4th Annual European Congress and Exhibition
RESTEK TECHNICAL POSTERS
B28, Tuesday, 10 a.m.
A Novel Solution for EtG/EtS Analysis in Human Urine by LC-MS/MS
Justin Steimling, Shun-Hsin Liang, Landon Wiest, Dan Li, Sharon Lupo, Frances Carroll (presenter), Ty Kahler, Sue Steinike, Paul Connolly
For more information, e-mail Frances Carroll.
Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are unique biomarkers of alcohol use. The analysis of EtG and EtS offers many advantages for abstinence monitoring including the detection window (~3 days), stability in stored specimens (nonvolatile), and specificity. EtG and EtS are both polar, which makes them difficult to retain via reversed-phase chromatography. Both compounds are also very sensitive to matrix interferences, which can result in being unable to achieve low limits of detection. Isobaric interferences can also make quantitation impossible. In this study, a simple dilute-and-shoot method was developed and validated for the analysis of EtG and EtS in human urine by LC-MS/MS.
Pooled human urine (prescreened to confirm absence of EtG/EtS) was fortified with EtG and EtS from 50-5,000 ng/mL for both analytes. Urine calibration samples, QC samples, and twenty selectivity lots were diluted 20-fold in the working internal standard (25 ng/mL for EtS-d5, and 100 ng/mL for EtG-d5 in 0.1% formic acid in water). The samples were vortexed and centrifuged prior to injection on a Raptor EtG/EtS column (100 x 2.1 mm, 2.7 μm). The mobile phases used were 0.1% formic acid in water (aqueous phase) and 0.1% formic acid in acetonitrile (organic phase) and the chromatographic separation was achieved with a gradient elution of 5-35% organic phase in two minutes. Primary method validation was performed on a Shimadzu XR coupled with a SCIEX API 4000 mass spectrometer using electrospray ionization in negative ion mode. In order to evaluate method ruggedness, precision and accuracy sets were also performed on a Shimadzu Nexera UHPLC coupled with a SCIEX Triple Quad 4500 mass spectrometer and a Waters ACQUITY I-Class UPLC system coupled with a Xevo TQ-S mass spectrometer using multiple column lots.
EtG and EtS were successfully resolved from matrix interference. The selectivity lots did not show additional interferences that would impact quantitation. The calibration linearity was acceptable for both analytes with r2 values ≥ 0.999 and % deviation of less than 10.0%. Three levels of QC samples were analyzed for accuracy and precision across multiple days, instrument platforms, and column lots. Mean accuracy values ranged from 90%-101% of the nominal concentration for QC low, mid, and high samples, and 89-105% for the QC LLOQ for both analytes. The %RSD did not exceed 10% for any set of QC samples throughout the study.
An easy dilute-and-shoot method was developed and validated for the quantitative measurement of EtG and EtS in human urine. The analytical method was demonstrated to be fast, reproducible, and rugged.
E10, Wednesday, 9:45 a.m.
Analysis of Free Metanephrine, Normetanephrine, and 3-Methoxytyramine in Plasma by Hydrophilic Interaction Liquid Chromatography
Shun-Hsin Liang, Frances Carroll, Ute Beyer (presenter)
For more information, e-mail Frances Carroll.
Metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (3-MT) are methylated metabolites of epinephrine, norepinephrine, and dopamine, respectively. These catecholamine metabolites are released from the adrenal medulla and sympathetic nervous cells and are maintained at very low concentrations in the bloodstream. Measurement of these metabolites in plasma is highly sensitive for the diagnosis of pheochromocytoma and paraganglioma as observed by elevated levels in circulation. Analysis of these polar compounds using typical reversed-phase LC is difficult due to limited chromatographic retention and lack of sensitivity. As a solution, an LC-MS/MS method was developed based on hydrophilic interaction liquid chromatography using a Raptor HILIC-Si column. Combined with a simple and fast solid-phase extraction procedure, an accurate and precise analysis of free metanephrine, normetanephrine, and 3-methoxytyramine in plasma can be achieved and applied to high-throughput clinical assays.
Charcoal-stripped human plasma (BioreclamationIVT) was fortified with three analytes to prepare calibration standards and QC samples. The linearity ranges were 0.051-20.28 nmol/L (10-4,000 pg/mL) for MN; 0.14-21.83 nmol/L (24-4,000 pg/mL) for NMN and 0.060-23.92 nmol/L (10-4,000 pg/mL) for 3-MT. Three QC levels were prepared at 40, 400, and 2,500 pg/mL for all three analytes. The fortified standards and QC samples were subjected to a solid phase extraction procedure. An internal standard (IS) solution was prepared at 4 ng/mL for metanephrine-d3; 8 ng/mL for normetanephrine-d3; and 3-methoxytyramine-d4 in methanol. A plasma sample aliquot (200 μL) was mixed with 10 μL of IS solution and 600 μL of 50 mM ammonium acetate. The mixture was loaded onto an EVOLUTE EXPRESS WCX 96-well plate (30 mg) and washed with 1 mL water and 1 mL methanol:acetonitrile (50:50). The elution was performed twice with 0.9 mL of 5% formic acid in methanol:acetonitrile (50:50) and evaporated to dryness at 55 °C under a gentle stream of nitrogen. Dried extracts were reconstituted with 100 μL of 10:90 100 mM ammonium formate in water (pH 3.0):acetonitrile and injected (10 μL) for analysis using a Raptor HILIC-Si 2.7 µm, 50 mm x 2.1 mm column.
A consistent and robust chromatographic performance was obtained using a 5-minute isocratic elution with 10:90 100 mM ammonium formate in water (pH 3.0):acetonitrile. No chromatographic interferences were observed from the analysis of blank plasma samples. Using 1/x weighted linear regression, all three analytes showed acceptable linearity with r2 values of 0.999 or greater and deviations <10%. The established limits of quantitation (LOQ) were 0.051 nmol/L (10 pg/mL), 0.14 nmol/L (24 pg/mL), and 0.060 nmol/L (10 pg/mL) for MN, NMN, and 3-MT, respectively. Precision and accuracy analyses were performed on three different days. The method accuracy was demonstrated with recovery within 5% of the nominal concentration for all QC levels. The %RSD was 0.2-4.5% and 1.1-4.2% for intraday and interday, respectively, indicating a good method precision.
It was demonstrated that the Raptor HILIC-Si column is excellent for sensitive and accurate analysis of MN, NMN, and 3-MT in human plasma, which is generally difficult to achieve using reversed-phase chromatography. With a fast and simple sample preparation procedure and 5 minutes of chromatographic analysis time, the established method provides a high-throughput assay for the clinical diagnosis of pheochromocytoma and paraganglioma.