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A sensitive and improved throughput UPLC-MS/MS quantitation method of total cytochrome P450 mediated arachidonic acid metabolites that can separate regio-isomers and cis/trans-EETs from human plasma.

ABSTRACT: A method for the detection and quantification of hydroxyl and epoxy arachidonic acid (AA) metabolites in human plasma was developed using liquid-liquid extraction, phospholipid saponification followed by derivatization of the acid moiety and liquid chromatographic tandem mass spectrometric detection. Derivatization with a pyridinium analog allowed for detection in the positive ion mode, greatly improving sensitivity and the stability of the more labile AA metabolites. The entire method utilizes a 96-well plate format, increasing sample throughput, and was optimized to measure 5-, 8-, 9-, 11-, 12-, 15-, 19-, and 20- hydroxyeicosatetraenoic acid (HETE), 5,6-, 8,9-, 11,12-, and 14,15- dihydroxyeicosatrienoic acid (DHET), and the regio- and cis-/ trans- isomers of 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). The method was validated for its applicability over the FA concentration range found in human plasma. Using 100??L aliquots of pooled human plasma, EET levels, particularly 5,6-EET, were observed to be higher than previously reported, with measured concentrations of 23.6?ng/ml for 5,6-EET, 5.6?ng/mL for 5,6-trans-EET, 8.0?ng/mL for 8,9-EET, 1.9?ng/mL for 8,9-trans-EET, 8.8?ng/mL for 11,12-EET, 3.4?ng/mL for 11,12-trans-EET, 10.7?ng/mL for 14,15-EET, and 1.7?ng/mL 14,15-trans- EET. This method is suitable for large population studies to elucidate the complex interactions between the eicosanoids and various disease states and may be used for quantitation of a wide variety of fattyacids beyond eicosanoids from small volumes of human plasma.

SUBMITTER: Zeigler M

PROVIDER: S-EPMC6269592 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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A sensitive and improved throughput UPLC-MS/MS quantitation method of total cytochrome P450 mediated arachidonic acid metabolites that can separate regio-isomers and cis/trans-EETs from human plasma.

Zeigler Maxwell M Whittington Dale D Sotoodehnia Nona N Lemaitre Rozenn N RN Totah Rheem A RA

Chemistry and physics of lipids 20180907

A method for the detection and quantification of hydroxyl and epoxy arachidonic acid (AA) metabolites in human plasma was developed using liquid-liquid extraction, phospholipid saponification followed by derivatization of the acid moiety and liquid chromatographic tandem mass spectrometric detection. Derivatization with a pyridinium analog allowed for detection in the positive ion mode, greatly improving sensitivity and the stability of the more labile AA metabolites. The entire method utilizes ...[more]

PMID: 30201384

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Project description:OBJECTIVE:With the widespread use of sex-steroid hormones in contraceptives and hormone replacement therapy, there is an increasing need for reliable analytical methods. We report the development of a sensitive and robust UPLC-MS/MS method for quantitation of both endogenous and synthetic sex-steroid hormones in human serum. STUDY DESIGN:We developed and validated a UPLC-MS/MS method to quantify progestogens (etonogestrel, levonorgestrel, medroxyprogesterone acetate, norethindrone, progesterone) and estrogens (estradiol and ethinyl estradiol) with good accuracy, high sensitivity, and excellent robustness. We then applied the method to the analysis of sex-steroid hormones in serum from 451 clinical research participants. RESULTS:Each UPLC-MS/MS analysis was 6.5 min. The lower limits of quantitation (LLOQs) were 25 pg/ml for the progestogens, and 2.5 and 5.0 pg/ml for estradiol and ethinyl estradiol, respectively. When estradiol was analyzed without assessment of progestogens, the LLOQ was reduced to 1 pg/ml. The calibration curves were linear from 25-50,000, 2.5-2000 (1-2000 for estrogens-only analysis) and 5-2000 pg/ml, respectively. Both the accuracy and precision were below±15% not only for routine validation (intraday and interday), but for long-term (>2 years) assay robustness with external controls, thereby, demonstrating the utility of this method for multi-year clinical trial assessments of progestogens and estrogens. We applied the method to quantify sex-steroid levels in 1804 clinical samples. CONCLUSIONS:We successfully developed a UPLC-MS/MS method, and overcame the matrix suppression to allow sensitive quantitation of both synthetic and endogenous sex-steroid hormones in human serum. IMPLICATIONS:We developed a sensitive and robust UPLC-MS/MS method to accurately measure the levels of sex-steroid hormones in serum. The method overcame matrix interference barriers and achieved excellent long-term stability and reproducibility (≥96.9% accuracy; ≤13.0% relative variability measured with external controls over 2 years), demonstrating its utility in clinical sample analysis.

Dataset Information

A sensitive and improved throughput UPLC-MS/MS quantitation method of total cytochrome P450 mediated arachidonic acid metabolites that can separate regio-isomers and cis/trans-EETs from human plasma.

Publications

A sensitive and improved throughput UPLC-MS/MS quantitation method of total cytochrome P450 mediated arachidonic acid metabolites that can separate regio-isomers and cis/trans-EETs from human plasma.

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