Protocol for untargeted multi-omics dried blood spot extraction using liquid chromatography-tandem mass spectrometry

Summary

Dried blood spots (DBS), most notably employed in newborn screening, present a valuable opportunity to analyze whole blood via a stable and simplified sample collection. DBS are generally used to evaluate one class of compounds at a time. Herein, we present a protocol to semi-quantitatively evaluate metabolites and lipids from the same DBS using a multi-omics approach. We describe steps for extracting both metabolites and lipids from the paper-based sample and analyzing them via liquid chromatography-tandem mass spectrometry.

For complete details on the use and execution of this protocol, please refer to Li et al.¹

Graphical abstract

Highlights

• •Biphasic extraction of lipids and metabolites from dried blood spots
• •LC-MS/MS-based analysis of lipid and metabolite dried blood-spot extracts
• •Guidance on reverse-phase chromatography for lipids and metabolites for LC-MS/MS

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

Dried blood spots (DBS), most notably employed in newborn screening, present a valuable opportunity to analyze whole blood via a stable and simplified sample collection. DBS are generally used to evaluate one class of compounds at a time. Herein, we present a protocol to semi-quantitatively evaluate metabolites and lipids from the same DBS using a multi-omics approach. We describe steps for extracting both metabolites and lipids from the paper-based sample and analyzing them via liquid chromatography-tandem mass spectrometry.

Before you begin

Dried blood spots (DBS) are a collection technique that offers several advantages, including good stability, low sample volume, and low cost. Currently, DBS research has successfully extracted a variety of analytes from the spots, including DNA, RNA, proteins, lipids, and metabolites.^2,3,4,5,6 The protocol below describes the specific steps for semi-quantitative metabolite and lipid extraction from a DBS collected from whole blood or plasma. Moreover, this protocol has been utilized with plasma samples.¹ The overall protocol yields semi-quantitative results, where qualitative identification of features are achieved through peak detection and ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowing the identification of analytes, such as amino acids, through tandem mass spectrometry (MS/MS) fragmentation patterned libraries and/or local compound libraries; additionally, relative quantitation is performed through comparison of peak heights or areas of the same features between sample groups. As this protocol is a semi-quantitative approach, absolute quantitation of analytes cannot be determined without additional standards and intervention prior to sampling discs. However, the protocol is intended to be implemented for pre-spotted clinical samples. If necessary, DBS sampling from a liquid sample is described for whole blood or plasma, as both matrices can utilize the protocol. After samples are acquired and dried, the protocol details how to perform a biphasic extraction for lipids and metabolites on DBS. The extraction is followed by a reconstitution for LC-MS/MS that is used for analysis. Finally, the raw data are analyzed using two open-source software tools, LipidMatch and mzmine, for analyte identification and annotation.^7,8 Generally, the data is normalized by sum, log transformed, and auto scaled prior to data analysis to assist in interpreting semi-quantitative results. This study was approved by the local ethics committee and conforms to regulatory standards; implementation of this protocol may require permission from relevant institutions.

The timing is dependent on the number of samples, with increased sample numbers increasing extraction time, analysis time via LC-MS/MS, and data analysis. Total procedure time for n=10 will take approximately 7 days.

Before extraction, verify all materials are present and not expired. Chloroform is utilized in the protocol, and glass should be used in all instances with chloroform present. Additionally, chloroform is hazardous and toxic and should be used in a fume hood, including when drying samples after the extraction. Materials can be obtained from other vendors; however, reagents and materials used in the LC-MS/MS can impact the chromatography. Once extraction begins, keep samples on ice when not directly working with them.

If spotting DBS, the samples should be prepared as described below before starting the protocol.

Innovation

Current DBS extractions are evaluating one class of analytes, i.e., metabolites, lipids, proteins, DNA, or RNA, at one time and from a single spot.^2,3,4,5,6 For metabolites and lipids, a different extraction technique is used for each, which can destroy the other analytes. The purpose of the biphasic extraction is to maximize molecular coverage from a limited and precious DBS sample by enabling simultaneous recovery of both metabolites and lipids from the same sample. Compared to other DBS extractions, where metabolites and lipids would require two samples. The protocol is designed to maximize analyte information from a limited sample. The DBS multi-omic protocol applies biphasic extraction similar to a Folch extraction.⁹ The Folch method applies a biphasic protocol to a liquid biological sample for the simultaneous extraction of metabolites and lipids using methanol and chloroform. Herein, a modified protocol is applied to DBS for the extraction of both metabolites and lipids from one sample.

Institutional permissions

All aspects of the extraction in this protocol must be performed in accordance with relevant regulatory and safety standards. Approval from the institution and following national guidelines and regulations are required when performing experiments on patient-derived samples. The local ethics committee approved this study (IRB202202277).

DBS sampling

Timing: 3 h

This section of the protocol covers spotting DBS discs for subsequent multi-omic extraction. The protocol can be used for plasma or whole blood. If using pre-spotted samples, skip to the Metabolite and Lipid Extraction from DBS. The protocol is written for a 10 μL volumetric disc contained in the Capitainer B10 microfluidic device; however, sheet sample discs can be used for method development and analysis. Additionally, different DBS can be punched out and used with the protocol.

• 1.Pipet 10 μL of whole blood or plasma onto the sampling disc.
• 2.
  Allow spots to dry at 25 °C for at least 3 h in the dark.

  • a.
    If storing discs for later extraction and analysis, allow the discs to dry, then store in a dry, dark, sealed container at 25 °C or sealed in a freezer (−80°C).

CRITICAL: Disc must be fully dried prior to any extraction.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Healthy Adult Whole Blood Dried Blood Spot	University of Florida	IRB202202277
Red Cross Plasma	Red Cross	N/A
Chemicals, peptides, and recombinant proteins
Chloroform, ACS 99.8+%	Fisher Scientific	Cat#AA32614K2
Water with 0.1% Formic Acid (Optima)	Fisher Scientific	Cat#LS118
Water (Optima)	Fisher Scientific	Cat#W6212
Acetonitrile (Optima)	Fisher Scientific	Cat#A955
Methanol (Optima)	Fisher Scientific	Cat#A456
2-Propanol (Optima)	Fisher Scientific	Cat#A461
Formic Acid Optima LC/MS Grade	Fisher Scientific	Cat#A117-50
Ammonium Acetate (Crystalline/HPLC)	Fisher Scientific	Cat#A639-500
Metabolomics Amino Acid Mix	Cambridge Isotope Laboratories, Inc.	Cat#MSK-A2-1.2
SPLASH LIPIDOMIX Mass Spec Standard	Avanti Research	Cat#330707
Software and algorithms
Mzmine 4.9	Schmid et al.7	https://mzmine.github.io/mzmine_documentation/
LipidMatch	Koelmel et al.8	https://innovativeomics.com/software/lipidmatch-suite
Other
1 mL Corning PYREX Disposable Individually Wrapped Glass Serological Pipets	Fisher Scientific	Cat#13-666-7F
Disposable Pasteur pipets	Fisher Scientific	Cat#K883350-0009
Capitainer®B10	Capitainer	Cat#B10
Glass LC vials with fused insert	Microsolv	Cat#9532S-E-0CV
5 mL Kimble Screw Thread Conical Glass Centrifuge Tubes	DWK Life Sciences	Cat#73785-5
Kimble, Black Phenolic Caps, 13-415	DWK Life Sciences	Cat#73800-13415
Hypersil GOLD™ C18 Selectivity HPLC Columns, 150 mm × 2.1 mm, 1.9 μm	Thermo Fisher Scientific	Cat#25002-152130
ACQUITY UPLC BEH C18 Column, 130Å, 1.7 μm, 2.1 mm × 50 mm	Waters	Cat#186002350
ACQUITY UPLC BEH C18 VanGuard Pre-column, 130Å, 1.7 μm, 2.1 mm × 5 mm, 3/pk	Waters	Cat#186003975
48 Position Multivap Nitrogen Dryer	Organomation	Cat#11848
Ultrasonic Cleaner	Fisherbrand	Cat#FB11201
Mini Vortex Mixer	Frisherbrand	Cat#14-955-151
Thermo Fisher Scientific Q Exactive Orbitrap Mass Spectrometer	Thermo Fisher Scientific	N/A
Thermo Fisher Scientific Vanquish Liquid Chromatography	Thermo Fisher Scientific	N/A

Materials and equipment

Mass spectrometer

In this protocol, a high-resolution Thermo Fisher Scientific Q Exactive Orbitrap Mass Spectrometer is utilized. An alternative high-resolution mass spectrometer can be used; however, the methods and parameters should be altered for that instrument. All metabolomic analyses are conducted with heated electrospray ionization for both positive and negative modes using a sheath gas flow rate of 55, aux gas flow rate of 10, sweep gas flow rate of 1, spray voltage of 3.5 kV, aux gas temperature at 375 °C, and a capillary temperature of 325 °C. The S-lens RF level is 30 with a full scan m/z range of 70–1000.0 at a resolution of 70,000. For lipidomic analysis, the same source is used with a sheath gas flow rate of 35, aux gas flow rate of 5, sweep gas flow rate of 1, spray voltage of 3.5 kV, aux gas temperature of 300 °C, and a capillary temperature of 300 °C. The S-lens RF level is 35 with a full scan m/z range of 200–2200.0 at a resolution of 70,000. For data-dependent tandem mass spectrometry, a normalized collision energy of 35 is applied for both analyses.

Liquid chromatography

In this protocol, a high-resolution Thermo Fisher Scientific Vanquish liquid chromatography (LC) is utilized. An alternative ultra-high resolution liquid chromatography can be used; however, the methods and parameters should be altered for that instrument. Gradients and mobile phases are described in the protocol.

Lipid Mobile Phase C

Reagent	Final concentration	Amount
Acetonitrile	59.9%	599 mL
Water	40%	400 mL
Formic Acid	0.1%	1 mL
Ammonium Acetate	10 mM	770.8 mg
Total	100%	1000 mL

Store at 25°C for up to a month.

Lipid Mobile Phase D

Reagent	Final concentration	Amount
2-Propanol	89.9%	899 mL
Acetonitrile	8%	80 mL
Water	2%	20 mL
Formic Acid	0.1%	1 mL
Ammonium Acetate	10 mM	770.8 mg
Total	100%	1000 mL

Store at 25°C for up to a month.

CRITICAL: All chemicals and reagents should be LC-MS grade but may be acquired from other vendors. 2-Propanol and acetonitrile are both flammable and must be stored away from ignition sources and used in well-ventilated areas. Acetonitrile and formic acid are the most hazardous of the group, being toxic and corrosive, respectively, and require strict fume hood use at all times. Formic acid and ammonium acetate are both respiratory irritants, particularly when heated or aerosolized. Water is the only non-hazardous chemical in the group. Appropriate PPE, adequate ventilation, and proper waste disposal protocols must be observed when handling all of these chemicals.

Step-by-step method details

Metabolite and lipid extraction from DBS

Timing: 3 h

This section of the protocol covers a modified Folch Split method applied to DBS discs, in which a 2:1 Chloroform (CHCl₃): Methanol (MeOH) ratio in the Folch method is retained.⁹ The metabolites and lipids are extracted at the same time via a biphasic extraction. The Folch is considered the gold standard of lipid extraction.¹⁰ Lipids are contained in the organic bottom layer, and metabolites are analyzed in the aqueous top layer.^1,9 The metabolite and lipid partitions are separated and dried down at the end of this section.

• 1.
  Add a 10 μL sampling disc to a 5 mL glass conical tube.

  • a.
    Use sterilized tweezers to gently remove the 10 μL sample disc from the back of the Capitainer B10 microfluidic device as shown in Figure 1A.
  • b.
    Sterilize tweezers between samples to prevent contamination.
  • c.
    Label tubes with sample ID and metabolite to denote that metabolites are dried down in this tube.
  • d.
    Prepare a solvent-only tube as an extraction blank that will be treated the same as all other samples.
• 2.
  Add ice-cold 2:1 CHCl₃: MeOH (400 μL of CHCl₃ and 200 μL of MeOH) to the conical tubes.

  • a.
    Use glass serological pipettes for measuring and depositing CHCl₃.
  • b.
    Distinct layers should be observed as shown in Figure 1B.

CRITICAL: All tubes and pipettes must be glass when working with CHCl₃ to prevent contamination caused by plastic.

CRITICAL: Keep samples on ice when not working with them.

• 3.Vortex for 10 s.
• 4.Sonicate samples for 10 min in an ice-cold bath (Figure 1C).
• 5.Add 100 μL of water to all samples.
• 6.Vortex for 10 s.
• 7.Rest samples on ice for 10 min and vortex every 5 min (Figure 1D).
• 8.Centrifuge samples at 3260 × g at 4°C for 10 min.
• 9.
  Remove the organic CHCl₃ layer (bottom layer) and add it to a new labeled tube using a glass Pasteur pipette.

  Note:Figure 1E demonstrates the division of layers before removal of the organic layer, and Figure 1F shows the removed organic layer and remaining aqueous layer. Methods video S1 demonstrates the process.

  • a.
    Label the new tube with the sample ID and organic layer.
  • b.
    Leave the aqueous MeOH and water layer (top layer) in the original conical tube.
  • c.
    If layers are cloudy and not separate, refer to troubleshooting problem 5.
• 10.
  Extract the aqueous layer and DBS again: add ice-cold 2:1 CHCl₃: MeOH (200 μL of CHCl₃ and 100 μL of MeOH) to the conical tubes containing the DBS and aqueous layer (Figure 2A).

  • a.
    Use glass serological pipettes for measuring and depositing CHCl₃.

    • i.
      Leave organic layer tubes on ice or at 4°C while performing the second extraction.

      Note: The re-extraction volumes are lower due to the majority of analytes being extracted in the first two extractions, and the volume in the second extraction is greater than the 1:20 sample-to-solvent ratio.⁹

  • b.
    Vortex aqueous samples for 10 s.
  • c.
    Sonicate aqueous samples for 10 min in an ice-cold bath.
  • d.
    Centrifuge aqueous samples at 3260 × g at 4°C for 10 min.
  • e.
    Remove the organic CHCl₃ layer (bottom layer) and add it to the current organic layer sample tube using a glass Pasteur pipette.

    Note:Figure 2B demonstrates the second extraction division of the aqueous and organic layers and the previously removed organic layer.

• 11.Centrifuge all samples at 3260 × g at 4°C for 10 min.
• 12.Dry organic layer samples under nitrogen at 30°C (Figure 2C).
• 13.
  Remove the supernatant from aqueous layer samples after centrifuging and place in a new tube. Leave any pellet that may be present.

  • a.
    Eppendorf tube and plastic pipette tips may be used.
  • b.
    Discard any protein pellet, and DBS left in a conical tube. See troubleshooting problem 3.
• 14.Dry aqueous layer samples under nitrogen at 30°C.
• 15.Store dried samples in a −80°C freezer until reconstitution and analysis. Figure 2D demonstrates a completely dry sample that is ready for storage or reconstitution.

Note: See troubleshooting problem 6 if experiencing low yield of analytes.

CRITICAL: Sample must be completely dry (Figure 2D).

Figure 1.

First extraction and layer partitioning

(A) DBS disc removal from Capitainer B10.

(B) Biphasic layers after CHCl₃:MeOH addition.

(C) Sonication of samples.

(D) Resting samples on ice.

(E) Partitioning of aqueous and organic layers.

(F) Separate organic and aqueous layers.

Figure 2.

Second extraction and nitrogen dry down

(A) Second extraction after the addition of CHCl₃:MeOH with the separated organic layer behind.

(B) Labeled separate layers prior to portioning and combining organic layers. The first extraction organic layer is shown with an arrow.

(C) Drying of samples under nitrogen.

(D) Fully dried samples that are ready for storage or reconstitution.

Reconstitution of metabolite and lipid extracts

Timing: 1 h

This section of the protocol discusses the reconstitution and preparation of the extracted samples for LC-MS/MS. Metabolites and lipids should be reconstituted and analyzed separately, depending on when samples are being analyzed via LC-MS/MS. Samples should be reconstituted on the day of analysis.

• 16.
  Metabolites: Reconstitute dried aqueous layer (Step 14) with 50 μL water (H₂O) with 0.1% formic acid (FA), adding 2 μL of metabolomic amino acid mix as an injection standard, or skip if not using injection standards.

  • a.
    Vortex for 10 s.
  • b.
    Centrifuge samples at 3260 × g at 4°C for 10 min.
  • c.
    Add supernatant to 300 μL glass-fused insert LC vials.

    • i.
      Plastic vials are acceptable.

Note: Injection standards are used to evaluate LC-MS performance to ensure consistent injections via LC.

Note: Prepare metabolomic amino acid mix as dictated by the packaging if reconstitution is needed. Follow expiration and storage as dictated. The stock solution is 500 μM of each compound except glycine, which is 2500 μM. The end concentration of 19.2 μM and 96.2 μM, respectively.

CRITICAL: Reconstitute samples on the day the samples are being analyzed via LC-MS/MS.

• 17.
  Lipids: Reconstitute dried organic layer (Step 12) with 100 μL 2-propanol (IPA).

  • a.
    Spike in 2 μL of stock SPLASH LIPIDOMIX standards as a lipid injection standard, if using. Refer to Table S1 for the SPLASH stock and working concentrations of individual compounds.
  • b.
    Vortex for 10 s.
  • c.
    Centrifuge samples at 3260 × g at 4°C for 10 min.
  • d.
    Add supernatant to 300 μL glass-fused insert LC vials.
• 18.
  Make quality controls for each analyte class if using standards, including extraction blanks, pooled samples, and neat quality controls.

  • a.
    Metabolites: Add 2 μL of metabolomic amino acid mix as an injection standard to 50 μL of H₂O with 0.1% FA.
  • b.
    Lipids: Add 2 μL of SPLASH LIDIOMIX lipid injection standard to 100 μL of IPA.
  • c.
    Vortex well before adding to LC vials.
• 19.
  Make blanks for each analyte class.

  • a.
    Metabolites: Add 300 μL of water with 0.1% FA to an LC vial.
  • b.
    Lipids: Add 300 μL of IPA to an LC vial.

Note: The reconstitution volume can be optimized per sample set, depending on the concentration of analytes of interest in samples. If the sample is too concentrated and saturates the LC column, refer to troubleshooting problem 1. If samples are too lowly abundant, refer to troubleshooting problem 2 or 6 as a possible solution. If a biphasic layer appears after adding reconstitution solution, refer to troubleshooting problem 4.

LC-MS/MS analysis of metabolite and lipid extracts

Timing: 90 min per sample

This section of the protocol discusses the analysis of samples via reverse-phase LC-MS/MS. Metabolites and lipids should be run separately with their corresponding parameters and mobile phases.

• 20.
  Prepare mobile phases.

  • a.
    Metabolites: Mobile phase A: H₂O with 0.1% FA; Mobile phase B: acetonitrile (ACN) with 0.1% FA (all Optima LC/MS grade). Mobile phases can be stored at 25 °C for up to a month.
  • b.
    Lipids: Mobile phase C: 60:40 ACN: H₂O with additives. Mobile phase D: 90:8:2 IPA:ACN:H₂O with additives (all Optima LC/MS grade), refer to Materials and Equipment for recipes. Mobile phases can be stored at 25 °C for up to a month.
• 21.Prepare LC gradient and MS parameters as shown in Figure 3 for metabolites and Figure 4 for lipids.
• 22.
  Calibrate MS and prepare LC column.

  • a.
    Metabolites: use a HyperSil Gold C18 2.1 mm × 150 mm with 1.9 μm particle size.
  • b.
    Lipids: use a Waters BEH C18 with 2.1 mm × 50 mm with 1.7 μm particle size and a matching guard column.
• 23.
  Analyze all samples in positive and negative modes and with MS/MS.

  • a.
    The run time is 20.5 min per sample for metabolites and 23.0 min per sample for lipids, with each analysis performed in each ionization mode.

CRITICAL: To reduce inter-run variability associated with column changes, mobile phase transitions, and ionization source variation, a fixed acquisition sequence is preferred. All samples are analyzed in the following order: (1) metabolites in positive ionization mode, (2) metabolites in negative ionization mode, (3) lipids in positive ionization mode, and (4) lipids in negative ionization mode. Deviation from this sequence, including alternating between analyte classes or ionization modes out of order, introduces additional sources of analytical error.

CRITICAL: Data-dependent MS/MS is needed to annotate lipids via LipidMatch and the spectral libraries available on mzmine.

Figure 3.

Metabolite LC gradient

Figure 4.

Lipid LC gradient

Data processing

Timing: 1-2 h per sample

This section of the protocol discusses the analysis of both lipid and metabolite data produced via reverse-phase LC-MS/MS. Metabolites are processed with mzmine and lipids are processed with LipidMatch.

• 24.Analyze metabolite LC-MS/MS data with mzmine.

Note: The mzmine parameter will change based on the individual LC-MS/MS and laboratory. Parameters should be based on raw chromatograms for more precise feature detection. Parameters listed should be used as a starting point and are utilized with the listed instruments.

• 25.Open mzwizard and add raw files for metabolites in one ionization mode.
• 26.
  Select UHPLC or HPLC, depending on the LC used.

  • a.
    Crop retention time to 0.15–17.50 min to reduce initial noises, disregarding the flush and equilibration time added at the end of the LC method.
  • b.
    Set max peaks in the chromatogram to 15 and the minimum consecutive scans to 6.
  • c.
    Set approximate feature FWHM to 0.08 min and RT tolerance (intra-sample) to 0.08 min and (sample-to-sample) to 0.15 min.
• 27.
  Select MS to Orbitrap, or MS used.

  • a.
    Select the ionization mode of the data files added.
  • b.
    Noise threshold for MS1 of 5.00E3 and MS2 of 2.50E2.
  • c.
    Minimum feature height of 5.0E4.
  • d.
    m/z tolerance (scan-to-scan) of 0.0020 m/z or 10.0 ppm, (intra-sample) 0.0015 m/z or 3.0 ppm, and (sample-to-sample) 0.0015 m/z or 5.0 ppm.
• 28.In the filters tab, keep “remove” for the original feature list, set 1 sample, and set 50.0% for minimum aligned samples.
• 29.
  Within the annotation tab, add local libraries and/or spectral libraries.

  • a.
    Compounds will be annotated via libraries added.

Note: Local libraries are laboratory-, instrument-, and column-dependent. Spectral libraries can be downloaded through mzmine and annotated via MS/MS.

• 30.Select DDA and export the path to the desired location.
• 31.Select “Create batch”, check, and then “OK”. The feature list appears when processing is finished.
• 32.Export the feature list with desired information for statistical analysis.
• 33.
  Analyze lipid data with LipidMatch, starting with preparing input files.

  • a.
    Convert raw files into .mzXML or .mzML and .MS2 files for MS/MS files using MSconvert, which is included with LipidMatch.
  • b.
    Rename files with the renaming tool included in the package.
• 34.
  Open the Flow interface and add files to the MS/MS, negative mode, and positive mode boxes.

  • a.
    Select the directory and name the project.
  • b.
    Select vendor for LC-MS/MS.
  • c.
    Start analysis with “start”.
• 35.Output files PosIDed_FIN and NegIDed_FIN contain annotated features and data to be used for statistical analysis.
• 36.
  For statistical analysis, normalize by sum with log transformation and auto scaling.

  • a.
    Blank feature filtering removes features for which the average intensity in blank injections is more than three times the average intensity in sample injections.
  • b.
    Annotation of features is performed in LipidMatch and mzmine.

Expected outcomes

This protocol was developed for untargeted lipidomic and metabolomic semi-quantitative analysis of DBS; as a result, different sample groups will have different profiles. However, certain chromatographic patterns should be retained when using reverse-phase liquid chromatography. As shown in Figure 5, lipids are grouped by their class, and certain lipids are expected in one polarity over the other. For example, in positive mode (Figure 5A), triglycerides (TG) are present near the end of the run but are not present in negative mode (Figure 5B). In contrast, fatty acids (FA) are present in negative mode (Figure 5B) and not in positive mode (Figure 5A). Several lipids are ionized in both polarities, such as phosphatidylcholines (PC), phosphatidylethanolamines (PE), and phosphatidylserines (PS). Results for the lipid extraction in this protocol should result in a similar lipid profile as shown in Figure 5, whereas certain lipids and intensities will vary; the pattern and certain lipids should be consistent across samples.

Figure 5.

Reverse-phase chromatography lipid patterns

(A) Positive mode. (B) Negative mode.

Similar to lipids, in the metabolomic analyses, some metabolites are likely to be retained across samples due to being readily available in blood, such as some amino acids. Figure 6 demonstrates the reproducibility of the metabolites extracted in three separate DBS extractions. Features were blank-filtered, and statistical analysis was performed on positive and negative modes separately. Kruskal-Wallis test was used to assess overall differences in groups, followed by Dunn’s multiple comparisons test for pairwise comparison. A significance threshold of p < 0.05 showed no significant difference in the total number of metabolite features between each trial. The LipidMatch level 1 IDs were analyzed with the same statistical analysis and demonstrated no significant difference between trials, except in trials 1 and 2 for positive mode and trials 1 and 3 in negative mode, as shown in Figure S1. The analysis was limited to level 1 IDs, which are not a complete lipid profile but contain the most confident identifications.

Figure 6.

Metabolite extraction reproducibility

(A) Positive mode. (B) Negative mode. Files were processed with more stringent mzmine MS parameters to incorporate all three trials in the processing. Data was statistically analyzed on GraphPad Prism 11 and represented as mean ± SD with p < 0.05.

In the research conducted by Li et al., the protocol was utilized for plasma samples in the extraction of metabolites and lipids from clinical samples.¹ The protocol demonstrated adequate extraction of analytes for meaningful insights into the metabolic alterations.

Limitations

This protocol details metabolite and lipid extraction from DBS. Inherently, some analytes will be lost when working with a dried sample compared to a liquid or tissue sample. Moreover, there are some differences between capillary and venous blood that need to be considered in the experiment design.¹¹ Furthermore, punching out spots with varying hematocrit levels can impact the amount of blood on a DBS.¹² A volumetric spot can be used to collect a specific volume of sample independent of hematocrit, which is applied herein. However, varying hematocrit levels and differing spot sizes have not been evaluated yet due to utilizing a standard volumetric disc. Finally, DBS are notable for their inherent stability, but the storage conditions of DBS prior to analysis should be considered. Various studies have examined the stability of different analytes on DBS at varying storage conditions.^13,14

Troubleshooting

Problem 1

Analytes are too concentrated and saturate the column on the LC. Refer to reconstitution steps 16 and 17.

Potential solution

• •Increase the reconstitution volume or dilute the samples. The injection standards should be modified at the same ratio.

Problem 2

Analytes are too weakly concentrated, and/or the injection standards are suppressing ionization. Refer to reconstitution steps 16 and 17.

Potential solution

• •Decrease the reconstitution volume, and the injection standards should be modified at the same ratio. If the injection standards are suppressing ionization, decrease the amount of volume or dilute the standards. If needed, run without injection standards.

Problem 3

Protein in extraction solutions. Refer to extraction step 13.

Potential solution

• •Centrifuge the samples (3260 × g at 4°C for 10 min) to create a protein pellet, then remove the supernatant. The removed supernatant should be dried down. We have noticed protein is more likely to be removed from the disc if the discs sit in solution for an extended period or when the discs are not completely dry to begin with.

Problem 4

Biphasic layers when reconstituting. Refer to reconstitution steps 16 and 17.

Potential solution

• •This will cause errors in the LC, mostly due to exceeding pressure limits or high pressure. The samples must be completely dry before reconstituting. When there are liquids remaining and reconstitution is performed, two layers are present in the LC vials. Dry samples again and reconstitute without injection standards.

Problem 5

Cloudy organic layer and incomplete division of organic and aqueous layers. Refer to extraction step 9.

Potential solution

• •Centrifuging the samples (3260 × g at 4°C) for an additional 10 min can assist in separating the layer, as well as adding an additional 100 μL of H₂O and repeating the steps after step 5.

Problem 6

Low yield of lipids and metabolites. Refer to extraction step 15.

Potential solution

• •Increasing the sonication time, preconditioning the disc, and performing an additional extraction can be used to try to remove more analytes. To precondition the disc, use methanol. To perform an additional extraction, repeat the steps of the second extraction (step 10).

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Timothy J. Garrett (tgarrett@ufl.edu).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Esabella R. Powers (powers.e@ufl.edu).

Materials availability

This study did not generate new unique reagents.

Data and code availability

See Li et al.¹ for data and protocol implementation. Further data with whole blood, including data used for reproducibility in Figure 6, are available upon request, but the data are not public due to an additional manuscript in preparation.

Acknowledgments

We would like to thank Wenqian Li for her previous work on multi-omics extractions on dried blood spots. This work was supported by a research grant from Capitainer to the University of Florida (PI: T.J.G.). The graphical abstract for this publication was created in BioRender. Powers, E. (2026) https://BioRender.com/h9ozvws.

Author contributions

E.R.P. designed/implemented experiments, analyzed the data, and constructed the protocol. T.J.G. served as a scientific advisor, provided resources, and reviewed the protocol.

Declaration of interests

The authors declare no competing interests.