This year at AAPS PharmSci 360, scientists from Medpace’s bioanalytical laboratory will be presenting two scientific posters regarding new methods for biomarker assay validation. You can learn more about the poster presentations below. Stop by Booth #605 at the conference for more information.
Scientific Posters
Poster No. M1330-04-027 on Monday November 5, 1:30 to 2:30 p.m.
Analysis of Arachidonic Acid as a Biomarker in Human Plasma using LC-MS/MS for Clinical Studies
Tian-Sheng Lu, Elise Snider, Nicole Greer, Joshua Froning and Yong-Xi Li
Medpace Bioanalytical Laboratories, 5365 Medpace Way, Cincinnati, OH 45227, USA
Purpose:
Arachidonic acid (AA, 20:4n-6), a polyunsaturated fatty acid, is the keystone fatty acid for the biosynthesis of eicosanoids in human body. The ratio of arachidonic acid (AA) to eicosapentaenoic acid (EPA, 20:5n-3) (omega-6/omega-3) is a measure of the body’s eicosanoid balance. Balancing the eicosanoids in the body is an excellent way managing heart disease, obesity and inflammatory processes. In order to support clinical studies, we have developed and validated LC-MS/MS methods for the assay of AA in plasma and in red blood cells.
Methods:
Due to the presence of endogenous arachidonic acid in human plasma, fatty acid free bovine serum albumin (BSA) was used as a surrogate matrix for preparation of calibration standards. Pooled human plasma was used for the preparation of quality control (QC) samples. For total arachidonic acid analysis in human plasma, the fatty acids were digested from triglyceride backbone using hydrochloride at for 45 min, followed by addition of sodium hydroxide for another 45 min. The digested samples were neutralized and extracted with hexane. The organic phase was transferred and evaporated to dryness. The residue was reconstituted for LC-MS/MS analysis. Separation was performed on a Shimadzu HPLC system with a reverse phase HPLC column. Detection was achieved using an API-4000 LC-MS/MS system with multiple reaction monitoring (MRM) in negative ESI MRM mode.
Results:
This assay was validated in a nominal range from 5 to 500 µg/mL for arachidonic acid, with correlation coefficients (r2) ≥0.9909. The intra-assay precision and accuracy ranged from 2.0% to 4.6% and from -1.0% to 9.3%, respectively. The inter-assay precision and accuracy ranged from 2.1% to 5.4% and 2.9% to 4.8%, respectively. Arachidonic acid was found stable in plasma on bench-top for 23 hours, through 3 cycles of freeze-thaw at -20oC and -70oC, 98 hours in an autosampler at room temperature and 55 hours in a refrigerator for 55 hours for processed samples. The method has been successfully applied to quantitate arachidonic acid in human plasma and for the measurement of AA/EPA ratios to support clinical studies.
Conclusions:
The method provides selective, precise, accurate, and reproducible measurements of arachidonic acid concentrations in human plasma and for the calculation of AA/EPA ratios.
Poster No. M1330-03-180 on Monday November 5, 1:30 to 2:30 p.m
Measurement of Squalene in Human Plasma by UPLC-APCI-MS/MS
Liang Feng, Guangchun Zhou, Morgan Byrd, and Yong-Xi Li
Medpace Bioanalytical Laboratories, 5365 Medpace Way, Cincinnati, OH 45227, USA
Purpose:
Squalene is a natural 30-carbon organic compound. It is the biochemical precursor to the whole family of steroids. Quantitative analysis of squalene has gained much interest recently. Currently available methods for the measurement of squalene in plasma are mainly based on HPLC or GC-MS. The aim of our study is to develop a simple and sensitive LC-MS/MS method for the quantitation of squalene in human K2EDTA plasma.
Methods:
Squalene and the internal standard squalene-d6 were spiked into 200 µL of human plasma. The analyte in human plasma was extracted by liquid-liquid extraction with mixed solvent which consists hexane and isopropanol. Calibration standards were prepared in surrogate matrix (0.1% Tween-20 in water), while QC samples were prepared by spiking appropriate amounts of squalene stock solutions in authentic human plasma. Separation was performed on a Shimazu HPLC system with a C8 HPLC column. Detection was achieved using an AB SCIEX Triple Quad 5500 system with multiple reaction monitoring (MRM) in positive APCI mode.
Results:
This assay was validated in a nominal range from 40 to 2000 ng/mL for squalene, with correlation coefficients (r2) ≥0.9991. The intra- and inter-day precision and accuracy for LLOQ, low, mid, and high QC samples meet the acceptance criteria of US FDA guidance. The method has been applied to quantitate the concentration of endogenous squalene in different lots of human plasma, and the result of concentration range coincide with the reported data. The method is successfully applied in clinical studies for drug development.
Conclusion:
Fast, easy method for a high non-polar compound, run time less than 5 minutes, without derivatization.
