Developing and Validating a Fast and Accurate Method to Quantify 18 Antidepressants in Oral Fluid Samples Using SPE and LC-MS-MS.

Antidepressant drugs are one of the most widely used medicines for treating major depressive disorders for long time periods. Oral fluid (OF) testing offers an easy and non-invasive sample collection. Detection of antidepressants in OF is important in clinical and forensic settings, such as therapeutic drug monitoring and roadside testing for driving under influence. We developed and validated a comprehensive liquid chromatography-tandem mass spectrometry method for 18 antidepressants (amitriptyline, bupropion, citalopram, clomipramine, cyclobenzaprine, desipramine, desvenlafaxine, doxepin, duloxetine, fluoxetine, imipramine, mirtazapine, nortriptyline, paroxetine, sertraline, trazodone, trimipramine, venlafaxine) in oral fluid collected by Quantisal® oral collection devices. One-half milliliter of Quantisal® OF (125 µL of neat OF) was submitted to solid-phase extraction. The chromatographic separation was performed employing a biphenyl column in gradient mode with a total run time of 5 min. The MS detection was achieved by multiple-reaction monitoring with two transitions per compound. The range for linearity of all analytes was from 10 to 1,000 ng/mL, with a limit of detection of 10 ng/mL. Intra and inter-day accuracy and precision (n = 15) were all within acceptable limits, ±20% error and ±15% relative standard deviation. Analyte recovery at 400 ng/mL concentration (n = 15) ranged from 91 to 129%. Matrix effect ranged from 73.7 to 157%. The internal proficiency test detected all antidepressants with accuracy ranging from 83.1 to 112.1%. The authentic patient sample showed a percentage difference compared to the previously calculated concentration of 86.3-111%. This method provides for the rapid detection of 18 antidepressants and metabolites in OF, which is readily applicable to a routine laboratory.

Retrospective Chart Review of Synthetic Cannabinoid Intoxication with Toxicologic Analysis.

INTRODUCTION: Use of synthetic cannabinoids (SC) has recently emerged as a new drug epidemic. Our emergency departments (EDs) received a surge of SC users presenting with lethargy and bradycardia, contrasting prior reports of SC-induced tachycardia and agitation. Our goal was to describe these novel presentations and characterize the compounds. METHODS: We present a case series of patients with SC intoxication who presented to our toxicology service covering two tertiary care EDs between 2/11/2015 and 6/23/2015. A retrospective chart review recorded initial vital signs, chief complaint and clinical course. Urine, blood and xenobiotic samples were analyzed using either liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry. We compared resulting spectra against databases containing numerous SCs or metabolites and scored based on a reference comparison. RESULTS: Between 2/11/2015 and 6/23/2015, we identified 141 visits. Males comprised 139 visits (age range 21-68 years; median 35, interquartile range 20). Sixty-eight percent presented with lethargy or loss of consciousness. Hypotension (SBP <90 mmHg) and bradycardia (HR<60 bpm) were seen in 10% and 24% of visits, respectively. While most patients were discharged after observation, three were admitted to the intensive care unit and seven to telemetry. Admissions were for vital sign instability, bradycardia requiring pacing, prolonged sedation and respiratory failure requiring mechanical ventilation.Laboratory analysis revealed SC in the XLR-11 family in 18/36 drug, 9/12 blood, and 23/31 urine samples. Carboxamide indazole derivative (CID) family compounds were detected in 13/36 drug samples, 21/31 urine samples, but no blood samples; 11/31 drug samples contained both XLR-11 and CID. Other compounds detected included PB-22 and nicotine. No JWH compounds, opiates, imidazoline receptor agonists, benzodiazepines or other sedative-hypnotics were detected. CONCLUSION: Unlike their predecessors, novel SC may be associated with significant central nervous system depression and bradycardia. While prior reports indicated that SC mostly contained JWH compounds, none were detected in these samples. The most commonly identified compounds in this series were CID and alkyl SC derivatives, such as INACA compounds and XLR-11. These tend to be full agonists at the cannabinoid receptor and are presumably more potent. The lack of other depressants suggests that the clinical findings are due to the combination of these compounds and not coingestants or adulterants. SC intoxication should be considered for patients with undifferentiated psychomotor depression and bradycardia.

Chiral Analysis of Methamphetamine in Oral Fluid Samples: A Method to Distinguish Licit from Illicit Drug Use.

Methamphetamine (MAMP) is a popular illicit drug abused for its central nervous system stimulating effects. MAMP is also used therapeutically in the treatment of overeating disorders, narcolepsy, attention deficit disorder, in over-the-counter (OTC) products to ease nasal congestion. MAMP exists in two enantiomeric forms, dextrorotary (d-MAMP) or levorotary (l-MAMP). The compounds are similar in chemical structure, simply differing in the orientation of functional groups around the asymmetric carbon. In part because of the availability of l-MAMP in OTC nasal inhalers, forensic guidelines require a sample to contain greater than 20% d-MAMP to consider illicit drug use when interpreting results. Standard analytical methods readily detect MAMP in biological specimens but cannot resolve the enantiomeric composition of the sample. Specialized analytical techniques based on chiral separation of the enantiomers are required to differentiate d-MAMP from l-MAMP. Our laboratory sought to develop and validate a method for the analysis of oral fluid specimens for d/l-MAMP using a chiral derivatizing agent and traditional reverse phase liquid chromatography tandem mass spectrometry (LC-MS-MS). MAMP was extracted from dilute oral fluid samples using Strata-XC solid phase extraction (SPE) cartridges and derivatized with Marfey's reagent. Chromatographic separation was achieved using Zorbax Eclipse Plus C18 columns. Linearity, accuracy and precision, recovery, matrix effects and specificity of the method were all within acceptable criteria. Intraday accuracy ranged from 93.3 to 103.4% and precision 0.1 to 1.6%. Interday accuracy ranged from 90.0 to 103.4% and precision 3.8 to 11.6%. Finally, having previously tested positive for MAMP using non-chiral analysis, 256 de-identified authentic oral fluid samples were analyzed using this validated method. 98% of all samples tested positive for d-MAMP at greater than 20%.

A Novel Chronic Opioid Monitoring Tool to Assess Prescription Drug Steady State Levels in Oral Fluid.

OBJECTIVE: Interpretation limitations of urine drug testing and the invasiveness of blood toxicology have motivated the desire for the development of simpler methods to assess biologically active drug levels on an individualized patient basis. Oral fluid is a matrix well-suited for the challenge because collections are based on simple noninvasive procedures and drug concentrations better correlate to blood drug levels as oral fluid is a filtrate of the blood. Well-established pharmacokinetic models were utilized to generate oral fluid steady state concentration ranges to assess the interpretive value of the alternative matrix to monitor steady state plasma oxycodone levels. METHODS: Paired oral fluid and plasma samples were collected from patients chronically prescribed oxycodone and quantitatively analyzed by liquid chromatography tandem mass spectrometry. Steady state plasma concentration ranges were calculated for each donor and converted to an equivalent range in oral fluid. Measured plasma and oral fluid oxycodone concentrations were compared with respective matrix-matched steady state ranges, using each plasma steady state classification as the control. RESULTS: A high degree of correlation was observed between matrices when classifying donors according to expected steady state oxycodone concentration. Agreement between plasma and oral fluid steady state classifications was observed in 75.6% of paired samples. This study supports novel application of basic pharmacokinetic knowledge to the pain management industry, simplifying and improving individualized drug monitoring and risk assessment through the use of oral fluid drug testing. Many benefits of established therapeutic drug monitoring in plasma can be realized in oral fluid for patients chronically prescribed oxycodone at steady state.

A Fast and Comprehensive Analysis of 32 Synthetic Cannabinoids Using Agilent Triple Quadrupole LC-MS-MS.

Synthetic cannabinoids are a group of psychoactive compounds that mimic the effects of Δ9-tetrahydrocannabinol, the primary psychoactive constituent of marijuana (Cannabis sativa L). The Drug Enforcement Administration has classified many of the most common cannabinoids as Schedule 1 controlled substances. As a result, several novel synthetic cannabinoid series have emerged in the illicit drug market, including PINACA, FUBINACA, PB-22, AKB-48 and multiple derivatives of these compounds. Our laboratory developed and validated an analytical method for the analysis 32 synthetic cannabinoid metabolites in urine samples. Included in this method are metabolites that are constituents of the new generation of synthetic cannabinoids. Following enzymatic hydrolysis, target analytes were recovered by liquid-liquid extraction utilizing 1-chlorobutane:isopropyl alcohol (70:30) as the organic ratio. Chromatographic separation and detection was achieved using an Agilent Technologies 1290 liquid chromatograph coupled to a 6460-triple quadrupole mass spectrometer with a Jetstream electrospray source. Linearity for all analytes was established along the range of 0.5-200 ng/mL. Both intraday and interday accuracy and precision data were all within acceptable limits, ±20% error and ±15% relative standard deviation, respectively. Recovery ranged from 48% to 104%. This method has shown to be selective and specific, providing no evidence of interference or carryover concerns. Finally, 11 distinct synthetic cannabinoids were detected in 23 of 25 donor samples analyzed with the method. The data presented here represents a validated liquid chromatography  tandem mass spectrometry method to accurately identify and quantitate synthetic cannabinoid metabolites in urine samples, incorporating new generation derivatives.

Assessment of the use of oral fluid as a matrix for drug monitoring in patients undergoing treatment for opioid addiction.

Drug testing is an important clinical tool that is available to physicians who are assessing the effectiveness of drug treatment as well as patient compliance to the administered program. While urine has traditionally been the matrix of choice for drug monitoring, oral fluid, a filtrate of the blood, has shown great promise as an alternative matrix for such applications. Oral fluid collection can be accomplished without the need for highly trained medical staff through the use of a simple, noninvasive oral fluid collection device, which obtains an adequate sample in only a few minutes. There has been a significant amount of research performed on the use of oral fluid for forensic toxicology application; however, more studies assessing the use of oral fluid drug testing are required to validate its ability to achieve clinical drug monitoring goals. Testing for various drugs in oral fluid may yield a different result when compared to the same drugs in urine, requiring an assessment of the utility of oral fluid for such practices. The purpose of this study was to examine the application of oral fluid drug testing in patients undergoing buprenorphine treatment for opioid dependence. A retrospective analysis of drug testing results obtained from 6,928 patients (4,560 unobserved urine collections and 2,368 observed oral fluid collections) monitored for heroin metabolite, amphetamine, benzodiazepines, buprenorphine, tetrahydrocannabinol, cocaine, codeine, hydrocodone, hydromorphone, methadone, morphine, oxycodone, and oxymorphone was completed. Results of this statistical exercise indicated that patients undergoing observed oral fluid collection tested positive more frequently than those unobserved urine collections for several illicit drugs and prescription medications targeted. Oral fluid was shown to detect illicit drug use as well as noncompliance in this patient population under the studied conditions more often than the urine specimens.

Validation of an EMIT® screening method to detect 6-acetylmorphine in oral fluid.

An automated assay was modified and validated to qualitatively screen for 6-acetylmorphine (6-AM) in oral fluid using the Siemens EMIT II(®) Plus 6-AM urine assay. The validation utilized an oral fluid calibrator at the currently proposed Substance Abuse and Mental Health Services Administration cutoff concentration of 4 ng/mL, as well as quality control material prepared and validated through liquid chromatography-tandem mass spectrometry. All calibrator, quality control and unknown specimens were analyzed based on the dilution and buffering system of the Quantisal(®) oral fluid collection device. Immunoassay parameters such as the pipetted sample and reagent volumes as well as photometric read times were adjusted as part of the assay modification process. Validation experiments included the determination of intra- and inter-day precision and reproducibility, limits of detection (LODs), assay selectivity, stability studies and a specimen agreement study (n = 132). The 6-AM assay performed well in all validation experiments, over multiple days and under various laboratory conditions. The LOD was determined to be 1.844 ng/mL. The assay sensitivity, specificity and overall misclassification rate were found to be 90, 100 and 6%, respectively.

Toxicity and bioaccumulation of the booster biocide copper pyrithione, copper 2-pyridinethiol-1-oxide, in gill tissues of Salvelinus fontinalis (brook trout).

This investigation studied the acute effects of copper pyrithione (CuPT) exposure on juvenile brook trout, Salvelinus fontinalis. Morphologic changes, copper bioaccumulation, and markers of oxidative stress in gill tissue were studied. Juvenile brook trout were treated with one of six experimental doses of CuPT (2-64 microg/L) for 2 hours. A seventh group served as a control population. Inductively coupled plasma atomic absorbance spectrophotometry (ICPAAS) analysis demonstrates significantly increased levels of copper in gill tissue (p < 0.001). Results from scanning electron microscopy and histological analysis demonstrate the formation of club-shaped lamella, edema, fusion of secondary lamella, loss of microridge structures and epithelial exfoliation. Transmission electron microscopy revealed altered morphology of chloride cells, including the swollen appearance of mitochondria with disruption of internal cristae and lipid membrane disruption. Thiobarbituric acid reactive substance (TBARS) assays demonstrated increased levels of lipid peroxidation products in gill tissue. Assays for the total antioxidant capacity of gill tissue revealed significantly lowered antioxidant levels. This data indicates that CuPT is potentially harmful to nontarget aquatic organisms at environmentally relevant doses.