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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Methods Mol Biol. 2021;2306:171–186. doi: 10.1007/978-1-0716-1410-5_12

Quantification of Plasma Oxylipins using Solid Phase Extraction and Reversed-phase Liquid Chromatography-Triple Quadrupole Mass Spectrometry

Guan-yuan Chen 1,2, Qibin Zhang 1,3,*
PMCID: PMC8672328  NIHMSID: NIHMS1762506  PMID: 33954947

Abstract

Oxylipins are an important class of bioactive lipids derived from polyunsaturated fatty acids. They can be both pro- and anti-inflammatory and function as important mediators in various pathological conditions. However, comprehensive analysis of oxylipins still remains a challenge because of their low abundance in plasma and the dominance of structurally similar isomeric species. Herein, we describe a simple and rapid method to comprehensively analyze oxylipins in blood plasma, which utilizes solid-phase extraction in 96-well format for efficient sample clean-up. Separation and detection of more than 130 oxylipins is accomplished by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring in negative ion mode. The absolute concentrations of oxylipins in human plasma are determined using the calibration curves constructed from internal standards. Detailed methods and precautions are presented for a successful adoption of this method in analytical laboratory.

Keywords: Oxylipins, LC-MS/MS, lipid mediators, MRM, human plasma, SPE

1. Introduction

Oxylipins belong to a subclass of lipid mediators [1] and play essential roles in regulation of local inflammation. They are originated from various polyunsaturated fatty acid (PUFA) precursors that are released from the membrane phospholipids by phospholipase A2 [2] and further metabolized by different enzymes including cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP), as well as some non-enzymatic pathways [3]. Various physiological conditions [4] and pathologies including inflammation [3], vascular diseases [5–8], metabolic syndrome [9], neurological diseases [10–12] and cancers [13,14] are reportedly related to the imbalance of oxylipins. To understand roles of oxylipins in enhancing and resolving inflammation and disease progression, it is necessary to provide an accurate quantification of their levels in human plasma and other biofluids.

Herein, we present a workflow for absolute quantification of oxylipins in human plasma. This workflow covers: 1) sample preparation, 2) data acquisition and 3) data analysis. Solid phase extraction (SPE) in 96-well format is employed to perform sample clean-up for high-throughput applications. Individual oxylipin species in the samples are separated and detected using a liquid chromatography-multiple reaction monitoring mass spectrometric (LC-MRM-MS) based method with good sensitivity and selectivity. Absolute concentrations of over 130 oxylipins can be quantified using this method.

2. Materials

2.1. Instruments and consumables

  1. HLB SPE 30 mg 96-well plate (Waters, Milford, MA).

  2. Biotage Pressure+ Manifold (Biotage, Uppsala, Sweden).

  3. TurboVap Evaporator (Biotage, Uppsala, Sweden).

  4. Vanquish UHPLC coupled with a Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific, Haverhill, MA).

  5. HSS T3 column (100 × 2.1 mm, 1.8 μm) with T3 VanGuard pre-column (5 × 2.1 mm, 1.8 μm, Waters, Milford, MA).

2.2. Standard stock solution and mixture

All the oxylipin and deuterated oxylipin standards (Cayman Chemical, Ann Arbor, MI USA) are prepared with MeOH in the concentration of 1 mg/mL as stock solution except polyunsaturated fatty acids (eicosapentaenoic acid, arachidonic acid, docosahexaenoic acid, adrenic acid), which are prepared as 500 μg/mL (see Note 1). All standards and standard mix are stored in −20°C freezer.

  1. MasterMix: Take desired amounts of individual standard into a 20-mL glass tube, gently dry under a steam of nitrogen, and reconstitute in 500 μL of 50% MeOH (see Note 2).

  2. 10 x ISTD: Take ten times desired amounts of ISTD into a 10-mL glass tube. Add MeOH to make 5 mL (see Note 3).

  3. QC standard: Take 6.25 μL of MasterMix and 20 μL of 10 x ISTD into a new tube. Add 173.75 μL of 50% MeOH to the mixture.

  4. Calibrators: Prepare two-fold dilution of the MasterMix using 50% MeOH, repeat the dilution sequentially 10 times. Take 90 μL from the MasterMix and each of the 11 sequential dilutions to 12 individual tubes and add to each tube 10 μL 10 x ISTD. Vortex prior to LC-MRM-MS analysis.

2.3. Solvents for SPE (for 1 plate of 96 samples)

  1. 5% MeOH solution: 1 L.

  2. MeOH: 500 mL.

  3. Deionized (DI) H2O: 500 mL.

  4. Phosphate buffered saline (PBS) solution: 250 mL.

2.4. Solvents for LC-MRM-MS

  1. Solvent A: 0.1% formic acid in H2O.

  2. Solvent B: 0.1% formic acid in ACN.

3. Methods

The workflow for quantitative analysis of oxylipins in human plasma (see Note 4) is illustrated in Figure 1. Same aliquot from each individual plasma samples is pooled to make it as a Pooled QC sample (see Note 5) and process it the same way as the individual plasma samples. Spike Internal standards (10 x ISTD) into each plasma sample prior to SPE cleanup, and the collected sample is subjected to LC-MRM-MS analysis in negative-ion mode (see Note 6).

Figure 1.

Figure 1.

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Workflow for LC-MRM-MS based quantification of oxylipins, covering the three major steps of this protocol.

3.1. SPE cleanup

The SPE cleanup method may vary depending on SPE packing materials and manufacturer’s instructions should be consulted. The following steps are to be applied to Biotage Pressure+ Manifold and 96-well format HLB SPE columns (see Note 7).

  1. Condition 96 well SPE cartridge by adding 1 mL MeOH, followed by 1 mL DI H2O with the Manifold pressure set around 6 psi.

  2. Mix 100 μL plasma (see Note 8) and 5 μL 10 x ISTD (see Note 9).

  3. Load the mixture onto the cartridge at a pressure of 3 psi.

  4. Wash with 1.5 mL of 5%MeOH at a pressure of 6 psi.

  5. Elute with 1.2 mL of MeOH at a pressure of 3 psi and collect the eluents.

  6. Dry the eluents in a 96-well TurboVap evaporator with nitrogen (see Note 10).

  7. Reconstitute in 50 μL of 50% MeOH.

3.2. Setting up MRM transitions

A heatmap-assisted strategy is employed to select the unique fragment ions as specific MRM transitions to monitor the various isomeric oxylipins as previously described (Table 1) [15] (see Note 11).

Table 1.

MRM transitions and retention times for oxylipins

Name Formula Type IS RT Precursor Product1 CE1 Product2 CE2
tetranor-PGDM C16H24O7 Analyte (d4) 6k PGF1α 1.13 327 309 10
20oh PGF 2α C20H34O6 Analyte (d4) 6k PGF1α 1.37 369 325 19 193 26
20oh PGE 2 C20H32O6 Analyte (d4) 6k PGF1α 1.5 367 331 10 349 10
d17 6k PGF 1α C20H32O6 Analyte (d4) 6k PGF1α 2.14 367 163 24 243 22
2,3-dinor 8-iso PGF 2a C18H30O5 Analyte (d4) 6k PGF1α 2.44 325 237 10
2,3-dinor TXB 2 C18H30O6 Analyte (d4) 6k PGF1α 2.49 341 167 10 141 14
6k PGF 1α C20H34O6 Analyte (d4) 6k PGF1α 2.6 369 163 25 245 24
(d4) 6k PGF 1α C20H30D4O6 IS - 2.61 373 167 25 249 24
2,3-dinor 11b PGF 2α C18H30O5 Analyte (d4) 6k PGF1α 2.62 325 145 15 163 10
20cooh LTB 4 C20H30O6 Analyte (d4) 6k PGF1α 2.72 365 347 16 169 20
Resolvin E 1 C20H30O5 Analyte (d4) 6k PGF1α 2.77 349 161 16 195 15
8-iso PGF 3a C20H32O5 Analyte (d4) 8-iso PGF2αVI 2.79 351 307 17 245 18
6k PGE 1 C20H32O6 Analyte (d4) 6k PGF1α 2.8 367 143 18
TXB 3 C20H32O6 Analyte (d4) TXB2 2.81 367 169 14 195 11
20oh LTB 4 C20H32O5 Analyte (d4) 8-iso PGF2αVI 2.84 351 195 16
TXB 1 C20H36O6 Analyte (d4) TXB2 3.12 371 171 17 197 14
PGF 3α C20H32O5 Analyte (d4) 8-iso PGF2αVI 3.14 351 307 16
8-iso PGF 2α III C20H34O5 Analyte (d4) 8-iso PGF2αVI 3.23 353 309 19 291 20
(d4) 8-iso PGF 2α VI C20H30D4O5 IS - 3.23 357 197 24 295 20
TXB 2 C20H34O6 Analyte (d4) TXB2 3.27 369 169 15 195 12
(d4) TXB 2 C20H30D4O6 IS - 3.27 373 173 15 199 12
6,15-dk-,dh-PGF 1α C20H34O6 Analyte (d4) 8-iso PGF2αVI 3.32 369 267 20 223 20
11β PGF 2α C20H34O5 Analyte (d4) 8-iso PGF2αVI 3.38 353 309 18 193 24
2,3-dinor-6k PGF 1a C18H30O6 Analyte (d4) PGF2α 3.39 341 323 14 161 20
PGE 3 C20H30O5 Analyte (d7) 5-oxoETE 3.41 349 313 10
5-iso PGF 2α VI C20H34O5 Analyte (d4) PGF2α 3.53 353 115 18
PGD 3 C20H30O5 Analyte (d7) 5-oxoETE 3.7 349 233 10
dhk PGE 2 C20H32O5 Analyte (d7) 5-oxoETE 3.75 351 333 10
PGF 2α C20H34O5 Analyte (d4) PGF2α 3.75 353 309 17 193 18
(d4) PGF 2α C20H30D4O5 IS - 3.75 357 313 17
PGF 1α C20H36O5 Analyte (d4) PGF2α 3.78 355 311 19 293 22
(d4) 15d PGJ 2 C20H24D4O3 IS - 4.11 319 275 14
(d4) PGE 2 C20H28D4O5 IS - 4.11 355 275 16 319 10
PGE 2 C20H32O5 Analyte (d4) PGE2 4.15 351 271 14 315 10
LXA 5 C20H30O5 Analyte (d7) 5-oxoETE 4.16 349 115 14 233 12
PGK 2 C20H30O5 Analyte (d7) 5-oxoETE 4.19 349 249 14 287 16
14,15-LTC 4 C30H47N3O9S Analyte (d5) LTC4 4.28 624 272 21 254 23
11d-TXB2 C20H32O6 Analyte (d4) TXB2 4.28 367 305 14 161 17
14,15-LTD 4 C25H40N2O6S Analyte (d7) 5-oxoETE 4.31 495 177 18 143 22
11βPGE 2 C20H32O5 Analyte (d4) PGE2 4.32 351 315 10 271 16
PGE 1 C20H34O5 Analyte (d4) PGE2 4.32 353 317 10 273 19
LXB 4 C20H32O5 Analyte (d7) 5-oxoETE 4.39 351 221 14 163 16
PGD 1 C20H34O5 Analyte (d7) 5-oxoETE 4.51 353 235 13
dh PGF 2α C20H36O5 Analyte (d4) dhk PGF2α 4.53 355 311 22 337 20
PGD 2 C20H32O5 Analyte (d7) 5-oxoETE 4.54 351 271 16 315 10
15k PGF 1α C20H34O5 Analyte (d4) dhk PGF2α 4.55 353 193 25
(d4) PGD 2 C20H28D4O5 IS - 4.58 355 319 10 275 16
Adrenic acid C22H36O2 Analyte (d7) 5-oxoETE 4.7 331 287 14 233 15
15k PGE 2 C20H30O5 Analyte (d7) 5-oxoETE 4.7 349 331 10 287 12
dhk PGF 2α C18H30O5 Analyte (d4) dhk PGF2α 4.78 353 195 15 113 22
15R-LXA 4 C20H32O5 Analyte (d7) 5-oxoETE 4.93 351 115 10 217 17
PGFM C20H34O5 Analyte (d4) dhk PGF2α 4.97 353 183 24 223 20
(d4) dhk PGF 2α C20H30D4O5 IS - 4.98 357 187 22 199 22
Resolvin D 1 C22H32O5 Analyte (d7) 5-oxoETE 4.98 375 141 13 215 17
LTD 4 C25H40N2O6S Analyte (d7) 5-oxoETE 5.01 495 177 18 143 23
6S-LXA 4 C20H32O5 Analyte (d7) 5-oxoETE 5.04 351 115 13 217 18
8-iso-15k PGF 2b C20H32O5 Analyte (d4) dhk PGF2α 5.05 351 219 14
dihomo PGF 2α C22H38O5 Analyte (d4) dhk PGF2α 5.05 381 337 20 319 21
PGEM C20H32O5 Analyte (d4) dhk PGF2α 5.06 351 333 10 315 18
LTC 4 C30H47N3O9S Analyte (d5) LTC4 5.06 624 272 21 254 22
(d5) LTC 4 C30H42D5N3O9S IS - 5.06 629 272 21 254 23
15d PGA 2 C20H28O3 Analyte (d7) 5-oxoETE 5.07 315 187 20
11t LTD 4 C25H40N2O6S Analyte (d7) 5-oxoETE 5.15 495 177 18 143 22
LTE 4 C23H37NO5S Analyte (d5) LTE4 5.2 438 333 17 351 14
(d5) LTE 4 C23H32D5NO5S IS - 5.2 443 338 17 356 15
11t LTC 4 C30H47N3O9S Analyte (d5) LTC4 5.2 624 272 21 254 22
dihomo PGE 2 C22H36O5 Analyte (d7) 5-oxoETE 5.28 379 343 12 361 10
11t LTE 4 C23H37NO5S Analyte (d5) LTE4 5.33 438 333 16 351 14
dhk PGD 2 C20H32O5 Analyte (d7) 5-oxoETE 5.37 351 333 10 315 12
(d4) dhk PGD 2 C20H28D4O5 IS - 5.38 355 337 10 319 11
PGA 2 C20H30O4 Analyte (d7) 5-oxoETE 5.5 333 315 10 271 14
PGJ 2 C20H30O4 Analyte (d7) 5-oxoETE 5.53 333 233 10
PGB 2 C20H30O4 Analyte (d7) 5-oxoETE 5.68 333 175 15 235 15
8,15-diHETE C20H32O4 Analyte (d6) 20-HETE 5.81 335 155 15 127 15
bicyclo PGE 2 C20H30O4 Analyte (d7) 5-oxoETE 5.85 333 235 20 204 20
5,15-diHETE C20H32O4 Analyte (d6) 20-HETE 5.9 335 173 14
Protectin D 1 C22H32O4 Analyte (d7) 5-oxoETE 5.92 359 153 15 206 15
7(R) Maresin-1 C22H32O4 Analyte (d7) 5-oxoETE 5.92 359 177 15 341 10
(d4) LTB 4 C20H28D4O4 IS - 5.96 339 321 14 153 16
LTB 4 C20H32O4 Analyte (d7) 5-oxoETE 5.98 335 195 14 317 13
15d PGD 2 C20H30O4 Analyte (d7) 5-oxoETE 6.01 333 271 14 315 10
12,13-EpOME C18H32O3 Analyte (d7) 5-oxoETE 6.15 295 195 15
12,13-diHOME C18H34O4 Analyte (d4) 12,13-diHOME 6.15 313 183 19
(d4) 12,13-diHOME C18H30D4O4 IS - 6.15 317 185 21
(d4) 9,10-diHOME C18H30D4O4 IS - 6.2 317 203 18
9,10-EpOME C18H32O3 Analyte (d7) 5-oxoETE 6.23 295 171 15
9,10-diHOME C18H34O4 Analyte (d4) 12,13-diHOME 6.23 313 171 27
12oxo LTB 4 C20H30O4 Analyte (d7) 5-oxoETE 6.25 333 179 15 153 15
tetranor 12-HETE C16H26O3 Analyte (d6) 20-HETE 6.33 265 109 10 165 12
5,6-diHETE C20H32O4 Analyte (d6) 20-HETE 6.33 335 317 19 317 10
19,20-DiHDPA C22H34O4 Analyte (d7) 5-oxoETE 6.36 361 273 15 229 15
14,15-diHETrE C20H34O4 Analyte (d7) 5-oxoETE 6.38 337 207 15
12-HHTrE C17H28O3 Analyte (d7) 5-oxoETE 6.41 279 179 11 217 10
11,12-diHETrE C20H34O4 Analyte (d7) 5-oxoETE 6.54 337 167 17 169 16
20cooh AA C20H30O4 Analyte (d7) 5-oxoETE 6.56 333 289 16 297 18
9-HOTrE C18H30O3 Analyte (d7) 5-oxoETE 6.68 293 171 14
8,9-diHETrE C20H34O4 Analyte (d7) 5-oxoETE 6.68 337 127 19 185 15
13-HOTrE C18H30O3 Analyte (d7) 5-oxoETE 6.78 293 195 12
18-HEPE C20H30O3 Analyte (d7) 5-oxoETE 6.78 317 215 10 259 13
13-HOTrE(y) C18H30O3 Analyte (d7) 5-oxoETE 6.86 293 113 19
5,6-diHETrE C20H34O4 Analyte (d7) 5-oxoETE 6.87 337 145 16 319 15
19-HETE C20H32O3 Analyte (d6) 20-HETE 6.89 319 231 10 177 15
(d6) 20-HETE C20H26D6O3 IS - 6.91 325 307 15 281 17
5,6-EET C20H32O3 Analyte (d7) 5-oxoETE 6.92 319 191 14
20-HETE C20H32O3 Analyte (d6) 20-HETE 6.92 319 289 15
15d PGJ 2 C20H28O3 Analyte (d7) 5-oxoETE 6.93 315 203 20
11-HEPE C20H30O3 Analyte (d7) 5-oxoETE 6.95 317 167 11 195 15
15-HEPE C20H30O3 Analyte (d7) 5-oxoETE 6.95 317 175 15 247 13
8-HEPE C20H30O3 Analyte (d7) 5-oxoETE 7.03 317 155 11
12-HEPE C20H30O3 Analyte (d7) 5-oxoETE 7.09 317 179 10
18-HETE C20H32O3 Analyte (d8) 15-HETE 7.12 319 261 18
9-HEPE C20H30O3 Analyte (d7) 5-oxoETE 7.13 317 149 12
17-HETE C20H32O3 Analyte (d8) 15-HETE 7.18 319 247 10
16-HETE C20H32O3 Analyte (d8) 15-HETE 7.19 319 233 12 189 11
5-HEPE C20H30O3 Analyte (d7) 5-oxoETE 7.19 317 115 10
(d4) 13-HODE C18H28D4O3 IS - 7.29 299 198 17
(d4) 9-HODE C18H28D4O3 IS - 7.3 299 172 18
13-HODE C18H32O3 Analyte (d7) 5-oxoETE 7.33 295 195 17
9-HODE C18H32O3 Analyte (d7) 5-oxoETE 7.34 295 171 16
20-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.38 343 241 10
(d8) 15-HETE C20H24D8O3 IS - 7.47 327 226 11 182 14
15-HETE C20H32O3 Analyte (d8) 15-HETE 7.56 319 175 13 219 10
16-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.59 343 233 10 189 13
17 HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.59 343 245 10
19(20)-EpDPE C22H32O3 Analyte (d7) 5-oxoETE 7.59 343 241 10
17(18)-EpETE C20H30O3 Analyte (d7) 5-oxoETE 7.68 317 215 10
11-HETE C20H32O3 Analyte (d8) 12-HETE 7.7 319 167 16
13-oxoODE C18H30O3 Analyte (d7) 5-oxoETE 7.73 293 113 22 179 10
10-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.73 343 153 14 181 10
14-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.73 343 205 10 234 10
(d8) 12-HETE C20H24D8O3 IS - 7.82 327 184 14 214 14
8-HETE C20H32O3 Analyte (d8) 12-HETE 7.83 319 155 10
11-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.85 343 149 10 165 11
13-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.87 343 193 10 221 10
12-HETE C20H32O3 Analyte (d8) 12-HETE 7.88 319 135 13
7-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.91 343 141 11 201 15
9-oxoODE C18H30O3 Analyte (d7) 5-oxoETE 7.93 293 185 18 197 21
15-oxoETE C20H30O3 Analyte (d7) 5-oxoETE 7.95 317 113 15 139 18
14(15)-EpETE C20H30O3 Analyte (d7) 5-oxoETE 7.95 317 207 12
8-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 7.99 343 189 10 109 13
9-HETE C20H32O3 Analyte (d8) 5-HETE 8 319 151 12 123 12
(d8) 5-HETE C20H24D8O3 IS - 8.05 327 116 14 210 16
15-HETrE C20H34O3 Analyte (d7) 5-oxoETE 8.06 321 221 16
5-HETE C20H32O3 Analyte (d8) 5-HETE 8.11 319 115 15
8-HETrE C20H34O3 Analyte (d7) 5-oxoETE 8.21 321 157 16 163 18
12-oxoETE C20H30O3 Analyte (d7) 5-oxoETE 8.26 317 153 16
4-HDoHE C22H32O3 Analyte (d7) 5-oxoETE 8.35 343 101 12
17k DPA C22H32O3 Analyte (d7) 5-oxoETE 8.51 343 247 16
(d11) 14,15-EET C20H21D11O3 IS - 8.65 330 219 10 175 13
14,15-EET C20H32O3 Analyte (d7) 5-oxoETE 8.7 319 219 10 175 12
(d7) 5-oxoETE C20H23D7O3 IS - 8.78 323 279 11 130 15
16(17)-EpDPE C22H32O3 Analyte (d7) 5-oxoETE 8.79 343 233 10 201 10
5-oxoETE C20H30O3 Analyte (d7) 5-oxoETE 8.82 317 203 17
(d11) 11,12-EET C20H21D11O3 IS - 8.9 330 179 11
11,12-EET C20H32O3 Analyte (d7) 5-oxoETE 8.94 319 208 10
(d11) 8,9-EET C20H21D11O3 IS - 8.98 330 155 12 190 15
8,9-EET C20H32O3 Analyte (d7) 5-oxoETE 9.03 319 155 12 151 10
5-HETrE C20H34O3 Analyte (d7) 5-oxoETE 9.12 321 115 13
15-oxoEDE C20H34O3 Analyte (d7) 5-oxoETE 9.21 321 223 22 195 20
10-Nitrooleate C18H33NO4 Analyte (d8) Arachidonic acid 9.74 326 181 15 279 14
9-Nitrooleate C18H33NO4 Analyte (d8) Arachidonic acid 9.75 326 195 27
Eicosapentaenoic acid C20H30O2 Analyte (d8) Arachidonic acid 9.76 301 257 10 203 13
Docosahexaenoic acid C22H32O2 Analyte (d8) Arachidonic acid 10.07 327 283 15 229 15
Arachidonic acid C20H32O2 Analyte (d8) Arachidonic acid 10.21 303 259 13 205 15
(d8) Arachidonic acid C20H24D8O2 IS - 10.21 311 267 10
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3.3. LC-MRM-MS analysis

  1. HPLC settings
    1. Thermostat for analytical column: 40 °C
    2. Flow rate: 0.3 mL/min
    3. Gradient: 0–0.5 min, 30% B; 0.5–1.0 min, 40% B; 1.0–2.5 min, 40%B; 2.5–4.5 min, 70% B; 4.5 −6.5 min, 70%B; 6.5–9.0min, 95%B; 9.0–12.0 min, 95%B (see Note 12)
    4. Injection volume: 10 μL/each (see Note 13)
  2. Mass spectrometry settings (unit arbitrary unless specified)
    1. Sheath gas: 45
    2. Aux gas:13
    3. Sweep gas: 1
    4. Ion transfer temperature: 350 °C
    5. Vaporizer temperature: 350 °C
    6. Electrospray voltage: 2,500 V in negative-ion mode
  3. Preparation of HPLC for LC-MS analysis
    1. Purge mobile phases
    2. Install analytical column (see Note 14)
    3. Equilibrate LC-MS/MS system for at least 20 min with initial gradient conditions (see Note 15)

3.4. Quality control and data analysis

  1. Check the overlaid pressure traces for all the runs in the same batch to evaluate the LC performance (see Note 16).

  2. Check the QC data with TraceFinder 4.0 (Thermo) (see Note 17).

  3. Examine the peak shapes and retention times of the targeted analytes in QCSTDs (see Note 18). A typical LC-MS chromatogram is shown in Figure 2, and the retention times of all oxylipin molecular species examined are listed in Table 1.

  4. Analyze the coefficient of variances (CV%) of QCSTD within the same batch and check the accuracy of QCSTDs (see Note 19).

  5. Perform calibration with TraceFinder as suggested by software manual (see Note 20).

  6. Calculate absolute concentrations for each analyte in each sample with embedded calibration curves.

  7. Evaluate performance of Pooled QC using statistic tools such as PCA with R statistical package or online tool such as MetaboAnalyst 4.0 [16] (see Note 21).

  8. Examine pooled QC clusters for reproducibility and export concentration table (see Note 22).

Figure 2.

Figure 2.

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Overlaid total ion chromatograms obtained from scheduled LC-MRM-MS analysis of oxylipin standards.

4. Notes

  1. Make sure standards are within expiration date. Some oxylipins are only good for 6-months. Detailed information can be obtained from vender’s website.

  2. Vortex the MasterMix for 30 sec prior to use, and the solution should be used within a month.

  3. 10 x ISTD is suggested to be stored in 500 μL aliquots and not to freeze and thaw repeatedly.

  4. This workflow also works for serum samples.

  5. Thaw and aliquot plasma samples in ice to avoid thermo degradation.

  6. It is highly desirable that 3–5 QCSTD and one pooled QC runs are added to every batch sample runs in order to monitor the instrument performance and the sample status.

  7. Positive pressure manifold requires pressurized nitrogen to operate. If unavailable, it can be replaced by a regular vacuum manifold but the flow has to be optimized before use.

  8. If the volume of the plasma/serum sample is less than 100 μL, make up the volume by adding PBS solution to prevent protein precipitation prior to add 10 x ISTD.

  9. The volume can be adjusted according to the amount of samples.

  10. Avoid drying sample above 30°C to prevent labile oxylipins, such as leukotrienes from degradation.

  11. The settings are applicable to a TSQ instrument, but may vary dependent on the type of the mass spectrometer used.

  12. A 1.5 min equilibrium time is required before the next injection to maintain repeatability.

  13. The injection volume can be adjusted to meet the quantitation need of the interested analytes.

  14. A guard columns with the same column packing is recommended for protection of the analytical column.

  15. Poor repeatability in the earlier few runs maybe an indication of insufficient equilibration in the LC system.

  16. Overlay pressure traces obtained from each LC-MS run with vendor’s software. If pressure lines are well matched among all the runs, the LC column is normally in good working condition. Otherwise, check column for clogging or leaking. If clogging, replace the guard column first.

  17. It can also be performed by other tools such as Skyline (https://skyline.ms/project/home/software/Skyline/begin.view).

  18. Although software can provide automated integration, it is still necessary to manually check to ensure that the correct peaks are integrated and identified.

  19. If CV% for most analytes in QCSTD is less than 15%, QC is passed and data analysis for samples can proceed, otherwise rerun the samples.

  20. The coefficient of determination (R2) should be ≥ 0.99, and a 1/x weighting factor be used for better fit.

  21. Concentrations can be obtained directly using the software. Apply dilution factor and concentration factor if used.

  22. The concentration table matrix is required for performing PCA analysis. Pooled QC samples clustered in the center of the PCA score plot is considered to be in good reproducibility and the analyses are valid for quantification.

Acknowledgement

This work was partially supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (R01 DK123499).

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