Cathinone stability in authentic urine specimens.

PURPOSE: Synthetic cathinones are encountered in a variety of antemortem and postmortem forensic toxicology investigations. Earlier experimental studies using fortified urine have evaluated analyte, temperature and pH-dependent variables associated with their stability. The purpose of this study was to compare experimental findings with those obtained using authentic urine from cathinone users. METHODS: In this report we compare cathinone concentrations in 180 authentic unpreserved urine specimens, following known periods of refrigerated storage. These findings are compared with previously published experimental data using fortified drug-free urine. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q/TOF-MS) was used to target 22 cathinones. Quantitative results were compared in urine specimens (pH 4.5-10) following 5-17 months of storage. RESULTS: The 180 specimens resulted in 164 quantitative findings involving α-PVP, ethylone, methylone, MDPV and pentylone. Initial drug concentrations ranged from 25ng/mL to over 100,000ng/mL. Upon reanalysis, the percentage of drug remaining (0-119%) was correlated with storage time and specimen pH. The ability to reconfirm original results was not correlated with storage time. Instead, specimen pH was far more predictive. The relationship between initial and final drug concentration was highly pH-dependent, yielding significant correlations for α-PVP, ethylone and methylone, particularly under acidic conditions. CONCLUSIONS: These results are in good agreement with experimental findings and highlight the critical importance of specimen pH, rather than conventional time dependent variables, when considering cathinone stability in biological samples. The potential for pre-analytical changes in cathinone concentrations must be carefully considered when interpreting their results.

Testing for designer stimulants: metabolic profiles of 16 synthetic cathinones excreted free in human urine.

The study of 34,561 urine specimens, submitted for designer stimulant testing between February 2011 and January 2013, provided an opportunity: to estimate the range of synthetic cathinones (SC) abused in the USA, to observe multiple examples of metabolic profiles for each drug in various stages of excretion in human urine, to evaluate the extent of metabolism of specific SC and to select metabolites or parent drugs for routine testing. Sixteen SC were found in random patient samples: buphedrone; butylone; 3,4-dimethylmethcathinone; ethcathinone; N-ethylbuphedrone; ethylone; flephedrone; mephedrone; 4-methylbuphedrone; 3,4-methylenedioxypyrovalerone (MDPV); 4-methyl-N-ethylcathinone; methylone; pentedrone; pentylone; α-pyrrolidinobutiophenone (PBP) and α-pyrrolidinopentiophenone (PVP). After liquid/liquid extraction and trifluoroacetylation, specimens were screened by gas chromatography-mass spectrometry (GC-MS) for drugs and metabolites excreted free in urine. Each SC exhibited a characteristic metabolic profile, as shown by multiple examples. Metabolites' structures were postulated on the basis of their mass spectra. A large group of SC appears to metabolize extensively by carbonyl reduction into respective substituted ephedrines and further by N-dealkylation into norephedrines. Abundant metabolites in this group are essential markers of the parent drug use. Unchanged drugs are far less abundant or not found at all. SC with methylenedioxy attachment to the aromatic ring, metabolize by carbonyl reduction to a much lesser extent and are best detected as such in free urine fraction. PBP and PVP can be detected either unchanged or as metabolites, resulting from pyrrolidine ring degradation into primary amine followed by carbonyl reduction. MDPV appears in urine as such with no apparent free metabolites.

Designer phenethylamines routinely found in human urine: 2-ethylamino-1-phenylbutane and 2-amino-1-phenylbutane.

2-Ethylamino-1-phenylbutane (EAPB) and 2-amino-1-phenylbutane (APB) were identified by gas chromatography-mass spectrometry in multiple urine samples submitted for stimulant drug testing and screened positive for amphetamines by enzyme immunoassay. Forty-two samples from all over the USA were found, containing both analytes during a 3-month period May-July 2013. A sports dietary supplement 'CRAZE' has been determined to be one of the sources of EAPB supply. EAPB along with its suggested metabolite APB were detected in a urine sample, obtained from a person known to use 'CRAZE'.

Detection of synthetic cannabinoids in oral fluid using ELISA and LC-MS-MS.

Synthetic cannabinoids are often referred to as 'Spice' or K2 compounds. Detection of these compounds in oral fluid has, to date, been limited to chromatographic procedures such as liquid chromatography with tandem mass spectrometry detection. We report the first analytical immunoassay for the screening of some synthetic cannabinoids in oral fluid specimens collected with the Quantisal™ device. JWH-200 was chosen as the calibration standard, because parent compounds, not metabolites, are predominantly detected in oral fluid. The immunoassay is capable of detecting JWH-200, JWH-018, JWH-073, JWH-022, AM-2201, AM-2232 and AM-1220. The assay was validated according to accepted laboratory protocols and applied to 32 authentic oral fluid specimens previously analyzed using LC-MS-MS at an accredited laboratory. The assay is sensitive, with a cutoff concentration of 0.25 ng/mL, and has a wide working range from 0.1 to 5 ng/mL. Intra- and interday precision were determined to be <10%. The screening method was completely validated and characterized; critical aspects of the screening included the incorporation of a preincubation step that improves the sensitivity of the assay to allow relevant concentrations of synthetic compounds in oral fluid to be detected.

Urine tested positive for ethyl glucuronide after trace amounts of ethanol.

AIM: Ethyl glucuronide (EtG) is used commonly as a marker for the detection of non-compliance of patients in alcohol withdrawal therapy in psychiatric hospitals in Europe and in work-place monitoring programmes in the United States. With the increased use of this new marker, questions related to an unintentional uptake of ethanol resulting in detectable EtG concentrations have been discussed. The aim of this study was to determine the concentration ranges of EtG and ethyl sulphate (EtS) after the consumption of very small amounts of ethanol (1 and 3 g), which are more likely to be incidental than intended. METHODS: Drinking experiments with ethanol amounts of 1 and 3 g, respectively, were performed on a total of 31 volunteers. EtG and EtS analysis in urine was performed by electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), and creatinine concentration was determined using the Jaffé reaction. Furthermore, data obtained from this experimentation were then compared to data from literature. RESULTS AND CONCLUSIONS: The maximum concentration of EtG normalized to creatinine after the uptake of 1 g and 3 g of ethanol was found to be 0.32 mg/l and 1.53 mg/l, respectively, and 0.15 mg/l and 1.17 mg/l for EtS; these peak concentrations are considered to be positive by many laboratories testing urine for ethanol conjugates in work-place testing progammes.

Influence of preservatives on the stability of ethyl glucuronide and ethyl sulphate in urine.

BACKGROUND: Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are specific and sensitive markers of ethanol consumption well established in monitoring withdrawal treatment in patients with chronic alcoholism. Recently, bacterial decomposition as well as in vitro and post-mortem formation of EtG was reported. The aim of this study was to investigate the influence of different preservatives on the stability of EtG and EtS concentrations in urine samples. METHODS: Urine samples were doped with glucuronidase-positive Escherichia coli after sterile filtration. The preservatives used were thymol, chlorhexidine, boric acid and the combination of chlorhexidine, ethylparabene and sodium propionate. Different aliquots of urine samples were stored refrigerated (4-8 degrees C), at room temperature (18+/-1 degrees C) and in an incubator (36+/-1 degrees C) for a period of 9 days with daily sampling. EtG and EtS analyses were performed by LC-ESI-MS/MS. The number of bacteria was detected by counting the colony forming units on Columbia blood agar plates. RESULTS AND CONCLUSIONS: Chlorhexidine on its own as well as in the aforementioned combination, and boric acid proved useful preservatives, while EtG degraded in samples doped with thymol. Addition of these preservatives did not interfere with the LC-MS/MS analysis.

A new method for simultaneous determination of cyclic antidepressants and their metabolites in urine using enzymatic hydrolysis and fast GC-MS.

A method for the simultaneous determination of six commonly prescribed cyclic antidepressants and their major metabolites in urine is presented. This method can be used for quantitation of amitriptyline, nortriptyline, imipramine, desipramine, doxepin, desmethyldoxepin, and maprotiline in human urine, in addition to the qualitative determination of their hydroxylated metabolites. This method is suitable for confirmation of drug abuse in health care professionals and overdose cases where the identity of the abused cyclic antidepressant may not be known. Samples are spiked with internal standard and hydrolyzed with beta-glucuronidase from Escherichia coli. Hydrolysis is found to be essential to the extraction procedure as the tertiary cyclic antidepressants are found to be extensively conjugated in urine. The secondary cyclic antidepressants, on the contrary, are found to be minimally conjugated. Drugs are extracted from alkalinized urine into solvent and derivatized with MSTFA/ammonium iodide/ethanethiol reagent. This reagent produces more stable derivatives compared to reagents previously employed. Gas chromatographic (GC)-mass spectrometric analysis is performed in electron ionization mode by selective ion monitoring, using hydrogen as a carrier gas, a short narrow bore GC capillary column, and fast temperature program, allowing for a rapid analytical cycle. While maintaining specificity for these drugs, concentrations in human urine ranging from 50 to 20,000 ng/mL can be measured with intraday and interday precisions, expressed as variation coefficient, of less than 2.8% for all analytes.

Detection of conjugated 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in oral fluid.

The presence of the conjugated marijuana metabolite 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THCA) glucuronide in oral fluid specimens is described for the first time. Oral fluid specimens were collected using a Quantisal device and analyzed for the presence of THCA using two-dimensional gas chromatography with mass spectrometric (GC-MS) detection both before and after hydrolysis. The nature of the conjugation was determined by analyzing specimens from a marijuana user without hydrolysis, with base hydrolysis, with beta-glucuronidase treatment, and hydrolysis using sulfatase only. Treatment with sodium hydroxide proved to be the most efficient hydrolytic procedure. Specimens collected over 48 h showed an average conjugation of over 64.5%. The specimens were also analyzed for the active component, tetrahydrocannabinol (THC), which was detected in the oral fluid, in most cases, for up to 24 h. Parent THC was not found to be glucuronide bound. Specimens were then subjected to commercially available immunoassays in order to determine their utility as screening procedures. The metabolite, THCA, was detected in all samples up to and including the specimen 48 h after smoking, using the more sensitive screening assay and two-dimensional GC-MS. Moreover, proof that the THCA is conjugated in oral fluid minimizes concerns associated with passive inhalation.

Simultaneous identification of 2-carboxy-tetrahydrocannabinol, tetrahydrocannabinol, cannabinol and cannabidiol in oral fluid.

Tetrahydrocannabinol (THC) is an important psychoactive ingredient in marijuana, which is the most widely used illegal recreational drug in the USA. Since it is generally smoked, the constituents of the plant material, as well as THC may be present in oral fluid specimens collected for the purposes of drug testing. We present an analytical procedure for the simultaneous determination of the pyrolytic precursor Delta(9)-tetrahydrocannabinolic acid A, tetrahydrocannabinol, cannabinol and cannabidiol in human oral fluid specimens using gas chromatography mass spectrometry (GC/MS). Solid phase extraction and GC/MS/EI with selected ion monitoring were used, and the linearity of the method ranged from 0-16 ng/mL of neat oral fluid. The recovery of the cannabinoids from the collection pad into the transportation buffer was greater than 70% for all cannabinoids tested at 4 ng/mL, and the intra- and inter-day precision was less than 10.3 and 15.2% for all analytes. The stability of the drugs in oral fluid and of the extracted derivatives was investigated. The procedure was applied to oral fluid specimens taken from habitual marijuana smokers. We have previously reported the presence of the metabolite 11-nor-Delta(9)-tetra-hydrocannabinol-9-carboxylic acid in oral fluid, but this is the first report of the plant constituent 2-carboxy-THC being detected in saliva.

Determination of meperidine, tramadol and oxycodone in human oral fluid using solid phase extraction and gas chromatography-mass spectrometry.

Analytical procedures for the determination of meperidine, tramadol and oxycodone in oral fluid have been developed and validated using gas chromatography-mass spectrometry (GC/MS) following initial screening with enzyme linked immunosorbent assay (ELISA). The oral fluid samples were collected using the Quantisal device, and any drugs present were quantified using mixed mode solid-phase extraction and electron impact GC/MS. For confirmation, three ions were monitored and two ion ratios determined, which were within 20% of those of the known calibration standards. The limits of quantitation were 10 ng/mL; the intra-day precision of the assays (n=5) was 2.33%, 1.00% and 7.61%; inter-day precision 2.48%, 2.44% and 5.8% (n=10) for meperidine, tramadol and oxycodone, respectively. The percentage recovery of the drugs from the collection pads was 86.7%, 87.7% and 96.6%, respectively (n=6). The methods were applied to specimens obtained during research studies in the USA.

Determination of propoxyphene in oral fluid.

The determination of propoxyphene in oral fluid using solid-phase extraction and gas chromatography-mass spectrometry is described for the first time. The method employs collection of oral fluid with the Quantisal device, immunoassay screening of the specimen, confirmation of the positive screened samples after extraction using cation exchange/hydrophobic solid-phase extraction columns, optimized derivative formation, and gas chromatography-mass spectrometry in electron impact mode. Validated parameters including selectivity, linearity, accuracy, intra- and interday precision, extraction efficiency, and limit of quantitation were all within acceptable limits. The method was applied to authentic specimens taken from an individual prescribed propoxyphene following surgery.

Analytical procedure for the determination of the marijuana metabolite 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in oral fluid specimens.

The determination of the marijuana metabolite 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THCA) in oral fluid specimens is described for the first time using a Quantisal oral fluid collection device and gas chromatography with single-quadrupole mass spectrometric detection. Oral fluid specimens were confirmed for the presence of THCA using two-dimensional gas chromatography-mass spectrometry in order to achieve the low concentration levels previously reported to be present in oral fluid. The extraction efficiency for THCA from the oral fluid collection pad was determined to be 80% at a concentration of 10 pg/mL with a coefficient of variation of 8.23%. The intraday precision of the assay ranged from 3.4% to 7.9% over four concentrations; the interday precision ranged from 8.3% to 18.5%. The limit of quantitation was 2 pg/mL. The method was applied to oral fluid specimens collected from a frequent user of marijuana. Samples were collected almost immediately after the subject smoked and then at intervals of 15 and 45 min and 1, 2, and 8 h after smoking. THCA was present in all the specimens, even the initial specimen taken almost immediately after smoking. The presence of THCA minimizes the argument for passive exposure to marijuana in drug-testing cases.

Detection of the marijuana metabolite 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in oral fluid specimens and its contribution to positive results in screening assays.

The detection of the marijuana metabolite 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid specimens is described, and its contribution to an immunoassay for the detection of cannabinoids is investigated. Oral fluid specimens, screened using an enzyme-linked immunosorbent immunoassay (ELISA), were carried forward to confirmation for both tetrahydrocannabinol (THC) and THC-COOH using gas chromatography-mass spectrometry (GC-MS). One hundred and fifty-three specimens were analyzed, of which 143 screened positive for cannabinoids. Ninety-five (66.4%) of these specimens were positive for both THC and THC-COOH; 14 (9.7%) were positive for THC-COOH only, and 27 (18.8%) were positive for THC only. The GC-MS assay for the detection of THC-COOH in oral fluid was linear to 160 pg/mL with a limit of quantitation of 2 pg/mL. The detection of the marijuana metabolite, THC-COOH, in 76.2% of oral fluid specimens screening positive for cannabinoids is reported. As a potential defense against passive exposure claims, proposed SAMHSA regulations may require the simultaneous collection of a urine sample when oral fluid samples are used. The detection of the metabolite, THC-COOH, is a significant alternative to this approach because its presence in oral fluid minimizes the argument for passive exposure to marijuana in drug testing cases.

Disposition of hydrocodone in hair.

The use of prescription drugs, including synthetic opiates, is increasing in the U.S., with emergency room reports showing a dramatic rise in prescription opiate abuse. As part of an ongoing study, the hair of admitted opiate users was analyzed for hydrocodone and hydromorphone, as well as codeine, morphine, and 6-acetylmorphine in order to determine if there was any correlation between self-reported frequency of opiate intake and the concentration of drug detected in hair. The hairs were confirmed using gas chromatography-mass spectrometry following screening by enzyme linked immunosorbent assay (ELISA). Twenty-four hair specimens collected from volunteers showed the presence of hydrocodone (130-15,933 pg/mg); four of those also contained hydromorphone (59-504 pg/mg). The specimens were also analyzed for morphine, codeine, and 6-acetylmorphine. Hair specimens from five self-reported codeine users showed concentrations of hydrocodone between 592 and 15,933 pg/mg. In addition, codeine was present at concentrations of 575-20,543 pg/mg, but neither morphine nor hydromorphone were present in any of those hair specimens. Though the analysis of some opiates in hair has been previously published, this is the first study where the hydrocodone and hydromorphone concentrations have been measured following self-reported opiate intake.

Application of two-dimensional gas chromatography with electron capture chemical ionization mass spectrometry to the detection of 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in hair.

The proposed federal regulations for the detection in hair of 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), a metabolite of marijuana, require a confirmatory detection level of 0.05 pg/mg. At present, the only way to achieve this on a routine basis has been with the use gas chromatography with tandem mass spectrometry (GC-MS-MS) technology. Tandem MS is an expensive approach and dissuades laboratories from attempting to enter the hair-testing market. A procedure for the determination of THC-COOH in hair using two-dimensional gas chromatography (GC x GC) coupled to mass spectrometry (GC-GC-MS) is described for the first time. The method makes use of several small improvements in the extraction, GC, and MS procedures to allow the required sensitivity to be achieved. The results of this approach demonstrate detection of THC-COOH in hair at a concentration level of 0.05 pg/mg with both a target quantitation ion and a unique confirming qualifier ion, using a single-quadrupole mass selective detector. These two ions and the enhanced separation of the GC-GC provide a high degree of confidence in the determinations. The method has been successfully applied to the detection of THC-COOH in hair specimens from known marijuana users, and it reaches the levels currently proposed in the Federal Register.