Routine application of SFC-MS in doping control: Analysis of 3 × 1000 urine samples using three different SFC-MS instruments.

Supercritical fluid chromatography-mass spectrometry (SFC-MS) has proved to be a beneficial tool for sample analysis for a wide variety of compounds and, as such, has recently gained the attention of the anti-doping community. We have tested the applicability of SFC-MS for routine doping control analysing approximately 3 × 1000 identical anti-doping samples utilising SFC-MS instruments from three different vendors: Agilent Technologies, Waters Corporation and Shimadzu Corporation. A 'dilute and inject' approach either without or after hydrolysis of glucuronide metabolites was applied. Most of the compounds included in our study demonstrated excellent chromatography, whereas some showed co-elution with endogenous interferences requiring MS discrimination. Retention times typically were very stable within batches (%CV ≤ 0.5%), although this appeared to be analyte and column dependent. Chromatographic peak shape was good (symmetrical) and stable over the period of the testing without any change of column. Our results suggest that SFC-MS is a sensitive, reproducible and robust analytical tool ready to be used in anti-doping laboratories alongside the currently applied techniques such as gas and liquid chromatography coupled to mass spectrometry. Even if instruments are designed slightly differently, all three setups demonstrated their fitness for the purpose in anti-doping testing.

Analysis of 17 fentanyls in plasma and blood by UPLC-MS/MS with interpretation of findings in surgical and postmortem casework.

The opioid crisis is linked to an increased misuse of fentanyl as well as fentanyl analogs that originate from the illicit drug market. Much of our current understanding of fentanyl and fentanyl analog use in our communities comes from postmortem toxicology findings. In the clinical settings of addiction medicine and pain management, where the opioid abuse potential is high, the use of fentanyl, as well as specific fentanyl analogs, may be underestimated due to limited plasma testing and limited availability of assays with suitable analytical sensitivity and selectivity to detect misuse of fentanyls. We report plasma and blood assays for 17 fentanyls (these include fentanyl, fentanyl analogs, fentanyl metabolites and synthetic precursors) in clinical, and medical examiner, casework. A mixed-mode solid phase extraction of diluted plasma or precipitated blood was optimized for maximum recovery of the fentanyls with minimized matrix effects. Analysis was performed using a Waters ACQUITY UPLC I-Class interfaced with a Waters Xevo TQ-S micro tandem quadrupole mass spectrometer. Method parameters were optimized and validated for precision, accuracy, carryover, linearity and matrix effects. Application studies were performed in postmortem blood obtained in 44 fentanyl-related fatalities and in serial plasma samples from 18 surgical patients receiving intravenous fentanyl therapy while undergoing parathyroidectomy. Fentanyls found in postmortem cases included fentanyl, norfentanyl, despropionyl-fentanyl (4-ANPP), beta-hydroxy fentanyl (ß-OH fentanyl), acetyl fentanyl, acetyl norfentanyl, methoxyacetyl fentanyl, furanyl fentanyl, cyclopropyl fentanyl, and para-fluorobutyryl fentanyl, with fentanyl, norfentanyl, 4-ANPP and ß-OH fentanyl predominating in frequency. Fentanyl concentrations ranged from 0.2 to 56 ng/mL and fentanyl was nearly always found with 4-ANPP, norfentanyl and ß-OH fentanyl. Concentrations of other fentalogs ranged from <1 to 84 ng/mL (extrapolated). In the surgical cases, fentanyl was detected and quantified along with norfentanyl and ß-OH fentanyl, but without detection of 4-ANPP in any of the samples. The association and relative concentrations of ß-OH fentanyl, fentanyl and norfentanyl in the postmortem and clinical studies indicated a metabolic, rather than an illicit, source of ß-OH fentanyl.

Matrix effects in metabolite quantification for MIST assessment: the impact of phospholipid removal and HPLC column particle size.

BACKGROUND: This Research article investigates the impact of phospholipid removal and high-performance liquid chromatography column particle size on the accuracy of determining the relative abundance of human metabolites using mass spectrometry peak areas in the context of assessing metabolite abundance for Metabolites in Safety Testing assessment. RESULTS/METHODOLOGY: Plasma samples spiked with 20 compounds, representing ten pairs of drugs and metabolites, were prepared using phospholipid removal plates (Ostro™) or standard protein precipitation techniques and analyzed by liquid chromatography-tandem mass spectrometry using high-performance liquid chromatography columns containing either 2.5 or 3.5 µm particles. Removal of phospholipids significantly reduced matrix effects for samples analyzed on the larger particle size columns while preventing phospholipid build up on the analytical columns. In addition, quantitative accuracy and linearity were not affected by phospholipid removal. CONCLUSION: Both sample preparation strategies and column particle sizes should be considered in order to reduce the inaccuracy as a result of matrix effects in assessing metabolite abundance using mass spectrometry peak areas.

Hydrophilic interaction chromatography (HILIC) for LC-MS/MS analysis of monoamine neurotransmitters.

BACKGROUND: Hydrophilic interaction chromatography (HILIC) is becoming an increasingly popular alternative to traditional reversed-phase chromatography for the analysis of polar compounds. The ability to retain the most polar compounds in HILIC makes it attractive for the analysis of certain large groups of compounds, such as monoamines, which are inherently very polar. RESULTS: This paper details the development of a HILIC LC-MS/MS method for the analysis of monoamine neurotransmitters. The emphasis is on method development; in particular, the factors influencing sensitivity, peak shape and resolution. Mobile-phase ionic strength, temperature and stationary phase functionality are shown to be key parameters for the successful development of HILIC methods. CONCLUSION: HILIC is shown to be an appropriate and suitable method for the analysis of monoamine neurotransmitters and an attractive alternative to reversed-phase analysis. The most polar analytes, which are essentially unretained by reversed-phase chromatography, demonstrate superior retention and resolution when analyzed by HILIC.

Use of a urine anastrozole assay to determine treatment discontinuation among women with hormone-sensitive breast cancer: a pilot study.

PURPOSE: Multiple studies have shown that adherence to adjuvant hormonal therapy in women with breast cancer is suboptimal. Measurements of compliance with self-report, pill counts, and/or pharmacy records are susceptible to bias. We assessed the feasibility of using a urine anastrozole assay as an objective biomarker of nonadherence to anastrozole treatment. PATIENTS AND METHODS: We recruited consecutive postmenopausal women, age ≥ 18 years, with hormone-sensitive nonmetastatic breast cancer who were prescribed anastrozole at least 3 months before enrollment. Each completed a short survey to gather information on demographics, anastrozole compliance history, and self-reported medication history, tumor characteristics, and treatment received. A single, random 15-mL urine sample was collected and tested for the presence of anastrozole using a previously validated assay. Patients were told they were part of a study to determine if anastrozole could be detected in the urine. RESULTS: Among 96 participants, mean age was 63.7 years (range, 51 to 70 years). The population was diverse, with 56.5% white, 57.6% US born, 59.8% unemployed, and 56.6% college educated. Prior treatment included chemotherapy (50%) and/or radiotherapy (58.7%). Mean duration of anastrozole treatment was 2.2 years (standard deviation, 1.6). Four participants reported nonadherence and declined to submit urine samples, and two had no detectable level of anastrozole (six of 96; 6.3%). Detectable levels among adherent women ranged from 49.3 to 632.8 ng/mL. CONCLUSION: We demonstrated that collection of urine to measure anastrozole levels is feasible and reliable. Identifying biomarkers to measure adherence is critical for studies investigating interventions to improve hormonal therapy compliance.

Simultaneous extraction and screening of diuretics, beta-blockers, selected stimulants and steroids in human urine by HPLC-MS/MS and UPLC-MS/MS.

Described herein are two general screening procedures for the simultaneous determination of 49 exogenous compounds (21 diuretics, 19 beta-blockers, eight stimulants and two steroidal drugs) in human urine by high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) and ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Urine samples were extracted using a simple and robust solid phase extraction (SPE) method. Samples were injected onto reversed phase HPLC and UPLC columns connected to tandem mass spectrometers capable of scan-to-scan polarity switching. The methods were validated according to the ISO 17025 international standard for the validation of a qualitative method. Sixty urine samples submitted for routine analysis were tested using both methods, the results of which correlated with results obtained from previously validated procedures. Both methods proved to be useful for routine urine analysis; most notably, the use of UPLC-MS/MS demonstrated that samples can be reliably screened with significantly reduced analysis times.

Quantitative confirmation of testosterone and epitestosterone in human urine by LC/Q-ToF mass spectrometry for doping control.

Testosterone (T) is the primary male sex hormone. In addition to the development of secondary sex characteristics, testosterone has anabolic effects including increases in muscle size and strength and increases in lean body mass, making it an attractive candidate to enhance athletic performance. In the case of exogenous administration of testosterone, the ratio of testosterone to its isomer, epitestosterone (E), is elevated. WADA has set a standard for T/E ratios of 4.0 as indicative of possible exogenous testosterone administration. Typically, a sample that screens for a T/E ratio above that threshold is then subjected to quantitative confirmation by GC/MS. This methodology, however, can limited due to sensitivity issues as well as a limited number of qualifying ions that can be used for unambiguous identification. We have developed a confirmation method which uses liquid/liquid extraction, followed by room temperature Girard P derivatization, and analysis using LC/MS-ToF. We observe a number of advantages over conventional GC/MS analysis. Analysis time is decreased. Sensitivity is increased, resulting in limits of detection of 2 and 0.5 ng/ml for testosterone and epitestosterone, respectively. The number of diagnostic qualifier ions is also increased allowing more confident identification of the analytes. Finally, while this method has been developed on a QToF instrument, it should be easily transferable to any tandem LC/MS/MS system.

Evaluation of ten oral fluid point-of-collection drug-testing devices.

Previously, the laboratory evaluations of six point-of-collection oral fluid (POC-OF) drug testing devices were reported. Four additional devices, Oralstat (American Bio Medica); SmartClip (Envitec); Impact (LifePoint); and OraLine IV s.a.t (Sun Biomedical Laboratories), were recently evaluated for their ability to meet the claimed (and proposed) cutoff concentrations set by the manufacturers for the detection of amphetamine(s), cocaine/metabolite, opiates, and cannabinoids (Oralstat also benzodiazepines). With the exception of the Sun Biomedical device, actual false-positive results were not encountered. Most devices performed well for the detection of opiates and amphetamine(s), but approximately half had amphetamine(s) cutoff concentrations greater than that proposed by the Substance Abuse and Mental Health Services Administration (SAMHSA). Only three devices had cocaine cutoffs less than or equal to 20 ng/mL (SAMHSA), and a number of false-negative results were obtained. The devices still were not capable of detecting Delta(9)-tetrahydrocannabinol at 4 ng/mL (SAMHSA). However, sensitivities improved since the initial studies, and approximately half of the devices met the THC-COOH cutoff proposed by SAMHSA. Results from the current and previous evaluations are presented in the paper and indicate that the sensitivity and performance of commercial OF drug testing devices is improving, but remains problematic for the reliable detection of cannabinoid use.

Mechanism of an exaggerated locomotor response to a low-dose challenge of methamphetamine.

Previous studies using phenylethylamine psychostimulants such as amphetamine (AMPH) have demonstrated that pretreatment with a high-dose of drug followed by a low-dose challenge injection (3 h later) results in an exaggerated behavioral response. In order to explore the mechanism of this exaggerated or what has been suggested to be a "sensitized" response, we investigated the effects of methamphetamine (METH) in a similar treatment paradigm. The current study found that, as suggested by previous studies, a low-dose challenge with METH substantially increased the locomotor response in animals that received a high-dose pretreatment (3.5 h prior to challenge). We also observed that rats displayed an increase in the concentrations of METH and its metabolite AMPH in the striatum following the low-dose challenge of METH if they were pretreated with METH versus saline. A similar pattern for METH and AMPH levels was measured in the plasma. Taken together, these results suggest that the accumulation of drug in animals pretreated with high-dose METH contributes to the overall enhanced behavioral response following challenges with low-doses of METH.

Persistence of tolerance to methamphetamine-induced monoamine deficits.

Methamphetamine is a highly addictive and potent stimulant, the use of which has increased significantly in recent years. In addition to the severe behavioral and societal consequences associated with methamphetamine abuse, methamphetamine can cause persistent damage to monoaminergic nerve terminals in rats, as measured by either monoamine concentrations or activity of the rate limiting synthetic enzymes, tyrosine hydroxylase and tryptophan hydroxylase. Repeated, sub-neurotoxic doses of methamphetamine, however, can cause rats to become resistant to the neurotoxic effects of multiple high-dose administrations of methamphetamine; a phenomenon known as tolerance. This study investigates the persistence of tolerance evoked by pretreatment with escalating-dose administrations of methamphetamine. Rats were pretreated over several days with low, escalating doses of methamphetamine, followed by high-dose methamphetamine challenge after variable recovery periods. Results revealed that tolerance to monoaminergic deficits persisted for at least one week, but was completely eliminated by 31 days. There were no differences in the distribution of methamphetamine or its major metabolite, amphetamine, between methamphetamine-pretreated animals and saline-pretreated animals 2 h after the final methamphetamine challenge injection, and there were no regional differences in methamphetamine concentrations between the frontal cortex, hippocampus or striatum. We also observed that while methamphetamine pretreatment attenuated the hyperthermia caused by the high-dose methamphetamine challenge, significant reductions in methamphetamine-induced hyperthermia were not required for the development of tolerance with this regimen.

A liquid chromatographic-tandem mass spectrometric method for the analysis of serotonin and related indoles in human whole blood.

This paper describes a high-performance liquid chromatographic-tandem mass spectrometric (HPLC-MS-MS) method for the determination of serotonin (5-HT) and the related indoles tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), indole-3-acetic acid (IAA), and indole-3-proprionic acid (IPA) in whole blood. Ionization was achieved by atmospheric pressure chemical ionization (APCI). The method uses a solvent gradient to achieve baseline separation of all analytes. Linearity was established for all components from 10 to 500 ng/mL, with the exception of TRP, which was calibrated from 0.4 to 20 microg/mL. Limits of quantitation were established that were well below the normal endogenous concentrations of 5-HT and its related indoles. Recoveries of 5-HT, TRP, 5-HIAA, IAA, and IPA were 95%, 91%, 98%, 79%, and 65%, respectively, from whole blood matrix and varied less than 2%. Serotonin, TRP, and IAA results were verified by cross-validation with an external laboratory using a previously validated HPLC method with fluorometric detection. A regression analysis of the two data sets resulted in correlation coefficients of 0.981, 0.964, and 0.987 for 5-HT, TRP, and IAA, respectively.