Adenine	2.6	25	[M + H]+	136.1	92	81	39	14
BEH AMIDE	(+)	Adenosine	2.6	35	[M + H]+	268	136.2 (Q)	61	23	10
BEH AMIDE	(+)	Adenosine	2.6	35	[M + H]+	268	119	61	65	14
BEH AMIDE	(+)	ADMA	4.7	30	[M + H]+	203.1	70.0 (Q)	56	31	10
BEH AMIDE	(+)	ADMA	4.7	30	[M + H]+	203.1	158.1	56	21	12
BEH AMIDE	(+)	Agmatine	4.9	35	[M + H]+	131.1	72.0 (Q)	61	21	12
BEH AMIDE	(+)	Agmatine	4.9	35	[M + H]+	131.1	60	61	15	10
BEH AMIDE	(+)	Alanine	4.1	25	[M + H]+	90.1	44.1	20	17	20
BEH AMIDE	(+)	Alanine	4.1	25	[M + H]+	90.1	45.0 (Q)	20	43	20
BEH AMIDE	(+)	Allantoic acid	4.2	25	[M + H]+	177.1	134.0 (Q)	66	11	10
BEH AMIDE	(+)	Allantoic acid	4.2	25	[M + H]+	177.1	61	66	15	10
HSS T3	(+)	Allantoin	0.6	60	[M + H]+	159	116.1 (Q)	61	11	14
HSS T3	(+)	Allantoin	0.6	60	[M + H]+	159	61.1	61	13	10
HSS T3	(−)	Alpha-ketoglutaric acid	0.6	60	[M−H]−	144.9	101.0 (Q)	−10	−12	−11
HSS T3	(−)	Alpha-ketoglutaric acid	0.6	60	[M−H]−	144.9	56.9	−10	−16	−7
HSS T3	(−)	Alpha-ketoisovaleric acid	1.6	60	[M−H]−	115	115.0 (Q)	−10	−5	−5
BEH AMIDE	(+)	Arachidonoylcarnitine	2.1	30	[M + H]+	448.4	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Arachidoylcarnitine	2	30	[M + H]+	456.4	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Arginine	5.1	35	[M + H]+	175.1	70.1 (Q)	51	35	12
BEH AMIDE	(+)	Arginine	5.1	35	[M + H]+	175.1	116.2	51	19	14
BEH AMIDE	(+)	Argininosuccinic acid	5.2	90	[M + H]+	291	70.1 (Q)	66	53	12
BEH AMIDE	(+)	Argininosuccinic acid	5.2	90	[M + H]+	291	116	66	27	14
BEH AMIDE	(+)	Asparagine	4.5	25	[M + H]+	133.1	74.1 (Q)	71	21	16
BEH AMIDE	(+)	Asparagine	4.5	25	[M + H]+	133.1	87	71	13	14
BEH AMIDE	(+)	Aspartic acid	4.8	90	[M + H]+	134	74.0 (Q)	61	19	12
BEH AMIDE	(+)	Aspartic acid	4.8	90	[M + H]+	134	88	61	15	12
HSS T3	(−)	Atrolactic acid	5.4	30	[M−H]−	165	120.9 (Q)	−20	−14	−7
HSS T3	(−)	Atrolactic acid	5.4	30	[M−H]−	165	118.9	−20	−14	−13
BEH AMIDE	(+)	Beta_alanine	3.9	25	[M + H]+	90	72.0 (Q)	61	11	10
BEH AMIDE	(+)	Beta_alanine	3.9	25	[M + H]+	90	45	61	47	20
HSS T3	(−)	Beta-hydroxyisovaleric acid	3.2	30	[M−H]−	117	59.0 (Q)	−15	−14	−7
HSS T3	(−)	Beta-hydroxyisovaleric acid	3.2	30	[M−H]−	117	41	−15	−34	−7
BEH AMIDE	(+)	Betaine	3.4	25	[M + H]+	118	58.1	60	20	26
BEH AMIDE	(+)	Betaine	3.4	25	[M + H]+	118	59.1 (Q)	60	15	10
HSS T3	(−)	Beta-muricholic acid	8.4	40	[M−H]−	407.2	407.2 (Q)	−120	−5	−5
BEH AMIDE	(+)	Butenoylcarnitine	2.7	30	[M + H]+	230.1	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Butyrylcarnitine	2.6	35	[M + H]+	232.1	85.0 (Q)	56	25	12
HSS T3	(−)	cAMP	3.5	30	[M−H]−	327.9	134.0 (Q)	−50	−32	−9
HSS T3	(−)	cAMP	3.5	30	[M−H]−	327.9	78.9	−50	−76	−11
BEH AMIDE	(+)	Carnitine	3.5	30	[M + H]+	162	103	56	18	14
BEH AMIDE	(+)	Carnitine	3.5	30	[M + H]+	162	85.0 (Q)	56	27	14
BEH AMIDE	(+)	Carnosine	5.3	55	[M + H]+	227	110.2 (Q)	51	31	10
BEH AMIDE	(+)	Carnosine	5.3	55	[M + H]+	227	210.2	51	17	16
BEH AMIDE	(+)	CDP-choline	5.6	40	[M + H]+	489	264.1 (Q)	46	37	18
BEH AMIDE	(+)	CDP-choline	5.6	40	[M + H]+	489	184.1	46	49	14
HSS T3	(−)	CDP-ethanolamine	0.6	40	[M−H]−	445	78.9 (Q)	−30	−104	−11
HSS T3	(−)	CDP-ethanolamine	0.6	40	[M−H]−	445	201.9	−30	−30	−15
HSS T3	(−)	cGMP	3.5	60	[M−H]−	343.9	149.9 (Q)	−55	−32	−11
HSS T3	(−)	cGMP	3.5	60	[M−H]−	343.9	78.9	−55	−82	−9
HSS T3	(−)	Chenodeoxycholic acid	9	40	[M−H]−	391.2	391.2 (Q)	−140	−5	−5
HSS T3	(−)	Cholesterol sulfate	10	40	[M−H]−	465.1	80	−40	−130	−11
HSS T3	(−)	Cholesterol sulfate	10	40	[M−H]−	465.1	96.9 (Q)	−40	−44	−13
HSS T3	(−)	Cholic acid	8.6	40	[M−H]−	407.1	407.1	−120	−5	−5
HSS T3	(−)	Cholic acid	8.6	40	[M−H]−	407.1	343.1 (Q)	−120	−46	−15
HSS T3	(−)	Cholic acid	8.6	40	[M−H]−	407.1	289.1	−120	−52	−21
BEH AMIDE	(+)	Choline	2.3	70	[M + H]+	104	60.1 (Q)	71	23	10
BEH AMIDE	(+)	Choline	2.3	70	[M + H]+	104	58.1	71	39	10
HSS T3	(−)	Citraconic acid	1.6	40	[M−H]−	129	84.9 (Q)	−10	−14	−9
HSS T3	(−)	Citraconic acid	1.6	40	[M−H]−	129	41	−10	−20	−7
HSS T3	(−)	Citramalic acid	1.2	60	[M−H]−	147.1	86.9 (Q)	−15	−20	−9
HSS T3	(−)	Citramalic acid	1.2	60	[M−H]−	147.1	85	−15	−20	−9
HSS T3	(−)	Citric acid	0.6	60	[M−H]−	190.9	111	−25	−18	−7
HSS T3	(−)	Citric acid	0.6	60	[M−H]−	190.9	85.0 (Q)	−25	−20	−9
HSS T3	(−)	Citric acid	0.6	60	[M−H]−	190.9	130.8	−25	−20	−15
BEH AMIDE	(+)	Citrulline	4.6	25	[M + H]+	176.1	159.1	26	13	12
BEH AMIDE	(+)	Citrulline	4.6	25	[M + H]+	176.1	70.1 (Q)	26	27	12
HSS T3	(−)	CMPF	8.2	40	[M−H]−	239	195.0 (Q)	−30	−18	−13
HSS T3	(−)	CMPF	8.2	40	[M−H]−	239	151	−30	−24	−13
BEH AMIDE	(+)	Creatine	4	25	[M + H]+	132	90.0 (Q)	66	17	14
BEH AMIDE	(+)	Creatinine	2.7	25	[M + H]+	114	44.1	51	15	20
BEH AMIDE	(+)	Creatinine	2.7	25	[M + H]+	114	86.0 (Q)	51	15	12
HSS T3	(−)	Cyclic-di-GMP	3.4	30	[M−H]−	688.9	78.9 (Q)	−135	−130	−9
HSS T3	(−)	Cyclic-di-GMP	3.4	30	[M−H]−	688.9	344	−135	−44	−21
BEH AMIDE	(−)	Cysteine sulfinic acid	4.8	35	[M−H]−	151.9	88.0 (Q)	−10	−18	−7
BEH AMIDE	(+)	Cystine	5.6	25	[M + H]+	241	152.0 (Q)	46	19	12
BEH AMIDE	(+)	Cystine	5.6	25	[M + H]+	241	74.1	46	39	12
BEH AMIDE	(+)	Cytidine	3.2	25	[M + H]+	244	112.0 (Q)	76	21	16
BEH AMIDE	(+)	Cytidine	3.2	25	[M + H]+	244	95	76	57	14
BEH AMIDE	(+)	Cytosine	3	25	[M + H]+	112	95.1 (Q)	71	25	14
BEH AMIDE	(+)	Cytosine	3	25	[M + H]+	112	52.1	71	41	8
BEH AMIDE	(+)	Decadienoylcarnitine	2.3	30	[M + H]+	312.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Decanoylcarnitine	2.3	30	[M + H]+	316.2	85.0 (Q)	66	29	12
BEH AMIDE	(+)	Decatrienoylcarnitine	2.4	30	[M + H]+	310.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Decenoylcarnitine	2.5	90	[M + H]+	314.2	85.0 (Q)	51	29	14
HSS T3	(−)	Deoxycholic acid	9	40	[M−H]−	391.2	391.2 (Q)	−140	−5	−5
BEH AMIDE	(−)	Dihydroorotic acid	3.5	55	[M−H]−	156.9	112.9 (Q)	−20	−12	−7
BEH AMIDE	(−)	Dihydroorotic acid	3.5	55	[M−H]−	156.9	42	−20	−34	−19
BEH AMIDE	(+)	Dodecanoylcarnitine	2.2	35	[M + H]+	344.2	85.0 (Q)	61	29	10
BEH AMIDE	(+)	Dodecenoylcarnitine	2.2	30	[M + H]+	342.1	85.0 (Q)	140	47	12
BEH AMIDE	(+)	Eicoseneoylcarnitine	2	30	[M + H]+	454.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Ethanolamine	3.5	25	[M + H]+	62	44.1 (Q)	51	13	20
BEH AMIDE	(+)	Ethanolamine	3.5	25	[M + H]+	62	45.1	51	19	8
HSS T3	(−)	Ethylmalonic acid	2.5	30	[M−H]−	130.9	87.0 (Q)	−10	−12	−9
HSS T3	(−)	Ethylmalonic acid	2.5	30	[M−H]−	130.9	69	−10	−32	−9
HSS T3	(−)	FAD	4.3	60	[M−H]−	783.9	78.9 (Q)	−115	−128	−9
HSS T3	(−)	FAD	4.3	60	[M−H]−	783.9	437	−115	−40	−17
HSS T3	(−)	Fumaric acid	0.8	60	[M−H]−	115.1	70.9 (Q)	−10	−10	−9
BEH AMIDE	(+)	Gamma-glutamylvaline	4	25	[M + H]+	247	72.0 (Q)	31	29	12
BEH AMIDE	(+)	Gamma-glutamylvaline	4	25	[M + H]+	247	183	31	21	12
BEH AMIDE	(+)	Glucosamine	4.7	25	[M + H]+	180	162.1 (Q)	26	11	12
BEH AMIDE	(+)	Glucosamine	4.7	25	[M + H]+	180	84	26	19	12
BEH AMIDE	(+)	Glutamic acid	4.3	35	[M + H]+	148	84	56	21	12
BEH AMIDE	(+)	Glutamic acid	4.3	35	[M + H]+	148	130.1 (Q)	56	13	16
BEH AMIDE	(+)	Glutamine	4.4	25	[M + H]+	147.1	130.2	56	13	10
BEH AMIDE	(+)	Glutamine	4.4	25	[M + H]+	147.1	84.0 (Q)	56	23	10
BEH AMIDE	(−)	Glutaric acid	1.7	55	[M−H]−	131.1	87.1	−15	−16	−7
BEH AMIDE	(−)	Glutaric acid	1.7	55	[M−H]−	131.1	112.9 (Q)	−15	−14	−7
BEH AMIDE	(+)	Glutarylcarnitine	3.4	30	[M + H]+	276.1	85.0 (Q)	86	29	12
HSS T3	(+)	Glutathione_disulfide	1.8	70	[M + H]+	613	484.1 (Q)	100	25	20
HSS T3	(+)	Glutathione_disulfide	1.8	70	[M+2H]2+	307.1	130.1	20	17	12
HSS T3	(−)	Glyceric acid	0.5	30	[M−H]−	104.9	43.0 (Q)	−20	−26	−19
HSS T3	(−)	Glyceric acid	0.5	30	[M−H]−	104.9	74.9	−20	−16	−9
BEH AMIDE	(+)	Glycerophosphocholine	4.5	25	[M + H]+	258	104.0 (Q)	66	23	14
BEH AMIDE	(+)	Glycerophosphocholine	4.5	25	[M + H]+	258	125.1	66	35	10
BEH AMIDE	(+)	Glycine	4.3	25	[M + H]+	76	30.1 (Q)	61	17	14
BEH AMIDE	(+)	Glycine	4.3	25	[M + H]+	76	48.1	61	9	8
BEH AMIDE	(+)	Guanidinoacetic acid	4.1	25	[M + H]+	118.1	101.0 (Q)	26	15	14
BEH AMIDE	(+)	Guanidinoacetic acid	4.1	25	[M + H]+	118.1	72	26	19	12
BEH AMIDE	(+)	Guanine	3.1	25	[M + H]+	152	135.1 (Q)	66	27	10
BEH AMIDE	(+)	Guanine	3.1	25	[M + H]+	152	110.1	66	29	10
BEH AMIDE	(+)	Guanosine	3.4	25	[M + H]+	284	152.1 (Q)	30	23	12
BEH AMIDE	(+)	Guanosine	3.4	25	[M + H]+	284	135.1	30	53	12
BEH AMIDE	(+)	Heptanoylcarnitine_glutaconylcarnitine	2.4	30	[M + H]+	274.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Hexadecadienoylcarnitine	2.1	30	[M + H]+	396.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Hexanoylcarnitine	2.5	30	[M + H]+	260.1	85.0 (Q)	66	27	14
BEH AMIDE	(+)	Hexenoylcarnitine	2.5	30	[M + H]+	258.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Histamine	4.6	70	[M + H]+	112.1	95.0 (Q)	36	19	14
BEH AMIDE	(+)	Histamine	4.6	70	[M + H]+	112.1	68.1	36	29	10
BEH AMIDE	(+)	Histidine	5.3	70	[M + H]+	156.1	110.1	36	21	8
BEH AMIDE	(+)	Histidine	5.3	70	[M + H]+	156.1	83.0 (Q)	36	35	12
BEH AMIDE	(+)	Histidinol	4.7	70	[M + H]+	142	124.0 (Q)	46	15	16
BEH AMIDE	(+)	Histidinol	4.7	70	[M + H]+	142	81	46	27	12
BEH AMIDE	(+)	Homoarginine	5.1	70	[M + H]+	189	84.0 (Q)	51	31	12
BEH AMIDE	(+)	Homoarginine	5.1	70	[M + H]+	189	130	51	23	10
BEH AMIDE	(+)	Homocitrulline	4.5	25	[M + H]+	190.1	173.1 (Q)	36	15	12
BEH AMIDE	(+)	Homocitrulline	4.5	25	[M + H]+	190.1	127.1	36	21	14
BEH AMIDE	(−)	Homocysteic acid	4.8	25	[M−H]−	182	79.9 (Q)	−25	−30	−9
BEH AMIDE	(−)	Homocysteic acid	4.8	25	[M−H]−	182	165	−25	−18	−11
BEH AMIDE	(+)	Homoserine	4.3	35	[M + H]+	120	44.1 (Q)	61	27	8
BEH AMIDE	(+)	Hydroxybutyrylcarnitine	3.3	30	[M + H]+	248	85.0 (Q)	61	27	12
BEH AMIDE	(+)	Hydroxybutyrylcarnitine	3.3	30	[M + H]+	248	189.2	61	19	16
BEH AMIDE	(+)	Hydroxydecanoylcarnitine	2.6	30	[M + H]+	332.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Hydroxydodecenoylcarnitine	2.2	30	[M + H]+	358.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Hydroxyisovalerylcarnitine	3.3	90	[M + H]+	262.1	85.0 (Q)	66	29	14
BEH AMIDE	(+)	Hydroxylysine	5.4	35	[M + H]+	163.1	128.0 (Q)	36	15	14
BEH AMIDE	(+)	Hydroxylysine	5.4	35	[M + H]+	163.1	82	36	23	12
BEH AMIDE	(+)	Hydroxyoctenoylcarnitine	2.3	90	[M + H]+	302.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Hydroxypalmitoylcarnitine	2.5	30	[M + H]+	416.3	85.0 (Q)	101	55	12
BEH AMIDE	(+)	Hydroxyproline	4.1	25	[M + H]+	132	86	46	19	12
BEH AMIDE	(+)	Hydroxyproline	4.1	25	[M + H]+	132	68.0 (Q)	46	27	10
BEH AMIDE	(+)	Hydroxytetradecenoylcarnitine	2.1	30	[M + H]+	386.2	85.0 (Q)	140	47	12
BEH AMIDE	(+)	Hydroxytryptophan	2.9	25	[M + H]+	221	204.1 (Q)	21	15	14
BEH AMIDE	(−)	Hypotaurine	4.1	25	[M−H]−	107.9	64.0 (Q)	−25	−18	−27
BEH AMIDE	(+)	Hypoxanthine	2.6	25	[M + H]+	137	110	66	29	14
BEH AMIDE	(+)	Hypoxanthine	2.6	25	[M + H]+	137	94.0 (Q)	66	29	12
BEH AMIDE	(+)	Imidazole	2.7	25	[M + H]+	69	42.1 (Q)	140	27	20
BEH AMIDE	(+)	Imidazole-4-acetic acid	3.9	25	[M + H]+	127	81.0 (Q)	36	21	12
BEH AMIDE	(+)	Imidazole-4-acetic acid	3.9	25	[M + H]+	127	109.1	36	13	8
HSS T3	(−)	Indole-3-carboxylic acid	6.4	40	[M−H]−	160	116.0 (Q)	−20	−20	−9
BEH AMIDE	(+)	Inosine	3	25	[M + H]+	269	137.1 (Q)	76	21	10
BEH AMIDE	(+)	Inosine	3	25	[M + H]+	269	119.1	76	57	14
HSS T3	(−)	Isocitric acid	0.6	60	[M−H]−	191	111	−25	−18	−7
HSS T3	(−)	Isocitric acid	0.6	60	[M−H]−	191	85.0 (Q)	−25	−20	−9
BEH AMIDE	(+)	Isoleucine	3.5	25	[M + H]+	132.1	86.1 (Q)	66	15	12
BEH AMIDE	(+)	Isoleucine	3.5	25	[M + H]+	132.1	69	66	23	10
BEH AMIDE	(+)	Isovalerylcarnitine	2.6	40	[M + H]+	246.1	85.0 (Q)	56	27	12
HSS T3	(−)	Itaconic acid	1.6	30	[M−H]−	128.9	85.0 (Q)	−45	−12	−41
HSS T3	(−)	Itaconic acid	1.6	30	[M−H]−	128.9	41	−45	−18	−19
HSS T3	(−)	Ketoleucine	3.8	40	[M−H]−	129.1	129.1 (Q)	−10	−5	−5
BEH AMIDE	(+)	Kynurenic acid	3	25	[M + H]+	190	144.0 (Q)	31	27	18
BEH AMIDE	(+)	Kynurenic acid	3	25	[M + H]+	190	89.1	31	53	14
BEH AMIDE	(+)	Kynurenine	3.5	25	[M + H]+	209.1	192.2 (Q)	41	13	14
BEH AMIDE	(+)	Kynurenine	3.5	25	[M + H]+	209.1	146.2	41	27	12
BEH AMIDE	(+)	LABELED_1-MeNAM	3	25	[M]+	144	81.0 (Q)	26	37	14
BEH AMIDE	(+)	LABELED_1-methylhistidine	4.8	100	[M + H]+	173.1	127.1 (Q)	36	21	10
HSS T3	(−)	LABELED_2KG	0.8	60	[M−H]−	152	59.9 (Q)	−25	−18	−9
HSS T3	(−)	LABELED_3HB	1.5	60	[M−H]−	107	61.0 (Q)	−15	−16	−3
BEH AMIDE	(+)	LABELED_4-hydroxyproline	4.1	35	[M + H]+	135	89.1 (Q)	51	21	12
BEH AMIDE	(+)	LABELED_Aspartic acid	4.8	90	[M + H]+	137.1	91.0 (Q)	51	15	14
BEH AMIDE	(+)	LABELED_C14	2.2	30	[M + H]+	381.2	85.0 (Q)	160	31	16
BEH AMIDE	(+)	LABELED_C16	2.1	30	[M + H]+	403.3	85.0 (Q)	121	31	10
BEH AMIDE	(+)	LABELED_C2	2.9	30	[M + H]+	207.1	85.1 (Q)	31	25	14
BEH AMIDE	(+)	LABELED_C3	2.7	30	[M + H]+	221.1	85.1 (Q)	41	27	12
BEH AMIDE	(+)	LABELED_C4	2.6	30	[M + H]+	235.2	85.1 (Q)	46	29	40
BEH AMIDE	(+)	LABELED_C5	2.5	30	[M + H]+	255.1	85.1 (Q)	56	25	12
BEH AMIDE	(+)	LABELED_C8	2.3	30	[M + H]+	291.1	85.0 (Q)	76	27	18
HSS T3	(−)	LABELED_cAMP	3.5	30	[M−H]−	332.9	134.0 (Q)	−45	−32	−9
BEH AMIDE	(+)	LABELED_Carnitine	3.5	25	[M + H]+	171.1	103.0 (Q)	31	25	16
HSS T3	(−)	LABELED_Cholic acid	8.6	40	[M−H]−	412.1	412.1 (Q)	−120	−5	−5
HSS T3	(−)	LABELED_Citric acid	0.8	60	[M−H]−	194.9	113.0 (Q)	−20	−18	−9
BEH AMIDE	(+)	LABELED_Citrulline	4.6	25	[M + H]+	180.1	163.0 (Q)	20	15	12
BEH AMIDE	(+)	LABELED_Creatinine	2.7	25	[M + H]+	117	47.1 (Q)	51	23	22
HSS T3	(−)	LABELED_CS	10	40	[M−H]−	472.1	97.0 (Q)	−135	−46	−11
BEH AMIDE	(+)	LABELED_Cytosine	3	25	[M + H]+	115.1	97.0 (Q)	76	25	14
HSS T3	(−)	LABELED_Deoxycholic acid	9	40	[M−H]−	397.2	397.2 (Q)	−140	−5	−5
BEH AMIDE	(+)	LABELED_Glutamine	4.4	25	[M + H]+	154.1	136.1 (Q)	36	13	10
BEH AMIDE	(+)	LABELED_Glycine	4.3	25	[M + H]+	78	32.1 (Q)	51	17	14
BEH AMIDE	(+)	LABELED_Histamine	4.6	70	[M + H]+	116.1	99.0 (Q)	46	21	16
BEH AMIDE	(+)	LABELED_Histidine	5.3	70	[M + H]+	162.1	115.1 (Q)	31	21	8
BEH AMIDE	(+)	LABELED_Isoleucine	3.5	25	[M + H]+	142.1	96.0 (Q)	76	17	12
HSS T3	(−)	LABELED_Ketoleucine	3.8	40	[M−H]−	135	135.0 (Q)	−10	−5	−5
HSS T3	(−)	LABELED_Ketovaline	1.6	40	[M−H]−	120	120.0 (Q)	−10	−5	−5
HSS T3	(−)	LABELED_Lactic acid	0.7	70	[M−H]−	92	42.0 (Q)	−15	−34	−19
BEH AMIDE	(+)	LABELED_Leucine	3.4	25	[M + H]+	138.1	91.1 (Q)	10	15	14
BEH AMIDE	(+)	LABELED_Methionine	3.6	25	[M + H]+	155	108.1 (Q)	16	15	10
BEH AMIDE	(−)	LABELED_NAA	2.6	40	[M−H]−	177	91.0 (Q)	−20	−22	−13
HSS T3	(+)	LABELED_N-acetylleucine	5.3	30	[M + H]+	184	96.0 (Q)	41	23	14
BEH AMIDE	(−)	LABELED_N-acetylserine	2.3	50	[M−H]−	149	116.9 (Q)	−20	−12	−7
HSS T3	(+)	LABELED_Nicotinamide	2	60	[M + H]+	129	85.0 (Q)	70	23	14
HSS T3	(+)	LABELED_Nicotinic acid	1	60	[M + H]+	128	84.0 (Q)	66	29	12
BEH AMIDE	(+)	LABELED_Ornithine	5.3	50	[M + H]+	138	121.0 (Q)	41	13	14
BEH AMIDE	(+)	LABELED_Phenylalanine	3.4	25	[M + H]+	171	106.1 (Q)	31	39	14
BEH AMIDE	(+)	LABELED_SAH	4.6	35	[M + H]+	395	141.1 (Q)	36	25	20
BEH AMIDE	(+)	LABELED_Serine	4.4	25	[M + H]+	109	63.1 (Q)	71	17	10
HSS T3	(−)	LABELED_Succinic acid	1.3	40	[M−H]−	120.9	76.9 (Q)	−20	−16	−9
BEH AMIDE	(−)	LABELED_Taurine	3.7	25	[M−H]−	128	80.0 (Q)	−30	−28	−9
BEH AMIDE	(+)	LABELED_TMAO	2.8	35	[M + H]+	85	66.1 (Q)	30	27	10
BEH AMIDE	(+)	LABELED_Urea	1.9	50	[M + H]+	64	46.0 (Q)	71	23	22
BEH AMIDE	(+)	LABELED_Valine	3.7	25	[M + H]+	126	80.1 (Q)	66	15	12
HSS T3	(−)	Lactic acid	0.7	40	[M−H]−	89	43.0 (Q)	−15	−16	−7
BEH AMIDE	(+)	Leucine	3.4	25	[M + H]+	132.1	86.1 (Q)	66	15	10
BEH AMIDE	(+)	Linoleoylcarnitine	2.1	70	[M + H]+	424.3	85.0 (Q)	60	27	15
HSS T3	(−)	Lipoic acid	8.2	40	[M−H]−	205	64.9 (Q)	−20	−36	−7
HSS T3	(−)	Lipoic acid	8.2	40	[M−H]−	205	93	−20	−18	−11
HSS T3	(−)	Litocholic acid	9.5	40	[M−H]−	376.2	376.2 (Q)	−120	−5	−5
BEH AMIDE	(+)	Lysine	5.3	55	[M + H]+	147	84.2	41	23	8
BEH AMIDE	(+)	Lysine	5.3	55	[M + H]+	147	130.2 (Q)	41	13	12
HSS T3	(−)	Maleic acid	0.8	60	[M−H]−	114.9	71.0 (Q)	−10	−14	−9
HSS T3	(−)	Malic acid	0.6	40	[M−H]−	133	114.9 (Q)	−15	−16	−7
HSS T3	(−)	Malic acid	0.6	40	[M−H]−	133	72.9	−15	−22	−11
BEH AMIDE	(+)	Malonylcarnitine	4.1	30	[M + H]+	248.1	85.0 (Q)	61	27	12
BEH AMIDE	(+)	Methionine sulfoxide	4.4	25	[M + H]+	166.1	74.0 (Q)	61	19	10
BEH AMIDE	(+)	Methionine sulfoxide	4.4	25	[M + H]+	166.1	56.1	61	33	8
BEH AMIDE	(+)	Methionine	3.6	25	[M + H]+	150	133.0 (Q)	61	13	16
BEH AMIDE	(+)	Methionine	3.6	25	[M + H]+	150	104.1	61	15	14
BEH AMIDE	(+)	Methylimidazole acetic acid	3.7	55	[M + H]+	141.1	122.9 (Q)	46	11	10
BEH AMIDE	(+)	Methylimidazole acetic acid	3.7	55	[M + H]+	141.1	95	46	21	14
HSS T3	(−)	Methylmalonic acid	1.1	30	[M−H]−	116.9	72.9 (Q)	−15	−14	−9
HSS T3	(−)	Methylmalonic acid	1.1	30	[M−H]−	116.9	54.9	−15	−34	−7
BEH AMIDE	(+)	Methylmalonylcarnitine	3.8	30	[M + H]+	262	85 (Q)	56	29	14
BEH AMIDE	(+)	Methylmalonylcarnitine	3.8	30	[M + H]+	262	218.2	56	21	16
HSS T3	(−)	Methylsuccinic acid	3.6	30	[M−H]−	131	87.0 (Q)	−10	−16	−11
HSS T3	(−)	Methylsuccinic acid	3.6	30	[M−H]−	131	112.9	−10	−16	−7
BEH AMIDE	(−)	Mevalonic acid	1.1	70	[M−H]−	147	58.9 (Q)	−18	−16	−7
BEH AMIDE	(+)	Myristoylcarnitine	2.2	30	[M + H]+	372.3	85.0 (Q)	60	27	15
BEH AMIDE	(+)	N,N-dimethylglycine	3.7	25	[M + H]+	104.1	58.1 (Q)	61	19	10
BEH AMIDE	(+)	N,N-dimethylglycine	3.7	25	[M + H]+	104.1	56.1	61	35	10
BEH AMIDE	(−)	N-acetylalanine	1.2	70	[M−H]−	129.9	88.0 (Q)	−10	−16	−9
BEH AMIDE	(−)	N-acetylaspartic acid	2.6	40	[M−H]−	174	88.0 (Q)	−15	−20	−11
BEH AMIDE	(−)	N-acetylaspartic acid	2.6	40	[M−H]−	174	114	−15	−14	−7
BEH AMIDE	(−)	N-acetylaspartylglutamic acid	3.7	35	[M−H]−	302.9	285.0 (Q)	−35	−14	−23
BEH AMIDE	(−)	N-acetylaspartylglutamic acid	3.7	35	[M−H]−	302.9	128	−35	−22	−9
BEH AMIDE	(+)	N-acetylglucosamine	3.3	75	[M + H]+	222.1	204.1 (Q)	36	11	24
BEH AMIDE	(+)	N-acetylglucosamine	3.3	75	[M + H]+	222.1	138.1	36	21	16
BEH AMIDE	(−)	N-acetylglutamic acid	2.4	40	[M−H]−	188	128.0 (Q)	−35	−18	−11
BEH AMIDE	(−)	N-acetylglutamic acid	2.4	40	[M−H]−	188	102	−35	−22	−11
BEH AMIDE	(+)	N-acetylglycine	1.7	40	[M + H]+	118.1	76.0 (Q)	61	11	32
BEH AMIDE	(+)	N-acetylglycine	1.7	40	[M + H]+	118.1	43.1	61	31	20
HSS T3	(+)	N-acetylisoleucine	5.2	30	[M + H]+	174.1	128.1 (Q)	46	13	10
HSS T3	(+)	N-acetylisoleucine	5.2	30	[M + H]+	174.1	86.1	46	23	10
BEH AMIDE	(+)	N-acetyl-L-citrulline	3	25	[M + H]+	218.1	201.2 (Q)	31	11	14
BEH AMIDE	(+)	N-acetyl-L-citrulline	3	25	[M + H]+	218.1	70.1	31	39	10
BEH AMIDE	(+)	N-acetyl-L-cysteine	1.4	35	[M + H]+	164	122.1 (Q)	41	13	16
BEH AMIDE	(+)	N-acetyl-L-cysteine	1.4	35	[M + H]+	164	76	41	25	12
HSS T3	(+)	N-acetylleucine	5.3	30	[M + H]+	174	128.1 (Q)	46	13	10
HSS T3	(+)	N-acetylleucine	5.3	30	[M + H]+	174	86.1	46	23	10
BEH AMIDE	(+)	N-acetyl-L-glutamine	2.9	70	[M + H]+	189	130.2 (Q)	66	19	10
BEH AMIDE	(+)	N-acetyl-L-glutamine	2.9	70	[M + H]+	189	84	66	33	12
BEH AMIDE	(+)	N-acetyl-L-histidine	4.1	25	[M + H]+	198.1	110.0 (Q)	41	29	16
BEH AMIDE	(+)	N-acetyl-L-histidine	4.1	25	[M + H]+	198.1	152.2	41	17	12
BEH AMIDE	(+)	N-acetyl-L-ornithine	4.3	25	[M + H]+	175	70.1 (Q)	46	33	12
HSS T3	(+)	N-acetylmethionine	4.1	30	[M + H]+	192	144.1 (Q)	36	15	12
HSS T3	(+)	N-acetylmethionine	4.1	30	[M + H]+	192	98	36	25	14
HSS T3	(+)	N-acetylphenylalanine	5.7	30	[M + H]+	208	120.1 (Q)	40	27	14
HSS T3	(+)	N-acetylphenylalanine	5.7	30	[M + H]+	208	166.1	40	15	12
HSS T3	(+)	N-acetylproline	3.6	60	[M + H]+	158	70.1 (Q)	36	27	12
HSS T3	(+)	N-acetylproline	3.6	60	[M + H]+	158	112.1	36	15	16
BEH AMIDE	(+)	N-acetylputrescine	3.4	25	[M + H]+	131	114.1 (Q)	41	15	14
BEH AMIDE	(+)	N-acetylputrescine	3.4	25	[M + H]+	131	72.1	41	21	12
BEH AMIDE	(−)	N-acetylserine	2.3	50	[M−H]−	145.9	116.0 (Q)	−15	−14	−7
BEH AMIDE	(−)	N-acetylserine	2.3	50	[M−H]−	145.9	74	−15	−20	−11
HSS T3	(+)	N-acetylthreonine	1.2	40	[M + H]+	162	120.0 (Q)	40	15	18
HSS T3	(+)	N-acetylthreonine	1.2	40	[M + H]+	162	74	40	23	12
HSS T3	(+)	N-acetyltryptophan	6.1	40	[M + H]+	247.1	201.1 (Q)	26	17	16
HSS T3	(+)	N-acetyltryptophan	6.1	40	[M + H]+	247.1	188.1	26	21	14
HSS T3	(+)	N-acetyltyrosine	4.1	30	[M + H]+	224	136.1 (Q)	36	23	16
HSS T3	(+)	N-acetyltyrosine	4.1	30	[M + H]+	224	178.1	36	13	14
HSS T3	(+)	N-acetylvaline	4.1	30	[M + H]+	160.1	72.0 (Q)	50	23	10
HSS T3	(+)	N-acetylvaline	4.1	30	[M + H]+	160.1	118.1	50	15	8
BEH AMIDE	(+)	Nalpha-acetyl-L-arginine	4	25	[M + H]+	217.1	158.2 (Q)	51	23	12
BEH AMIDE	(+)	Nalpha-acetyl-L-arginine	4	25	[M + H]+	217.1	70.1	51	43	10
BEH AMIDE	(+)	Nalpha-acetyllysine	4	25	[M + H]+	189.1	84.0 (Q)	61	29	12
BEH AMIDE	(+)	Nalpha-acetyllysine	4	25	[M + H]+	189.1	129.1	61	19	12
HSS T3	(+)	N-butyrylglycine	3.2	40	[M + H]+	146	76.0 (Q)	71	13	10
HSS T3	(+)	N-butyrylglycine	3.2	40	[M + H]+	146	43	71	27	20
HSS T3	(+)	N-butyrylglycine	3.2	40	[M + H]+	146	71	71	15	14
HSS T3	(+)	N-caprylylglycine	8	40	[M + H]+	202	76 (Q)	86	13	12
HSS T3	(+)	N-caprylylglycine	8	40	[M + H]+	202	57	86	25	10
HSS T3	(+)	N-caprylylglycine	8	40	[M + H]+	202	127	86	15	14
BEH AMIDE	(+)	Nepsilon-acetyllysine	4.2	25	[M + H]+	189.1	84.1 (Q)	51	29	14
BEH AMIDE	(+)	Nepsilon-acetyllysine	4.2	25	[M + H]+	189.1	126.2	51	17	10
BEH AMIDE	(+)	N-formylkynurenine	3.7	25	[M + H]+	237	146.1 (Q)	36	33	10
BEH AMIDE	(+)	N-formylkynurenine	3.7	25	[M + H]+	237	118.2	36	39	10
HSS T3	(+)	N-furoylglycine	3.5	30	[M + H]+	170	95.0 (Q)	46	27	14
HSS T3	(+)	N-furoylglycine	3.5	30	[M + H]+	170	124	46	15	14
HSS T3	(+)	Nicotinamide	2	60	[M + H]+	122.9	80	66	27	12
HSS T3	(+)	Nicotinamide	2	60	[M + H]+	122.9	78.0 (Q)	66	31	12
HSS T3	(+)	Nicotinic acid	1	60	[M + H]+	123.9	80.0 (Q)	71	29	14
HSS T3	(+)	Nicotinic acid	1	60	[M + H]+	123.9	78	71	29	12
BEH AMIDE	(+)	N-methylaspartic acid	4.7	55	[M + H]+	148.1	88.1 (Q)	56	19	8
BEH AMIDE	(+)	N-methylaspartic acid	4.7	55	[M + H]+	148.1	102	56	17	12
HSS T3	(+)	N-myristoylglycine	9.3	40	[M + H]+	286	76.0 (Q)	36	15	12
HSS T3	(+)	N-myristoylglycine	9.3	40	[M + H]+	286	57	36	45	26
HSS T3	(+)	N-myristoylglycine	9.3	40	[M + H]+	286	95	36	23	14
HSS T3	(+)	N-oleoylglycine	9.6	40	[M + H]+	340.1	76.1 (Q)	41	19	12
HSS T3	(+)	N-oleoylglycine	9.6	40	[M + H]+	340.1	55.1	41	69	8
HSS T3	(+)	N-oleoylglycine	9.6	40	[M + H]+	340.1	265.2	41	17	18
HSS T3	(+)	N-palmitoylglycine	9.5	40	[M + H]+	314.1	76.1 (Q)	31	17	12
HSS T3	(+)	N-palmitoylglycine	9.5	40	[M + H]+	314.1	239.2	31	17	28
HSS T3	(+)	N-palmitoylglycine	9.5	40	[M + H]+	314.1	57.1	31	41	10
HSS T3	(+)	N-propionylglycine	1.4	40	[M + H]+	132	76.0 (Q)	71	13	12
HSS T3	(+)	N-propionylglycine	1.4	40	[M + H]+	132	57	71	19	9
BEH AMIDE	(+)	Octadecanoylcarnitine	2.1	90	[M + H]+	428.2	85.0 (Q)	56	33	12
BEH AMIDE	(+)	Octanoylcarnitine	2.3	30	[M + H]+	288.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Octenoylcarnitine	2.4	30	[M + H]+	286.2	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Oleoyl L-carnitine	2.1	90	[M + H]+	426.3	85.0 (Q)	66	31	10
HSS T3	(−)	O-phosphotyrosine	0.5	40	[M−H]−	260	78.9 (Q)	−20	−20	−9
HSS T3	(−)	O-phosphotyrosine	0.5	40	[M−H]−	260	63	−20	−128	−9
BEH AMIDE	(+)	Ornithine	5.3	50	[M + H]+	133	70.0 (Q)	31	27	12
BEH AMIDE	(+)	Ornithine	5.3	50	[M + H]+	133	116.1	31	13	14
BEH AMIDE	(+)	Palmitoleoylcarnitine	2.1	30	[M + H]+	398.3	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Palmityol-L-carnitine	2.1	90	[M + H]+	400.3	85.1 (Q)	61	31	12
HSS T3	(−)	Pantothenic acid	3.8	30	[M−H]−	217.9	87.9 (Q)	−30	−18	−11
HSS T3	(−)	Pantothenic acid	3.8	30	[M−H]−	217.9	146	−30	−22	−9
BEH AMIDE	(+)	Phenylacetylglutamine	2.1	35	[M + H]+	265.1	130.1 (Q)	81	21	10
BEH AMIDE	(+)	Phenylacetylglutamine	2.1	35	[M + H]+	265.1	84	81	43	10
BEH AMIDE	(+)	Phenylalanine	3.4	25	[M + H]+	166.1	103.1	20	38	18
BEH AMIDE	(+)	Phenylalanine	3.4	25	[M + H]+	166.1	120.2 (Q)	20	19	10
HSS T3	(−)	Phenylpyruvic acid	4.5	40	[M−H]−	162.9	91.0 (Q)	−10	−14	−11
HSS T3	(−)	Phosphoenolpyruvic acid	0.5	40	[M−H]−	166.9	78.9 (Q)	−10	−18	−9
HSS T3	(−)	Phosphoenolpyruvic acid	0.5	40	[M−H]−	166.9	62.9	−10	−90	−9
HSS T3	(+)	Picolinic acid	0.9	60	[M + H]+	124	78.0 (Q)	46	25	12
HSS T3	(+)	Picolinic acid	0.9	60	[M + H]+	124	106	46	15	16
HSS T3	(+)	Pivaloylglycine	3.7	40	[M + H]+	160	57.0 (Q)	71	19	8
HSS T3	(+)	Pivaloylglycine	3.7	40	[M + H]+	160	85	71	13	12
BEH AMIDE	(+)	Proline	3.7	25	[M + H]+	116	70.1	71	21	12
BEH AMIDE	(+)	Proline	3.7	25	[M + H]+	116	68.1 (Q)	71	37	10
BEH AMIDE	(+)	Propionylcarnitine	2.7	25	[M + H]+	218	85.0 (Q)	51	25	12
HSS T3	(+)	Purine	2.6	70	[M + H]+	120.9	94.0 (Q)	81	29	12
HSS T3	(+)	Purine	2.6	70	[M + H]+	120.9	67	81	39	10
BEH AMIDE	(+)	Putrescine	5.1	55	[M + H]+	88.9	72.0 (Q)	56	15	12
BEH AMIDE	(+)	Pyridoxamine	4.3	35	[M + H]+	169	152.1 (Q)	26	17	12
BEH AMIDE	(+)	Pyridoxamine	4.3	35	[M + H]+	169	134.2	26	29	12
BEH AMIDE	(+)	Pyridoxine	2.6	35	[M + H]+	170	134.1 (Q)	31	29	10
BEH AMIDE	(+)	Pyridoxine	2.6	35	[M + H]+	170	152.1	31	19	10
BEH AMIDE	(+)	Pyroglutamic acid	2.2	55	[M + H]+	129.9	84.0 (Q)	56	19	12
BEH AMIDE	(+)	Pyroglutamic acid	2.2	55	[M + H]+	129.9	56	56	31	10
HSS T3	(−)	Pyruvic acid	0.5	30	[M−H]−	86.9	86.9 (Q)	−10	−5	−15
HSS T3	(−)	Pyruvic acid	0.5	30	[M−H]−	86.9	43	−10	−12	−19
BEH AMIDE	(+)	Riboflavin	3	25	[M + H]+	377	243.0 (Q)	66	31	20
BEH AMIDE	(+)	Riboflavin	3	25	[M + H]+	377	172.1	66	51	12
BEH AMIDE	(+)	S-adenosyl-L-homocysteine	4.6	25	[M + H]+	385	136.2 (Q)	61	25	12
BEH AMIDE	(+)	S-adenosyl-L-homocysteine	4.6	25	[M + H]+	385	134.1	61	25	10
BEH AMIDE	(+)	S-adenosyl-L-methionine	5.4	25	[M + H]+	399.1	250.2 (Q)	81	19	22
BEH AMIDE	(+)	S-adenosyl-L-methionine	5.4	25	[M + H]+	399.1	298.1	81	19	18
BEH AMIDE	(+)	Sarcosine	3.9	25	[M + H]+	90.1	44.1 (Q)	61	15	20
BEH AMIDE	(+)	SDMA	4.7	30	[M + H]+	203	70	51	31	10
BEH AMIDE	(+)	SDMA	4.7	30	[M + H]+	203	172.1 (Q)	51	19	14
BEH AMIDE	(+)	Serine	4.4	25	[M + H]+	106	60.0 (Q)	81	15	10
BEH AMIDE	(−)	Shikimic acid	2.5	35	[M−H]−	172.9	93.0 (Q)	−20	−22	−11
BEH AMIDE	(−)	Shikimic acid	2.5	35	[M−H]−	172.9	111	−20	−14	−7
BEH AMIDE	(+)	S-methylcysteine sulfoxide	4.3	35	[M + H]+	152.1	88.0 (Q)	61	13	14
BEH AMIDE	(+)	S-methylcysteine sulfoxide	4.3	35	[M + H]+	152.1	42.1	61	29	8
BEH AMIDE	(+)	S-methylcysteine	3.7	25	[M + H]+	136	119	31	13	16
BEH AMIDE	(+)	S-methylcysteine	3.7	25	[M + H]+	136	73.0 (Q)	31	21	12
HSS T3	(+)	S-methylglutathione	1.7	60	[M + H]+	322	176.0 (Q)	56	23	14
HSS T3	(+)	S-methylglutathione	1.7	60	[M + H]+	322	193.2	56	17	16
HSS T3	(+)	S-methylthioadenosine	4.6	40	[M + H]+	298.1	136.1 (Q)	61	25	12
HSS T3	(+)	S-methylthioadenosine	4.6	40	[M + H]+	298.1	119	61	67	18
HSS T3	(−)	Succinic acid	1.3	40	[M−H]−	117	72.9	−15	−16	−9
HSS T3	(−)	Succinic acid	1.3	40	[M−H]−	117	98.9 (Q)	−15	−14	−11
BEH AMIDE	(+)	Succinoadenosine	3.6	35	[M + H]+	384	252.1 (Q)	61	27	18
BEH AMIDE	(+)	Succinoadenosine	3.6	35	[M + H]+	384	162.1	61	51	12
BEH AMIDE	(+)	Succinylcarnitine	3.6	30	[M + H]+	262.1	85.0 (Q)	56	27	10
BEH AMIDE	(+)	Succinylcarnitine	3.6	30	[M + H]+	262.1	203	56	21	16
BEH AMIDE	(−)	Taurine	3.7	25	[M−H]−	124	80.0 (Q)	−35	−26	−11
BEH AMIDE	(+)	Tetradecadienoylcarnitine	2.2	30	[M + H]+	368.3	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Tetradecenoylcarnitine	2.2	30	[M + H]+	370.3	85.0 (Q)	60	27	15
BEH AMIDE	(+)	Thiamine	3.5	25	[M]+	265	122.1 (Q)	71	21	8
BEH AMIDE	(+)	Thiamine	3.5	25	[M]+	265	144	71	19	18
BEH AMIDE	(+)	Threonine	4.2	35	[M + H]+	120.1	74	56	15	12
BEH AMIDE	(+)	Threonine	4.2	35	[M + H]+	120.1	102.1 (Q)	56	11	14
HSS T3	(+)	Thymidine	3.6	30	[M + H]+	243	127.1 (Q)	76	17	10
HSS T3	(+)	Thymidine	3.6	30	[M + H]+	243	117.1	76	17	16
HSS T3	(+)	Thymine	2.2	40	[M + H]+	126.9	109.9 (Q)	56	21	14
HSS T3	(+)	Thymine	2.2	40	[M + H]+	126.9	84	56	23	12
HSS T3	(−)	Trans-aconitic acid	1	60	[M−H]−	173.1	85.0 (Q)	−10	−16	−11
HSS T3	(−)	Trans-aconitic acid	1	60	[M−H]−	173.1	129	−10	−8	−15
BEH AMIDE	(+)	Trimethylamine_N-oxide	2.8	25	[M + H]+	76	58.2	51	25	10
BEH AMIDE	(+)	Trimethylamine_N-oxide	2.8	25	[M + H]+	76	59.1 (Q)	51	17	10
BEH AMIDE	(+)	Tryptophan	3.4	25	[M + H]+	205	188.1 (Q)	26	15	14
BEH AMIDE	(+)	Tryptophan	3.4	25	[M + H]+	205	146.2	26	25	12
BEH AMIDE	(+)	Tyrosine	3.8	25	[M + H]+	182	136.1 (Q)	41	19	16
BEH AMIDE	(+)	Tyrosine	3.8	25	[M + H]+	182	91	41	39	16
HSS T3	(−)	UDP-GlcNac	0.5	40	[M−H]−	605.9	78.9	−70	−128	−11
HSS T3	(−)	UDP-GlcNac	0.5	40	[M−H]−	605.9	272.9 (Q)	−70	−46	−15
HSS T3	(−)	UDP-GlcNac	0.5	40	[M−H]−	605.9	384.9	−70	−38	−25
BEH AMIDE	(+)	Uracil	1.9	55	[M + H]+	113	70.0 (Q)	51	21	10
BEH AMIDE	(+)	Uracil	1.9	55	[M + H]+	113	96	51	23	12
BEH AMIDE	(+)	Urea	1.9	50	[M + H]+	61	44.0 (Q)	66	23	20
HSS T3	(−)	Uric acid	1.1	70	[M−H]−	167	123.9 (Q)	−25	−20	−13
HSS T3	(−)	Uric acid	1.1	70	[M−H]−	167	42	−25	−44	−19
BEH AMIDE	(+)	Uridine	2.5	25	[M + H]+	245	113.1 (Q)	76	15	14
BEH AMIDE	(+)	Uridine	2.5	25	[M + H]+	245	70	76	45	12
HSS T3	(−)	Ursodeoxycholic acid	8.6	40	[M−H]−	391.2	391.2 (Q)	−140	−5	−5
BEH AMIDE	(+)	Valine	3.7	25	[M + H]+	118	72.1	20	15	12
BEH AMIDE	(+)	Valine	3.7	25	[M + H]+	118	55.1 (Q)	20	27	8
HSS T3	(+)	Xanthine	1.6	40	[M + H]+	153	110.0 (Q)	46	27	16
HSS T3	(+)	Xanthine	1.6	40	[M + H]+	153	136	46	23	12
BEH AMIDE	(+)	Xanthosine	3.2	25	[M + H]+	285	153.1 (Q)	71	15	12
BEH AMIDE	(+)	Xanthosine	3.2	25	[M + H]+	285	136	71	43	16
HSS T3	(−)	Xanthurenic acid	3.8	30	[M−H]−	203.9	160.0 (Q)	−20	−22	−11
HSS T3	(−)	Xanthurenic acid	3.8	30	[M−H]−	203.9	115.9	−20	−34	−13

Abbreviations: CE, collision energy; CXP, collision cell exit potential; DP, declustering potential; ID, identifier; min, minutes; MRM, multiple reaction monitoring; m/z, mass-to-charge; s, seconds; (Q), quantifier ion; Q1, first quadrupole; Q3, third quadrupole; V, voltage.

The MRM instrument settings for metabolites detected as protonated species have been collected by direct infusion of synthetic standards in a methanol-water 50:50%(v/v) 0.1% formic acid solution, at concentrations between 1 and 500 ng/mL. Solutions in methanol-water 90:10%(v/v) 0.1% ammonium hydroxide at concentrations between 1 and 1000 ng/mL were prepared for direct infusion of metabolites detected as deprotonated species. Optimal MRM settings were found using the automatic compound optimization function of Analyst 1.7.2. The MRM settings should be reoptimized on a different mass spectrometer.

Expected outcomes

Our protocol targets 260 small polar metabolites. The protocol is tailored to internal interests that focus primarily on profiling amino acids and derivatives, acylcarnitines, tricarboxylic acid cycle intermediates, ketone bodies, and ketoacids. Figure 3 shows the distribution of the targeted metabolites according to their chemical taxonomy. The raw data encompass an assembly of mass-to-charge signals as a function of retention time. Figure 4 shows the superimposed extracted ion chromatograms of the IS quantifier MRMs in the BEH AMIDE assay, to provide an example of chromatographic profiles. The processed results, after data integration and quality controls, are formatted as a data matrix with relative concentrations (or normalized area counts) of all the reported metabolites (columns) for each experimental sample (rows).

Note: Not all targeted metabolites are detected in all biological matrices. It is also important to consider the characteristics of the samples and the population from which the samples are drawn, as differences in the metabolite profile can be expected. In clinical trials, plasma or serum samples are usually collected at fasting state. Differences due to dietary intake can be expected at fed states. Circadian rhythms introduce biological variations in metabolite concentrations as well. Differences are also expected due to age, gender, ethnicity, state of the disease, use of prescription drugs. Differences are expected between serum and EDTA plasma samples.²⁸ Hence, it is important to consistently analyze the same sample type within a study, control pre-analytical variables,²⁹ and randomize the samples based on population characteristics of relevance for the study to avoid biased statistical results.³⁰

Figure 3.

Targeted metabolites

Number of targeted metabolites grouped by SuperClass RefMet classification (A Reference Set of Metabolites Names, https://www.metabolomicsworkbench.org/databases/refmet/browse.php). Data presented as counts.

Figure 4.

Extracted ion chromatogram of the quantifier MRMs for the labeled IS monitored in the BEH AMIDE assay

Data generated from unpublished sources. Graphs were created using Analyst 1.7.2. Top: Positive Ion Mode MRMs [LABELED_1-MeNAM, 3.04 min, m/z 144.0 > 81.0; LABELED_1-methylhistidine, 4.80 min, m/z 173.05 > 127.1; LABELED_4-hydroxyproline, 4.11 min, m/z 135.0 > 89.1; LABELED_Aspartic acid, 4.76 min, m/z 137.1 > 91.0; LABELED_C14, 2.18 min, m/z 381.2 > 85.0; LABELED_C16, 2.13 min, m/z 403.3 > 85.0; LABELED_C2, 2.94 min, m/z 207.1 > 85.1; LABELED_C3, 2.73 min, m/z 221.1 > 85.1; LABELED_C4, 2.59 min, m/z 235.15 > 85.1; LABELED_C5, 2.54 min, m/z 255.1 > 85.1; LABELED_C8, 2.30 min, m/z 291.1 > 85.0; LABELED_Carnitine, 3.52 min, m/z 171.1 > 103.0; LABELED_Citrulline, 4.60 min, m/z 180.1 > 163.0; LABELED_Creatinine, 2.74 min, m/z 117.0 > 47.1; LABELED_Cytosine, 3.02 min, m/z m/z 115.1 > 97.0; LABELED_Glutamine, 4.42 min, m/z 154.1 > 136.1; LABELED_Glycine, 4.27 min, m/z 78.0 > 32.1; LABELED_Histamine, 4.76 min, m/z 116.1 > 99.0; LABELED_Histidine, 5.28 min, m/z 162.1 > 115.1; LABELED_Isoleucine, 3.48 min, m/z 142.05 > 96.0; LABELED_Leucine, 3.40 min, m/z 138.1 > 91.1; LABELED_Methionine, 3.60 min, m/z 155.0 > 108.1; LABELED_Ornithine, 5.30 min, m/z 138.0 > 121.0; LABELED_Phenylalanine, 3.39 min, m/z 171.0 > 106.1; LABELED_SAH, 4.63 min, m/z 395.0 > 141.1; LABELED_Serine, 4.44 min, m/z 109.0 > 63.1; LABELED_TMAO, 2.77 min, m/z 85.0 > 66.1; LABELED_Urea, 1.88 min, m/z 64.0 > 46.0; LABELED_Valine, 3.69 min, m/z 126.0 > 80.1]. Bottom: Negative Ion Mode MRMs [LABELED_NAA, 2.55 min, m/z 177.0 > 91.0; LABELED_N-acetylserine, 2.34 min, m/z 149.0 > 116.9; LABELED_Taurine, 3.72 min, m/z 128.0 > 80.0]. MRM data acquisition settings are listed in Table 4. Abbreviation legend for the ISs can be found in Table 1. Y-axis represents absolute counts and X-axis represents time in minutes.

Quantification and statistical analysis

Timing: 1 day+

This step describes the logic we deploy to check data quality and to advance from raw to processed data.

Note: The time needed to complete this task is highly variable and depends on the total number of samples and the number of metabolites detected. Integrations are managed independently for each assay (BEH AMIDE and HSS T3).

Note: Not all metabolites have ideal peak shapes and are free from interfering signals. The peak integration is therefore a more cumbersome process for less performing metabolites, requiring higher percentage of manual curation of the area under the curve (AUC).

Note: Peak areas are integrated using the instrument software Multiquant (Ab Sciex, sciex.com). Integrations are curated on all quality controls, blank ISs, calibrators, and experimental samples acquired before advancing to the quality control step. A flowchart of the process is presented in Figure S2.

• 1.
  Integrate the quality control plasma.

  • a.
    Integrate the peak areas of all MRMs for the internal standards and a subset of certified and ‘information value’ metabolites (see metabolite list for NIST SRM 1950 plasma in Table S7).
• 2.
  Integrate the blank IS.

  • a.
    Integrate all the IS MRMs.
  • b.
    Integrate noise for the quantifier MRM of each targeted metabolite at the expected RT.
  • c.
    Average the blank IS noise areas for each targeted metabolite.
  • d.
    Set signal thresholds as area counts greater than the average blank IS noise area for each targeted metabolite.
• 3.
  Integrate the ISs in the experimental samples.

  • a.
    Integrate all the IS MRMs. Area counts are used to identify sample outliers (e.g., partial or missed injections) as described in the quality control section.
• 4.
  Identify the ‘not-detected’ and ‘detected’ metabolites.

  • a.
    Integrate peak areas of each quantifier MRM for each targeted metabolite in all experimental samples. Integrate noise if the signal is not distinguishable from the blank.
  • b.
    Calculate the fraction of experimental samples with peak areas for the quantifier ions greater than the signal thresholds:

    • i.
      If the fraction is <25%, label the metabolite as ‘not-detected’ and exclude it from further quality control steps and statistical analysis.
    • ii.
      If the fraction is ≥ 25%, label the metabolites as ‘detected’. Integrate the qualifier ions for the ‘detected’ metabolites in all experimental samples.
• 5.
  Label the ‘detected’ metabolites as ‘not-regressed’ or ‘regressed’

  • a.
    Integrate the peak areas of all MRMs for each ‘detected’ metabolite in the calibrators.
  • b.
    Label the ‘detected’ metabolites that have average peak areas for the quantifier ions lower than signal thresholds in at least the lower calibrator (5%) as ‘not-regressed’.
  • c.
    Label the ‘detected’ metabolites passing the signal threshold rule for all calibrators as ‘regressed’.

Note: The rationale described above allows us to identify metabolites (labeled as ‘not-regressed’) which could be selectively detected in a subset of the sample cohort (as low as 25%) but sufficiently diluted when pooling all the experimental samples (to prepare the ‘100% pool calibrator’) such that average signal might not be distinguishable from noise in the calibrators (created by dilution of the 100% pool). Metabolites identified as ‘not regressed’ are submitted to statistical analysis using normalized area counts (rather than regressed relative concentration to pooled calibrators) to test if significant differences in the distribution of normalized signal is associated with factors of interest (e.g., gender, treatment, etc.). This situation has been very rarely encountered in our studies.

• 6.
  After data integration is completed, check data assembly.

  • a.
    For each batch, export all peak areas and RTs of all ‘regressed’ metabolites and all ISs for all experimental samples, blank ISs and calibrators ran from the instrument software.
  • b.
    Verify in the data export that there are no ‘N/A’ values present. The Multiquant software will output ‘N/A’ if no peak has been integrated, hence this check verifies that no peak has been accidentally skipped during the integration step.
  • c.
    Verify that the total number of area counts in the export file is equal to the total number of samples ran per batch (sum of all experimental samples and calibrators, including blanks) multiplied by the number of MRMs integrated (e.g., 200 MRMs for 71 experimental samples, two blank ISs, and 14 calibrators equate to 17,400 data points in the export file). This check verifies that the export file is correct, and no data is missing (e.g., the output file includes only a portion of the batch).

Note: The protocol relies on a series of custom-made quality controls applied to the experimental samples and the targeted metabolites. Experimental samples and/or calibrators identified as outliers (e.g., missed or partial injections, sample preparation errors) are removed from the dataset that advances to the quantitation step. Metabolites are also removed from the dataset if one or multiple quality controls fail.

Note: The quality controls are performed independently for the BEH AMIDE and HSS T3 assays.

Only the quality controls applied to the IS and ‘regressed’ metabolites are described below. The quality controls are performed independently on separate batches of experimental samples and calibrators ran, unless otherwise specified.

• 7.
  Build IS quality control charts for each IS.

  • a.
    Calculate the average area count and standard deviation (SD) using all experimental samples and calibrators (including blank ISs).
  • b.
    Calculate the warning limits as average area count ±2 SDs.
  • c.
    Calculate the action limits as average area count ±3 SDs.
  • d.
    Build the quality control charts that display the absolute area counts for each experimental sample and calibrator (including blank ISs) overlaid with the warning and action limits (Figure 5).
  • e.
    Identify outliers as samples having IS area counts outside the action limits:

    • i.
      If >5% of the ISs falls outside the action limits for the sample (either an experimental sample or a calibrator), remove the sample from the dataset.
    • ii.
      If ≤5% of the ISs falls outside the action limits for the sample, check the peak integrations. If no corrections are needed, flag the sample for further review.

Note: We have observed that missed or partial injections are the most frequent outliers (still occurring less than 1%) giving peak area counts significantly lower than average for all ISs, thus easy to identify upon inspection of the quality charts. Improper sealing of the LC-MS 96-well plates with the aluminum foil, causing the organic solvent mixture to evaporate overtime, has been identified as the main cause of missing or partial injections.

• 8.
  Calculate the IS RT coefficient of variations for each IS:

  • a.
    Calculate the RT average and SD using all experimental samples and calibrators (including blank ISs) that ran in the batch and were not identified as outliers.
  • b.
    Calculate the coefficient of variation (CV%) as SD divided by average RT, multiplied by 100.
  • c.
    Verify to ensure that the CV% is <2%. If the CV% is ≥ 2%:

    • i.
      Calculate the difference between the average RT and each individual sample RT (either experimental sample or calibrator or blank IS) and identify the samples that deviate outside the average ± 3 SD limit.
    • ii.
      Inspect the peak integrations to verify no mistake has occurred and flag the outlier samples for further review.
• 9.
  Calculate ‘regressed’ metabolite RT coefficient of variations for each ‘regressed’ metabolite:

  • a.
    Calculate the RT average and SD for the quantifier ion using all experimental samples and calibrators (excluding the blank ISs) that were not identified as outliers.
  • b.
    Calculate the CV% as SD divided by average RT, multiplied by 100.
  • c.
    Verify to ensure that the CV% is <2%. If the CV% is ≥ 2%:

    • i.
      Calculate the difference between the average RT and each individual sample RT (either experimental sample or calibrator, excluding the blank ISs) and identify the samples that deviate outside the average ± 3 SD limit.
    • ii.
      Inspect the peak integrations to verify no mistake occurred and flag the outlier samples for further review.

Note: The overall accuracy and precision of the RTs for both the ISs and the ‘regressed’ metabolites is monitored by comparing the RT averages and SDs across all batches. No significant differences and shifts over time are expected.

• 10.
  Calculate the qualifier-to-quantifier ion RT difference for each ‘regressed’ metabolite:

  • a.
    Calculate the difference between the RTs of the qualifier and quantifier ions in each sample (experimental samples and calibrators, excluding blank ISs, that were not identified as outliers).
  • b.
    Verify to ensure that the difference is < 0.01 min. If the rule fails:

    • i.
      Inspect the peak integrations to verify that no mistake has occurred.
    • ii.
      Flag the outlier samples for further review.
• 11.
  Calculate ‘regressed’ metabolite MRM coefficient of variations for each ‘regressed’ metabolite:

  • a.
    Calculate the ratio of qualifier to quantifier MRM peak areas in all experimental samples and calibrators (excluding blank ISs) that have not been identified as outliers.
  • b.
    Calculate the average MRM ratio, SD, and CV%.
  • c.
    Verify to ensure that the CV% is <15%. If CV% is ≥ 15%:

    • i.
      Calculate the difference between the average MRM ratio and the MRM ratio of each individual sample (either experimental samples or calibrators, excluding the blank ISs, that were not identified as outliers) and identify the samples that deviate outside the average ± 3 SD limit.
    • ii.
      Inspect the peak integrations to verify no mistake occurred and flag the outlier samples for further review.

Note: The combination of the above rules helps identify if the imprecision of the MRM ratios is high overall and or if a particular subset of samples drives the high CV%. In the latter case, provided no errors are found in the peak integrations, careful revision of the chromatograms is warranted to understand if unknown matrix effects and/or interferences affect the data. Low signal-to-noise has been found to be the most common driver of high CV%.

Note: The overall accuracy and precision of the MRM ratios for all ‘regressed’ metabolites is monitored by comparing the MRM ratio averages and SDs across all batches. No significant differences and shifts are expected over time.

Note: Similar quality controls are applied to the certified quality control plasma and are used to assess the instrument and assay repeatability over time (across studies). Similarly, to what described above, consistency of RTs and absolute signal for the ISs, as well as consistency of RTs, absolute and normalized signal of reference analytes (Table S7), and MRM ratios are monitored accross studies ran over time using independent aliquots of certified plasma extracted with different batches of extractions solutions.

• 12.
  Calculate linear regressions for each ‘regressed’ metabolite:

  • a.
    Normalize the quantifier ion area count for each calibrator (excluding the blank ISs) against the area count of each IS.
  • b.
    For each IS, compute a linear regression where the dependent variable (Y) is the normalized area count in a calibrator and the independent variable (X) is the dilution percentage of the ‘100% pool calibrator’ (5%, 10%, 15%, 30%, 50%, 75%, and 100%).
  • c.
    For each IS, tabulate the slope, intercept, correlation coefficient, residuals, and root mean square error (RMSE), see example in Table S8.
  • d.
    For each IS, calculate the average and SD of the correlation coefficient, and RMSE across all batches.
  • e.
    Overlay calibrator responses and regression lines on a scatter plot for all batches to visually observe consistency across a study.
  • f.
    Plot a bar graph that ranks the ISs from best to worst regression fit (from largest to smallest correlation coefficient ±SD) as shown in Figure 6 as an example.

Note: The protocol relies on a custom-made strategy for data normalization. Quality controls are built into the data normalization strategy to drive final decisions on which ‘regressed’ metabolites advance to statistical analysis. Data normalization is performed independently for the BEH AMIDE and HSS T3 assays. Calculations are performed independently on separate batches, unless otherwise specified.

• 13.
  Select the matching IS for each ‘regressed’ metabolite to normalize the data of all batches, following all criteria below:

  • a.
    Same polarity of molecular ions between the ‘regressed’ metabolite and the IS,
  • b.
    Largest correlation coefficient for the regression line,
  • c.
    Smallest RMSE average and SD across batches,
  • d.
    Closest RT between the ‘regressed’ metabolite and the IS,
  • e.
    Closest chemical taxonomy (e.g., stable-labeled amino acids to normalize endogenous amino acids such as aspartate-d₃ for glutamate, or stable-labeled acylcarnitine to normalize endogenous acylcarnitines such as O-octanoyl-L-carnitine-d₃ for decanoylcarnitine),
  • f.
    Whenever possible, match endogenous metabolites with their stable-labeled IS (e.g., leucine with ¹³C₆-leucine), which is expected to provide the optimal regression fit (see Table S8; Figure 6),
  • g.
    If multiple ISs are equally suited, select the pairing that is most consistently being used across studies for the same biological matrices.
  • h.
    If none of the matches provides an average correlation coefficient >0.85, remove the metabolite from the final dataset and exclude it from statistical analysis.

Note: The same ISs are used to normalize the area counts of ‘regressed’ metabolites across all batches.

Note: The repeatability of the assay has proven to be very high within the same biological matrices across studies, with most of the targeted metabolites showing optimal matching with the same ISs consistently. Table S9 lists the preferred IS matches for a subset of targeted metabolites consistently detected across different biological matrices.

• 14.
  Regress the normalized area counts for each ‘regressed’ metabolite:

  • a.
    Normalize the quantifier ion area count with the area count of the chosen IS.
  • b.
    Calculate the slope and intercept of the linear regression using the normalized area counts as dependent variable (Y) and the dilution percentage of the pool calibrators as independent variable (X). Blank ISs are excluded. Do not force intercept through the origin.
  • c.
    Use the linear regression equation to calculate the percentage of dilution for each experimental sample (excluding outliers).
  • d.
    Export the results as regressed relative concentrations (%) in a datamatrix (samples by metabolites).

Note: We recommend not imputing relative concentrations of >100% and between 0-5%. Negative values are imputed with a fixed value (2.5) if less than 5% of the total number of data points, otherwise a random distribution centered around half of the lowest calibrator (2.5) and fixed SD (similar to that of the lowest calibrator) is used.

Note: As described in steps 30 and 31, the experimental samples are diluted 1:1 with the extraction solution while transferring them to the LC-MS 96-well plate, after an aliquot of the supernatant is pooled to create the ‘100% pool calibrator’. This shifts the average regressed concentration of all experimental samples around 50% (±regression uncertainty). The 1:1 dilution step is performed to center the experimental samples within the calibration range (5–100%), see Figure S3. Extrapolations >100% and <5% for individual experimental samples that are either significantly more concentrated or diluted than the 100% pool are expected and more likely to occur as the number of experimental samples in a study increases.

Note: It is important to remember that the metabolite signal at the lowest calibrator is not necessarily close to the limit of detection for a specific MRM. The signal-to-noise of the lowest calibrator can be very high depending on the actual concentration of the metabolite in the 100% pool, the ionization efficiency of the metabolite, and the noise level in blanks. On the other hand, some metabolites could have lower signal-to-noise in all calibrators.

Note: As the pool calibrators run throughout the entire data acquisition, they can be used as quality controls to monitor the repeatability of the assay over time. Quantifying the experimental samples relatively to the pooled calibrators also allows to normalize plate-to-plate differences.

CRITICAL: This protocol targets 260 small polar metabolites and provides relative quantitation compared to pool calibrators. Accuracy and precision in quantifying all targeted metabolites simultaneously are not the utmost achievable using mass spectrometry. Matrix effects, interferences from isomers and isobaric species, poor chromatographic behavior, poor ionization efficiency, and variable concentration and signal-to-noise ranges are the most common issues that lower the accuracy and precision of the quantitation in targeted metabolomics protocols compared to developing methods to quantify each targeted metabolite independently.

Figure 5.

Quality control chart for LABELED_Taurine (RT: 3.72 min, [M−H]⁻m/z 128.0 > 80.0)

Warning limit (average area counts ±2 standard deviations [SD]): yellow line. Action limit (average area counts ±3 SD): red line. Each gray dot represents an injection. Y-axis represents absolute counts and X-axis represents counts.

Figure 6.

Linear regression

Bar graph of correlation coefficients for Alanine (top) and Isoleucine (bottom) against all combinations of ISs measured in the BEH AMIDE assay. Internal standards are ranked in order of decreasing average correlation coefficient. Data are from unpublished sources. Data presented as mean ± standard deviation (SD).

The normalized data are regressed against calibrators built using the pool of all the experimental samples. The results are expressed as percentages, making them not directly comparable across studies. Namely:

For each metabolite, a calibrator is assigned a relative percentage concentration based on the dilution factor from the ‘100% pool calibrator’ (e.g., calibrator 5% represents a dilution factor of 20) regardless of the metabolite absolute concentration in the 100% pool. Thus, one cannot state that a metabolite in calibrator 30% is more concentrated than another metabolite in calibrator 5%.

Due to ion suppression, matrix effects, and differences in ionization efficiencies, we cannot state that within the same calibrator, a metabolite with greater area counts is more concentrated than the metabolite with lower area counts.

Calibrators across studies are assigned the same relative dilution factor (e.g., 30%) if they have been diluted to the same extent from the ‘100% pool calibrator’. However, if the pool calibrators have been built pooling different experimental samples, we cannot state that the concentration of any metabolite in the two 30% calibrators is the same, or greater, or lower compared to each other.

Metabolite relative concentrations can be compared directly if the regressed data have been calculated against the same pool calibrators. An experimental sample can be directly compared to the pool calibrators. For example, it is appropriate to state that an experimental sample with a regressed concentration of 50% for valine has a concentration equal to (±regression uncertainty) the average valine concentration of all the experimental samples used to create the 100% pool. We remind the reader that the experimental samples are diluted 1:1 for the preparation of the LC-MS 96-well plates after an equal volume of the supernatant is pooled to prepare the ‘100% pool calibrator’.

Limitations

Some limitations apply to targeted metabolomics approaches in general. Briefly, the detection of the metabolites using LC-MS/MS is highly dependent on their actual concentration in a biological matrix, therefore, not all the metabolites targeted are detected in all biological matrices. The sample preparation requires extraction of soluble metabolites in organic solvent mixtures that are optimized based on the targets of interest; inevitably, loss of less soluble metabolites occurs depending on the organic solvent mixture used to extract the samples, thus biasing the list of detectable metabolites. It is also important to state that the metabolite coverage in targeted metabolomics assays is small compared to the number of polar metabolites identified in the human metabolome. We acknowledge that the use of targeted metabolomics applied to drug discovery and development has therefore the risk of being agnostic to relevant metabolites which are not included in the assay. Another limitation lies in the analysis of stereoisomers, which are intrinsically difficult to separate using conventional separation techniques. Chiral separation strategies are usually needed to reach such structural differentiation of metabolites (e.g., L- vs. D- stereoisomers), which could have highly significant clinical impact. Also noteworthy to note that targeted metabolomics provides a static snapshot of metabolism and does not reveal dynamic metabolic changes. Metabolic flux analysis and isotope enrichment studies are needed for such purpose. In addition to that, translation of metabolomics-derived findings into mechanistic insights requires a multidisciplinary approach and extensive validation of findings using in vivo assays, replication cohorts, animal model knockouts, etc.; all resources that might not be available. Lastly, the biological interpretation of findings can be hindered by poor or missing metabolite annotations and knowledge of biological pathways. Just the same, (pre-)analytical issues and biases, as well as flawed experimental designs can compromise the quality of the samples used for analysis, hinder the reliability of the results, and undermine the ability to answer relevant and specified biological questions.

Specifically, to this protocol, the quantitation strategy relies on the preparation of experimental sample pool calibrators rather than using surrogate matrices like charcoal stripped plasma, water, bovine serum albumin, phosphate buffered saline, and mixtures thereof, into which synthetic standards are spiked. Primary reason for this choice is the inaccurate and cumbersome operation of spiking 260 synthetic standards in the surrogate matrix in variable concentration ranges that closely mimic the range of metabolite concentrations in the experimental samples. Also, the protocol provides only relative rather than absolute quantitation of metabolites in biological samples. We note, anyhow, that the protocol can detect selective patterns of metabolic alterations in response to stimuli (e.g., a pharmacological intervention compared to placebo) which we leverage to investigate and better understand mechanism of action of drugs, disease progression, metabolic phenotypes, and patient stratification. Although only relative concentrations to pooled calibrators are calculated in our protocol, our quantitation strategy relies on a multipoint calibration strategy to express the results rather than using only normalized area counts or single-point calibration to the 100% pool or to the IS concentration in solution, for the following main reasons:

Both using normalized area counts or a single-point calibration strategy to express results assume that a zero-concentration sample (blank) would give a response of zero. Therefore, use the origin in order to linearly model the response to the sample concentration, which is frequently inaccurate.

They both assume that equal fold changes in the response between samples equate to the same fold chance in concentrations for different metabolites, which is not accurate if the analytical sensitivities are different.

Interfering background noise from the extraction solution can be easily identified (as opposed to true signal that originates from the extracted experimental samples) if the signal does not proportionally decrease from high to low calibrators.

Another limitation is that relative concentrations to the pool calibrators are calculated using linear regressions. The pool calibrators span only across one order of magnitude (dilution factor of 20 between calibrators 100% and 5%). Conditions of linearity are almost always met. However, quadratic and weighted 1/x regressions as also commonly used for mass spectrometry datasets. It is possible to build an unbiased approach where linear, quadratic, and weighed regressions are compared to find the optimal regression fit. Lastly, our protocol relies on normalizing all regressed metabolites against all available ISs to experimentally verify the optimal match for data regression. We developed a custom-made data processing pipeline to be able to perform such computations (not in scope for this protocol). The lack of statistical and informatic support can limit other users’ ability to adopt such strategy. Since the same targeted metabolite-IS matchings have been largely observed as optimal across studies, we provide here pre-defined list of matches for the portion of targeted metabolites that is more consistently detected in different biological matrices as a simpler alternative strategy (Table S9).

Troubleshooting

Problem 1

There is not sufficient volume of extraction solution to complete the preparation of the experimental samples and fresh batches of extraction solutions might need to be prepared, potentially causing analytical biases and batch effects (related to step 4).

Potential solution

• •We recommend storing the extraction solution in small aliquots to avoid accidental loss of large amounts of solution. Also, as described in Table S4, we recommend preparing twice the amount of extraction solution needed to prepare all experimental samples, calibrators, and quality controls to account for possible reruns.
• •If the volume of the extraction solution was sufficient to extract all experimental samples, but it is not sufficient to prepare the pooled calibrators and blank ISs, we recommend recalculating the number and the level of the calibrators to prepare (e.g., 5%, 15%, 30%, 60%, and 100% instead of 5%, 10%, 15%, 30%, 50%, 75%, and 100%) and/or reduce the frequency of calibration curves to require less extraction solution overall.
• •If the volume of the extraction solution is not sufficient to extract all experimental samples, and multiple batches must be prepared, we recommend randomizing the experimental samples such that different batches of extraction solution are not used to extract subsets of experimental samples that need to be statistically compared (e.g., wild-type vs. knockout). We also recommend comparing blank ISs prepared using the different batches of the extraction solution to verify the IS responses are not significantly different.

Problem 2

There is insufficient plasma aliquot, i.e., < 25 μL (related to step 2).

Potential solution

It is possible to use as little as 10-μL aliquots to run our assays. The sample preparation requires 1:6 dilution of plasma with extraction solution, hence 60 μL of extraction solution to be added in the well of the plate containing the 10-μL aliquot. We warn that since 20–25 μL of supernatant are needed to prepare the final LC-MS 96-well plates, and variable amounts (typically between 5-20 μL) of supernatant are used to prepare the 100% pool calibrator, sample reruns might not be possible. In such case, we recommend the use of resealing pierceable silicone mats to cover the LC-MS 96-well plate rather than the use of aluminum heat seals to avoid evaporation of the supernatant after LC injection and allow for long term storage of the LC-MS 96-well plate at −20°C. Alternatively, consider using autosampler vials for LC injection capped with resealing pierceable screw caps, which however can be extremely laborious when the number of samples is large.

Problem 3

Clots can form in the sample 96-well plate containing the plasma aliquots which can cause inaccuracies when mixing the extraction solution in the well and/or potentially contaminate surrounding wells (related to step 9).

Potential solution

We recommend using a small pipette tip (< 20 μL dispensing volumes) to mix the sample thoroughly. If a large clog is present, gently tap the solution inside the well and then slowly mix the extraction solution with the plasma by aspirating only small volumes (≤ 10 μL) multiple times (10×).

Problem 4

To prepare the ‘100% pool calibrator’, the sample 96-well plate is centrifuged and then the adhesive aluminum foil removed to transfer the supernatant into the LC-MS 96-well plate (related to step 15). When gently peeling off the adhesive foil from one corner of the plate, one could observe that supernatant is wetting the bottom surface of the foil and peeling off the entire adhesive foil could cause sample cross-contamination. This typically happens if the plate is not handled carefully after the centrifugation.

Potential solution

Do not attempt to remove the adhesive foil. Reseal the corner of the foil peeled off and re-centrifuge the plate for 5 min at 4000 rpm (3200 × g) at +4°C.

Problem 5

Less than 25 μL of plasma is available for a subset of experimental samples (related to step 2).

Potential solution

Transfer 10 μL of plasma in the sample 96-well plate, instead of 25 μL, annotate the plate number and well coordinates, and add 60 μL of extraction solution, to maintain a dilution ratio of 1:6. Skip these samples from the preparation of the ‘100% pool calibrator’ to spare supernatant for the preparation of the LC-MS 96-well plates.

Problem 6

While preparing the ‘100% pool calibrator’, a small subset of samples is accidentally skipped or, conversely, added twice (related to step 17).

Potential solution

There is no need to reprepare the ‘100% pool calibrator’, unless the total volume is now insufficient to prepare the rest of the calibrators (see an example of how to calculate volumes in the Table S4). As long as aliquots of the same calibrators are systematically injected during the LC-MS/MS analysis, our quantitation strategy can be applied correctly, although the pooled calibrators are not exactly representative of all the experimental samples.

Problem 7

The concentration range of the endogenous metabolites in the extracted biological matrix can be widely different depending on the matrix itself (e.g., urine vs. adipose tissue), the species (e.g., human vs. rat), the disease/treatment state (e.g., wild type vs. knockout), in a manner that is somewhat unpredictable. Highly concentrated metabolite might have signals that are close to saturating the detector of the mass spectrometer, which could compromise the quality of the data acquired (related to step 14, quantification and statistical analysis section).

Potential solution

To lower saturating signals of highly concentrated metabolites different solutions can be adopted:

• •detune the collision energy to reduce the efficiency of the CID fragmentation and lower the signal of the fragment ion isolated in the third quadrupole.
• •choose lower abundant fragments as quantifier ions.
• •as operators become familiar with the protocol and consistently observe that certain metabolites are highly concentrated in specific biological matrices (e.g., creatine in skeletal muscle), we recommend adding matching IS to the extraction solution to help model a linear response of the normalized area counts in the calibration curve.

Problem 8

Highly concentrated metabolites can also affect the performance of the chromatographic separation, thereby altering chromatographic peak shapes and potentially causing carry-over issues, as no solutions are available to avoid column overload other than additionally diluting the supernatant and/or reducing the volume of the supernatant injected into the LC system (related to step 14, quantification and statistical analysis section).

Potential solution

• •Prior to starting data acquisition of calibrators and experimental samples, inject an aliquot of a blank IS, followed by an aliquot of the ‘100% pool calibrator’ and then a double blank to estimate the signal-to-noise of all detected metabolites and observe their carry-over in the double blank. List all metabolites that might require detuning of the instrument settings (as mentioned in problem 7).
• •Further diluting the experimental samples (and the calibrators accordingly) or decreasing the LC injection volume is another possible solution, but needs to be carefully evaluated considering the totality of the metabolites of interest, as some could become undetected if signal-to-noise is lowered.
• •If instrument time is available, experimental samples and calibrators could be injected multiple times using different injection volumes and customizing the list of MRMs acquired with each injection so that the most abundant signals are collected when injecting the smaller volumes.

Problem 9

The ‘instrument queque’ might stop at random points in a sequence if various LC-MS issues arise, requiring troubleshooting and/or maintenance (related to step 14, quantification and statistical analysis section).

Potential solution

We recommend preparing additional aliquots of the calibrators to restart the data acquisition with a set of a blank IS and calibrators before resuming the acquisition of experimental sample data when the instrument is ready to restart. Just the same, if scheduled maintenance of the instrument is needed, we recommend adding a set of a blank IS and calibrators before stopping the data acquisition and upon resuming it.

Problem 10

The number of MRMs is large and could overall exceed the threshold of total cycle time (250 ms) set to assure that enough data points is collected to accurately trace the chromatographic peaks of the metabolites as they elute from the UPLC columns (related to step 53).

Potential solution

• •Employ the advanced MRM scheduling option in Analyst 1.7.2 (Ab Sciex) that allows one to customize the MRM window of every metabolite and therefore optimize as much as possible the scan MRM time (see Table 4). The MRM windows can be adjusted based on the peak shape and peak width at baseline, precision of the RTs, and known matrix effects that might contribute to minor shifts of the RTs. Smaller MRM windows can be set for sharper peak shapes.
• •Set a target cycle time across MRMs rather than a target scan time per MRM (see Table 2) in Analyst 1.7.2. By doing so, the dwell time per MRM is calculated not to exceed the total cycle time threshold set (250 ms). The instrument software calculates a dwell to assign to each MRM based on the total number of MRMs falling at any given retention time. Be aware that the minimum dwell time is 3 ms, therefore, a 3-ms dwell will be set for every MRM for which the theoretically calculated value would have been ≤3 ms. This means that if the number of MRMs is too high, the total cycle time will exceed the threshold regardless of the method settings.
• •If instrument time is available, run multiple injections of the same samples to collect different MRMs separately and merge the data after acquisition. It is critical to split evenly the MRMs monitored across the entire chromatographic run to avoid overpopulating any portion of the chromatographic run anyhow. The use of different instrumentation allowing for faster data acquisition (e.g., minimum dwell of 1 ms), can also help mitigate this problem.

Problem 11

Our protocol relies on running two chromatographic assays, BEH AMIDE and HSS T3, both requiring protein precipitation with organic solvent mixtures and direct injection of the supernatant. The extraction solvent for the HSS T3 assay is a mixture of 80:20%(v/v) methanol-water. The LC gradient of the HSS T3 assay starts with high percentage of the aqueous mobile phase (Table 2). The injection of a highly organic supernatant into a mobile phase with high water percentage can result in poor chromatographic behavior of the first eluting polar metabolite (related to step 53).

Potential solution

We recommend using a pre-column mixer (see key resources table) to ensure the highest possible mixing efficiency of the organic and aqueous solutions, resulting in sharper and more symmetrical peak shapes for the first eluting polar metabolites.

Problem 12

The pool calibrators are prepared as serial dilution of the pool of all experimental samples (the ‘100% pool calibrator’) into the neat extraction solution, hence increasingly diluting the biological matrix into an organic solvent mixture. A few metabolites in our panel show minor shifts of retention time and peak shape because of variable matrix composition (referred to step 16).

Potential solution

We suggest adding matching ISs for the metabolites more susceptible to matrix effects to help increase confidence in peak identification and integration.

Problem 13

The pool calibrators are prepared as serial dilution of the pool of all experimental samples into the neat extraction solution, which increasingly dilutes the biological matrix. Matrix effects can affect the ionization efficiency, either suppressing or enhancing it, which can lead to nonlinear responses of area counts across the calibration range. The quality control charts built on absolute area counts of the ISs can be affected by ionization effects. Figure S4 shows the example of an IS response that decreases at higher concentrations of the calibrators due to ion suppression. The variability of the response in the calibrators can lead to the blank IS and more concentrated calibrators (e.g., the 100% pool) falling outside the action limits, thus more prone to be identified as outliers (referred to step 16).

Potential solution

For ISs in which response is sensitive to matrix and ionization effects, we recommend building a separate series of quality control charts excluding the blank IS and all calibrators to assess the variability of the signal only across experimental samples. We also recommend adding ‘matching IS’ to the extraction solutions for endogenous metabolites that are prone to such matrix effects to help normalize signal response and increase the accuracy and precision of the regression fit.

Resource availability

Lead contact

Further information and requests on procedures, resources and reagents should be directed to and will be fulfilled by the lead contact, Valentina Pirro (pirro_valentina@lilly.com).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Valentina Pirro (pirro_valentina@lilly.com).

Materials availability

Materials were purchased from publicly available vendors or prepared in-house from commercial reagents. Key resources table details all the preferred materials used in the laboratory for the execution of the protocol.

Data and code availability

Part of data presented in this protocol is published.^1,18,19,20 Part of the data is unpublished and generated during method development.