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Society of Forensic Toxicologists—Annual Meeting

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SOFT

RESTEK TECHNICAL POSTERS


Presented Wednesday, January 10, 12:00-1:30 p.m.

P112 The Analysis of Common Antiepileptic Drugs in Human Urine by LC-MS/MS
Landon Wiest (presenter), Frances Carroll, Sharon Lupo, Shun-Hsin Liang, Paul Connolly, Carrie Sprout, Rick Lake, Rob Freeman, Ty Kahler
Restek Corporation
For more information, email Landon Wiest.
Download a PDF of the full presentation.
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Introduction
The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) in therapeutic drug monitoring and toxicology labs has increased significantly over the years. LC-MS provides sensitivity, speed, and the ability to simplify sample preparation. The Raptor Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles (SPP) with the resolution of Ultra Selective Liquid Chromatography (USLC) technology. In this example, a simple dilute-and-shoot method was developed for 14 common antiepileptic drugs in urine using a Raptor Biphenyl column.

Methods
Human urine samples were diluted in 0.1% formic acid in water and injected into a Shimadzu Nexera UHPLC equipped with an AB SCIEX API 4500 MS/MS. Detection was performed using electrospray ionization in positive ion mode with scheduled multiple reaction monitoring (MRM). The separation was performed using water and methanol mobile phases modified with 0.1% formic acid under gradient conditions on a Restek Raptor Biphenyl 2.7 µm, 100 x 2.1 mm column.

Results
Linearity, precision, and accuracy experiments were performed during method development. Purchased human urine was fortified with 14 drug analytes and their deuterated internal standards. The calibration range for most analytes was from 10 to 1,000 ng/mL; R values were all greater than 0.990. Accuracy and precision were determined by fortifying human urine at a concentration of 800 ng/mL prior to dilution. Mean values at this level ranged from 88% to 110% of nominal concentrations for all analytes. Coefficient of variation (CV) was calculated for the determination of precision and ranged from 6.2% to 10.5%.

Conclusions
The Raptor Biphenyl column was excellent for the simultaneous analysis of 14 antiepileptic drugs in human urine. Accurate and reproducible results were achieved in less than 5.5 minutes of chromatographic analysis time, making this column and method applicable for low-cost and high-throughput analysis in therapeutic drug monitoring and toxicology labs.

P113 The Analysis of Common Drugs of Abuse in Human Urine by LC-MS/MS
Susan Steinike (presenter), Sharon Lupo, Shun-Hsin Liang, Frances Carroll, Ty Kahler, Paul Connolly
Restek Corporation
For more information, email Susan Steinike.
Download a PDF of the full presentation.
Read abstract

Introduction
The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) in forensic toxicology labs has increased significantly over the years. LC-MS/MS provides sensitivity, speed, and the ability to simplify sample preparation. The Raptor Biphenyl column was developed to complement high-throughput LC-MS/MS analyses by combining the increased efficiency of superficially porous particles with the resolution of Ultra Selective Liquid Chromatography technology. In this example, a simple dilute-and-shoot method was developed for 10 common drugs of abuse and their metabolites in urine using a Raptor Biphenyl 5 µm column. The low backpressure of the 5 µm particle column allows even conventional 400 bar LC systems to take advantage of this high-speed separation with a total analysis time of 5 minutes.

Methods
Human urine samples were diluted 5x in mobile phase A and injected into a Shimadzu Prominence UFLCXR HPLC equipped with an AB SCIEX API 4000 MS/MS. Detection was performed using electrospray ionization in positive ion mode using scheduled multiple reaction monitoring (MRM). The separation was performed using water and methanol mobile phases modified with 0.1% formic acid under gradient conditions on a Restek Raptor Biphenyl 5 µm, 50 x 2.1 mm column.

Results
Linearity, precision and accuracy, and matrix effects experiments were performed during method development. Purchased human urine was fortified with 10 drug analytes and their deuterated internal standards. The calibration range for codeine and 6-MAM was from 5.00 to 750 ng/mL. The calibration range for benzoylecgonine was from 1.00 to 500 ng/mL. The calibration range for the remaining seven analytes was from 1.00 to 750 ng/mL. Accuracy and precision were determined by fortifying human urine at a concentration of 50.0 ng/mL prior to dilution. Mean values at this level ranged from 91.9% to 103% of nominal concentrations for all analytes. Coefficient of variation (CV) was calculated for the determination of precision and ranged from 2.90% to 6.54%. Matrix effects were assessed by infusing a solvent standard (post column) prepared at a concentration of 50 ng/mL while injecting a blank matrix sample diluted 5x in mobile phase A. Areas of ion suppression would result in dips in the response of the solvent standard. There was no evidence of ion suppression during the time period of analyte elution.

Conclusions
Innovations in SPP column technology allow for faster and more sensitive LC-MS/MS assays. The Raptor Biphenyl 5 µm column provides highly retentive, selective, and rugged reversed-phase separations with ultra-low backpressure. Now, even conventional LC systems can benefit from the speed and efficiency of a Raptor Biphenyl column.

P114 The Analysis of Emerging Drugs of Abuse: Updating an Existing Method with New Compounds
Sharon Lupo, Frances Carroll (presenter), Shun-Hsin Liang, Justin Steimling, Ty Kahler, Susan Steinike, Paul Connolly
Restek Corporation
For more information, email Frances Carroll.
Download a PDF of the full presentation.
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Introduction
The determination of psychoactive drugs and their metabolites has become routine in many forensic toxicology laboratories. The optimization of analysis time, resolution between metabolites, method robustness, and the ability to add emerging compounds is of ultimate importance when developing an efficient method for validation. The Raptor Biphenyl column combines the speed of superficially porous particles (SPP) with the resolution of highly selective USLC technology to give analysts the ability to produce fast, dilute-and-shoot methods while staying current with the ever-changing landscape of illegal drugs.

Methods
The method investigations were performed on a Waters ACQUITY I-class equipped with a Xevo TQ-S and a Shimadzu Nexera equipped with a SCIEX 4500. Both systems utilized electrospray ionization in positive ion mode. Standards were prepared in human urine and were diluted 3x in a 0.2 μm PVDF Thomson SINGLE StEP filter vial with 50:50 water:methanol prior to analysis. Data were collected with MRM windows of approximately ± 30 seconds. Chromatographic optimization resulted in complete resolution of isobars and separation from major matrix interferences of a representative pooled urine sample. Water and acetonitrile mobile phases modified with 0.1% formic acid were used under gradient conditions on a Restek Raptor Biphenyl 2.7 µm, 50 x 3.0 mm column equipped with a Raptor Biphenyl EXP 2.7 µm, 5 x 3.0 mm guard.

Results
Chromatographic separation is essential for analyzing synthetic cannabinoids JWH-018 and JWH-073 and their metabolites due to the presence of multiple positional isomers among the monohydroxylated metabolites. Since these positional isomers have identical molecular weights and very similar fragmentation patterns, they are indistinguishable by MS/MS detectors and chromatographic resolution is required for positive identification.

Previously, a method was presented for the comprehensive screen of 17 synthetic cannabinoids, 12 metabolites and 5 internal standards with a cycle time of 5 minutes. All positional isomers were resolved on the Raptor Biphenyl column, making it possible for the most abundant metabolites from a given parent compound to be identified in authentic samples.

Today, laboratories are faced with the difficult task of keeping up with the ever-growing list of synthetic cannabinoids illicit drugmakers produce to avoid legal classification and detection. In an effort to determine the ability of the original method to keep pace with the rapidly changing list, five emerging synthetic cannabinoids (i.e., AB-PINACA, AB-FUBINACA, PINACA, 5F-PB-22, and PB-22) and salvinorin A were prepared in human urine and analyzed using the same methodology.

Conclusions
The analysis of synthetic cannabinoids and their metabolites can be a difficult and challenging task. The Raptor Biphenyl column provides solutions to the chromatographic and validation issues surrounding this analysis. It has the ability to provide highly retentive, selective, and rugged reversed-phase separations, allowing for the simultaneous analysis of 22 synthetic cannabinoids, 12 metabolites, and salvinorin A. It has been demonstrated that analyte lists can easily be expanded as new synthetic cannabinoids are introduced. The speed of SPP allows analysis times to become shorter. The unique selectivity of Restek’s Biphenyl phase allows isomer separation to be easily achieved.

P115 A Novel Solution for EtG/EtS Analysis in Human Urine by LC-MS/MS
Justin Steimling, Frances Carroll, Shun-Hsin Liang (presenter), Sharon Lupo, Ty Kahler, Sue Steinike
Restek Corporation
For more information, email Shun-Hsin Liang.
Download a PDF of the full presentation.
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Introduction
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.

Methods
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 20 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.

Results
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.

Conclusions
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.


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