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SOFT 2019

Society of Forensic Toxicologists, Inc. (SOFT 2019)

SOFT
SOFT

RESTEK TECHNICAL POSTERS

WEDNESDAY, OCTOBER 16, BOOTH# 511, 12:00-2:00 p.m.
P18 Quantitative Analysis of 58 Antipsychotics and Antidepressants in Human Urine by LC-MS/MS
Frances Carroll (presenter), Shun-Hsin Liang, Ravali Alagandula, Justin Steimling, Ty Kahler
Restek Corporation
For more information, email Frances Carroll.
Read abstract

Introduction
Human mental health disorders have become modern-day living diseases and contribute significantly to worldwide morbidity and mortality. This is reflected in fast growing numbers of antipsychotics (APs) and antidepressants (ADs) entering the market and the rising rate of prescription. In the forensic setting, due to their abusive potential, the detection of these drugs is critical in determining their involvement in intoxications and suicides. In the clinical setting, analysis of APs and ADs in blood or urine is necessary to ensure suitable therapeutic concentration and to monitor patients’ compliance. It has been reported that the patient’s adherence to prescribed APs is quite low, and thus new clinical recommendations have been released regarding when and how to use urine testing procedures to help monitor adherence. By combining a simple sample preparation procedure and a fast chromatographic elution with the Raptor Biphenyl column, a highly specific and accurate method was established for simultaneous measurement of 58 AP and AD drugs in human urine.

Objectives
Develop a comprehensive method for simultaneous quantification of 58 AP/AD drugs and metabolites using the combination of a simple urine hydrolysis procedure and a fast chromatographic method.

Methods
Drug-free human urine (BioIVT) was fortified with 58 analytes to prepare the calibration standards and QC samples. Bupropion-D9 was used as the internal standard for quantification of all 58 compounds. The urine sample was treated with IMCSzyme® for hydrolysis followed by protein precipitation with acetonitrile. The supernatant was injected onto a Raptor Biphenyl 2.7 µm, 50 mm x 3.0 mm column for analysis using a Shimadzu Nexera X2 LC System coupled with a SCIEX Triple Quad 4500 MS/MS.

Results
In addition, the existence of isobaric compounds (maprotiline vs. amitriptyline; protriptyline vs. nortriptyline) is an added difficulty as chromatographic separation is required for these analytes. By specifically evaluating these issues, a gradient elution was designed to greatly reduce the carryover while maintaining chromatographic separation of the isobaric compounds. Using additives of 0.1% formic acid and 5mM ammonium formate in both aqueous and organic (methanol) mobile phases, 28 APs and 30 ADs could be analyzed with a total run time of 5.5 minutes with no matrix interference.

The linearity test showed that the majority of compounds (43 out of 58) could be quantified in the range of 10-2500 ng/mL with either quadratic or linear regression (1/x weighted). Other compounds could be quantified in the range of 20-2500 ng/mL, 10-500 ng/mL, or 10-1000 ng/mL. Chromatographic carryover was initially problematic for accurate measurement of AP and AD drugs. Due to their relatively high carryover, olanzapine and desmethylolanzapine were quantified in the range of 25-2500 and 35-2500 ng/mL, respectively. All compounds showed good linearity with r values of 0.998 or greater, and the % deviations were <15%. Three QC levels were prepared at suitable concentration per the different quantification ranges of the compounds. The method accuracy was demonstrated with %recovery <15% of the nominal concentration for all QC levels. The %RSD was <10% for acceptable method precision.

Conclusions
It was demonstrated that simultaneous measurement of 58 AP and AD drugs and their metabolites in urine can be achieved with a simple sample preparation procedure and a fast 5.5-minute LC-MS/MS analysis using the Raptor Biphenyl column. The major carryover issue was addressed and resolved in this study with proper injection needle rinsing and LC elution conditions. The established method provides high-throughput and accurate determination of the majority of mental health drugs on the market, and is suitable for both clinical and forensic monitoring of AP and AD drugs in human urine.

P19 Analysis of Phosphatidylethanol in Human Whole Blood by LC-MS/MS
Frances Carroll (presenter), Shun-Hsin Liang, Ravali Alagandula, Frances Carroll, Justin Steimling, Ty Kahler
Restek Corporation
For more information, email Frances Carroll.
Read abstract

Introduction
Phosphatidylethanol (PEth) is a group of phospholipids formed through enzymatic reaction between ethanol and phosphatidylcholine on the cell membrane. Among multiple homologues of PEth, PEth-16:0/18:1 (palmitic acid/oleic acid) is the predominant molecule extracted from human erythrocytes and can be measured in whole blood as specific biomarker of alcohol consumption with a detection window of up to 3-4 weeks. Previously PEth was considered a biomarker for high and sustained alcohol consumption, but with the application of highly sensitive LC-MS/MS techniques, it is now possible to use PEth concentration in blood to differentiate chronic drinking from social drinking or as a marker of absolute abstinence. In this study, a fast chromatographic analysis was developed using a Raptor FluoroPhenyl column. Specific and sensitive measurement of PEth-16:0/18:1 in whole blood was achieved with a combination of simple protein precipitation and fast 3.5-minute LC cycle time.

Objectives
The intent of this study was to develop a fast chromatographic analysis of PEth-16:0/18:1 in whole blood using a Raptor FluoroPhenyl column. Specific and sensitive measurement of PEth-16:0/18:1 in whole blood was achieved with a combination of simple protein precipitation and fast 3.5-minute LC cycle time.

Methods
The PEth-free pooled human whole blood (BioreclamationIVT) was fortified with PEth-16:0/18:1 (RedHot Diagnostics AB) to prepare calibration standards and QC samples. The linearity ranges were from 0.025 - 4µM (18-2810 ng/mL). Three QC levels were prepared at 0.075, 0.75, and 2.5µM. Following the sample preparation procedure described by RedHot Diagnostics, the blood sample (50µL) was mixed with 50µL of internal standard (0.4µM PEth-d5 in 2-propanol) and 150 µL of 4:1 2-propanol: tetrahydrofuran. The mixture was vortexed for 20 seconds at 3000rpm and centrifuged for 10 minutes at 4300rpm. The supernatant (2µL) was injected onto a Raptor FluoroPhenyl 2.7µm, 50x2.1mm column and analyzed using a Waters ACQUITY UPLC coupled to a Xevo TQ-S MS/MS system. The separation was performed using 5mM ammonium acetate in water and 9:1 methanol: 2-propanol as mobile phases with a 3.5 minute cycle time.

Results
Detection range was established from 0.025 to 4µM with standard curves showing r2 values of 0.999 or greater and the %deviations (from nominal concentration) were <10% (<20% for 0.025 µM standard). The method accuracy was demonstrated from the %recovery of within 5% of the nominal concentration for all QC levels. The %RSD was from 0.117-1.33% and 2.30-5.08% for intra-day and inter-day, respectively, indicating acceptable method precision. Consistent chromatographic performance (retention, peak shape, and sensitivity) was observed upon continuous 500 injections demonstrating good method robustness.

Conclusions
The analytical conditions outlined in our study indicate that the method was specific and sensitive for PEth analysis in human whole blood. The accurate and reproducible analysis can be achieved with a simple protein precipitation procedure and a fast 3.5 minutes of chromatographic run time. This method is thus applicable for low-cost and high throughput analysis to monitor alcohol consumption.

P20 Analysis of d and l-Amphetamines Using Reversed-Phase LC-MS/MS
Ravali Alagandula (presenter), Shun-Hsin Liang, Frances Carroll, Justin Steimling, Ty Kahler
Restek Corporation
For more information, email Ravali Alagandula.
Read abstract

Introduction
Amphetamine (AMP) and methamphetamine (MAMP) are psychostimulant drugs and occur as two enantiomers, dextrorotary and levorotary due to their chiral center. The dextro-methamphetamine (d-isomer) form is highly abused and typically found in illicit preparations. However, detection of abuse is complicated because consumption of some over-the-counter and prescription medications may yield positive results if the analytical method cannot distinguish between the enantiomers. Chiral separation of d- and l-methamphetamine and their metabolites d- and l-amphetamine can help determine whether the source was licit or illicit, but chiral columns can be expensive, may necessitate a dedicated instrument, and are not as broadly useful as ubiquitous C18 columns.

Objectives
The intent of this study was to develop and validate a high throughput LC-MS/MS method for the separation and quantitation of l- and d- methamphetamine and amphetamine enantiomers in urine, using a reversed-phase column with pre-column derivatization followed by dilution in urine, with a total run time of 7 minutes.

Methods
In order to provide labs with a high-throughput assay that effectively separates d- and l- amphetamine and methamphetamine enantiomers in urine without the use of a costly and specialized chiral column, a LC-MS/MS method was developed using a standard reversed-phase Raptor C18 column. The method employs a simple pre-column derivatization followed by dilution and results in a selective, specific analysis of d- and l-amphetamine and methamphetamine enantiomers that is free from sample matrix interferences. The LC-MS/MS method was developed using a Raptor C18 2.7 μm, 100 mm × 2.1 mm column with water and methanol mobile phases modified with 0.1% formic acid. The LC gradient resulted in a total analysis time of 7.0 minutes, including column equilibration time with good baseline resolution of the target compounds, allowing easy peak identification and quantitation. Carryover was not observed.

Results
Separation was achieved within a total run time of 7 minutes and quantitation in urine was performed across a linear range of 50-5000 ng/mL. Validation across this range demonstrated good linearity with r2 values of 0.998 or greater and acceptable method accuracy and precision with %RSD <10% and %recovery within 10% of the nominal concentrations for low, mid, and high QC levels and within 15% for the LLOQ showing acceptable method accuracy and precision. This method provides reliable analysis of d- and l-amphetamine and methamphetamine enantiomers in a workflow and time frame suitable for high-throughput clinical and forensic toxicology labs.

Conclusions
An enantioselective method for d- and &l- methamphetamine and amphetamine determination in urine was successfully developed. To accomplish AMPs chiral resolution, pre-column derivatization with CDR (Marfey's reagent) was performed with minimal (dilute and shoot) sample preparation. Although CDR utilization in the present method increased the sample preparation time by approximately 2 h, conventional reversed-phase conditions on a C18 column were utilized and the chromatographic separation time was limited to 7 min. Additionally, the use of deuterated internal standards for each enantiomer allowed the method to maintain accurate and reliable quantitative results and able to accurately identify/ quantify licit vs. illicit methamphetamine.

A Novel Solution for EtG/EtS Analysis in Human Urine by LC-MS/MS
Ravali Alagandula (presenter), Justin Steimling, Frances Carroll
Restek Corporation
For more information, email Ravali Alagandula.
Read abstract

Introduction
Ethyl Glucuronide (EtG) and Ethyl Sulfate (EtS) are stable phase II metabolites of ethanol and unique biomarkers for alcohol intake and abuse. Analysis of these markers offers many advantages for abstinence monitoring including a longer detection window, stability in stored specimens, and specificity. EtG and EtS are polar analytes, making 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. In some troublesome urine samples, isobaric interferences elute close to EtG and EtS, resulting in poor peak shapes and ultimately leading to potentially biased results. In this study, a simple solvent precipitation method was developed for the analysis of EtG and EtS in human urine by LC-MS/MS.

Objective
The intent of this study was to develop and validate a method that provides a simple, fast, and sensitive measurement of EtG and EtS in human urine.

Methods
MS gold human urine (pre-screened to confirm absence of EtG/EtS) was fortified with EtG and EtS ranging from 50-5,000 ng/mL for both analytes. MS gold human urine calibrators and QC samples, blank pooled human urine spiked at QC concentrations and UTAK level 1 and 2 QC samples were prepared using a simple solvent precipitation method, where a 50 μL aliquot is taken from the spiked samples, mixed with internal standard and 150 µL of acetonitrile, vortexed at 3000 rpm for 10 seconds and centrifuged at 4300 rpm for 10 minutes at 10 °C. After centrifugation, 100 µL of the supernatant was diluted with 900 µL (40-fold dilution) of 0.01% formic acid in water and injected on a Raptor EtG/EtS column (100x2.1mm, 2.7 μm). The mobile phases used were 0.01% 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 3 minutes with a total analysis time of 4.5 minutes, including column re-equilibration time. Experiments were performed on both a Shimadzu Prominence HPLC coupled with a SCIEX API 4000™ and a Shimadzu Nexera X2 UHPLC coupled with a Shimadzu LCMS-8045 using electrospray ionization in negative ion mode.

Results
EtG and EtS were successfully resolved from matrix interferences in troublesome urine samples. The method sensitivity was significantly improved with sample cleanup, decreased formic acid concentrations in the aqueous mobile phase, MS source parameters optimization and source cleaning, thereby making the method accessible for older instruments with lower sensitivity. Excellent calibration linearity was achieved for both analytes with R2 values ≥ 0.998 and % deviation < 12.0%. Four levels of QC samples were analyzed for accuracy and precision across multiple days, and column lots. Mean accuracy values ranged from 93%-105% of the nominal concentration, for the QC LLOQ, low, mid, and high samples and 98-133% for both UTAK level 1 and 2 QC samples for both analytes. The %RSD did not exceed 10% for any set of QC samples throughout the study.

Conclusions
A simple protein precipitation method was developed for the quantitative measurement of EtG and EtS in human urine. The analytical method was demonstrated to be fast, reproducible, and rugged.

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