(T1330-06-38) Rapid Quantitation of Docosahexaenoic Acid and Its Hydroxy Metabolites in Liver Cells Using Differential Mobility Separation (SelexION) Coupled with Liquid Chromatography Tandem Mass Spectrometry

Purpose: Docosahexaenoic acid (DHA) is a crucial lipid for brain function. DHA is obtained from diet or synthesized from the essential fatty acid named alpha-linolenic acid within the body. DHA can undergo enzymatic metabolism to produce mono-, di- and tri-hydroxy docosahexaenoic acid (HDHA) derivatives, epoxy- and oxo-DHA metabolites, or modified by non-enzymatic mechanism to neuroprostanes. DHA metabolites have a range of biological functions like cytoprotection, anti-hypertension, anti-hyperalgesia, anti-arrhythmia, anti-tumorigenesis etc. Alteration in the levels of DHA and/or its metabolites may be an early indication of pathogenesis which makes understanding the concentration of these metabolites critical. Additionally, DHA-based therapies are under development as anti-cancer agents. In the current study, we focused on developing a sensitive and selective bioanalytical method using differential mobility separation (DMS) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitate seven mono-hydroxy docosahexaenoic acids (i.e. 4-HDHA, 7-HDHA, 10-HDHA, 13-HDHA, 14-HDHA, 17-HDHA, 20-HDHA), two di-hydroxy docosahexaenoic acids (i.e. Protectin D1 or PD1, and Maresin 1 or Mars1) and one tri-hydroxy docosahexaenoic acid (i.e. Resolvin D1 or RVD1).

Methods: As the HDHAs exist as positional isomers, it is challenging to resolve them by standard LC; therefore, an additional orthogonal separation tool employing DMS (Sciex’s SelexION⁺ instrument) was coupled with standard LC-MS/MS system to achieve enhanced separation of the HDHAs. Additionally, the DMS allows for rapid analysis. The LC-MS/MS system comprised Shimadzu Nexera X2 series, SCIEX SelexION⁺ device coupled to SCIEX QTRAP® 5500 mass spectrometer equipped with a Turboionspray™ source. The LC column used was Luna 5 mm C18 column (150 × 2 mm) from Phenomenex with mobile phase A consisting of 70:30 methanol: water with 0.01% acetic acid, and mobile phase B of methanol with 0.01% acetic acid using a gradient separation. Mass spectrometric conditions involved negative ion electrospray ionization with MS/MS detection in MRM mode. Calibration curve standards were prepared from 0.50 to 500 ng/mL in ethanol (apparent pH 3) with quality control (QC) standards at 0.5, 1.5, 50, and 250 ng/mL for LLOQ, LQC, MQC, and HQC standards respectively. Internal standard used was 13-hydroxyoctadecadienoic acid (13-HODE) at 275 ng/mL. The developed method was validated as per US FDA guidance, “Bioanalytical Method Validation Guidance for Industry.” Validation studies involved testing for selectivity, sensitivity, intra-run and inter-run accuracy and precision, extraction recovery, matrix effect, and stability (autosampler, benchtop, freeze/thaw, and long-term). The developed and validated bioanalytical method was used for quantitation of DHA and its hydroxy metabolites in malignant murine liver cell line TIB-75. Untreated cells and cells treated with low density lipoprotein (LDL) - DHA (20 µM) nanoparticles for 24 hr were examined in this study. Cell lysates were prepared by taking 5 million cell pellet and homogenizing in 1 mL of acidified water (pH 3), adding additional 1 mL of acidified water, adjusting the resulting solution to 15% methanol (v/v) and adding 13-HODE at 10ng to each sample. Cell lysate samples were processed by solid phase extraction (SPE) procedure using Waters C18 SPE cartridges.

Results: Using the SelexION+device the analytes resolved efficiently (Fig.1) with the exception of few overlapping peaks, and these overlapping pairs were differentiated by MS/MS with distinct precursor to product ion transitions. The method run time was 12 min and all peaks eluted between 3.52 and 9.84 min. The accuracy of the method ranged from 84.0% to 113% while the precision values were between 2.70% and 15.3%. The recovery values ranged from 15.0 ± 4.0% in case of RVD1 to 72.4 ± 21% in case of 10-HDHA. The matrix factor (MF) values were between 98.4 ± 1.8 and 115 ± 8.2 for most of the analytes except RVD1 which had a MF value of 43.0 ± 2.2. Although recovery and matrix effect values were low for RVD1, it was sufficient to achieve adequate analyte peak intensity for quantitation of the LLOQ standard. The method was stable across all tested stability studies with relative error percentage from 81.1% to 121% and relative standard deviation percentage from 0.90% to 19.0%. With respect to concentrations of analytes in cell lysate samples, all mono-hydroxy docosahexaenoic acids were present in concentrations ranging from 0.01 to 2.07 ng/mL, the di-hydroxy and tri-hydroxy docosahexaenoic acids (i.e. PD1, Mars 1, and RVD1) were below detection level (therefore data not presented), and the DHA levels ranged from 4.07 to 104 ng/mL (Fig.2).

Conclusion: A sensitive, selective, accurate and reproducible bioanalytical method was developed and validated for analysis of DHA and its hydroxy metabolites using DMS SelexION+device coupled to QTRAP 5500 LC-MS/MS system. The method was validated using FDA methodology and was used to quantitate concentrations of DHA and its hydroxy metabolites in cell lysates prepared from untreated and LDL-DHA (20 µM) treated TIB-75 cells.

Fig.1. Representative chromatogram of DHA, and the HDHAs in a 100 ng/mL solution prepared in ethanol and spiked with 5 ng/ml of internal standard (13-HODE).

Fig.2. Bar graphs indicating concentrations of DHA and mono-hydroxy docosahexaenoic acids in control and LDL-DHA treated cells from malignant murine liver cell line TIB-75. Data was statistically analyzed using Unpaired t-test with Welch’s correction, where ** indicates P value < 0.01, and * indicates P value < 0.05.