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Continuing our studies for the analyses of drugs of abuse in municipal wastewater, a method was developed for the analysis of benzodiazepines in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Ten benzodiazepines and metabolites were analyzed (structures were found), including alprazolam, α-OH-alprazolam (the primary urinary metabolite of alprazolam), chlordiazepoxide, flurazepam, 2-OH-ethylflurazepam (the primary urinary metabolite of flurazepam), 7-NH2-flunitrazepam, nordiazepam, oxazepam, temazepam and α-OH-triazolam (the primary urinary metabolite of triazolam) (representative chromatograms were found). These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Wastewater Treatment Plant (WWTP) in Oxford, Mississippi (MS) and the University WWTP in University, MS. These wastewater samples were collected on weekends in which the Ole Miss Rebel football team held home games at the Vaught-Hemingway Stadium, University, and one weekend on which there was no game. The collected samples were analyzed using a validated method and found to contain alprazolam, α-OH-alprazolam, nordiazepam, oxazepam and temazepam. None of the samples contained chlordiazepoxide, flurazepam, 2-hydroxyethyl-flurazepam, 7-NH2-flunitrazepam and α-OH-triazolam.

There has been a recent rise in the number of cases of athletes being banned from competition because of positive tests for prohibited substances in their biological specimens. Most of these substances are on the World Anti-Doping Agency (WADA) prohibited list, while others are not specifically named on the list. N-Ethyl-α-ethyl-phenethylamine (ETH), a derivative of phenethylamine (PEA), is one of these unlisted substances and shares chemical and biological effects to the amphetamines, which are listed on the WADA prohibited substances list. It is classified as Category 6B stimulant on the list. This study was directed toward the development of an liquid chromatography tandem mass spectrometry (LC-MS-MS) method for the analysis of ETH in performance-enhancing dietary supplement. A standard was prepared and confirmed by spectroscopic analysis, which was then used to develop the analytical procedure. The procedure was validated and found to have an limit of detection of 2.5 ng/mL, limit of quantification of 5 ng/mL and upper limit of linearity of 500 ng/mL, with within-day variability at the 10-ng/mL level range of 3.88-7.89% (n = 6) and 1.39-3.36% (n = 6) for the 100-ng/mL level. The day-to-day variability was 9.8% for the low control and 3.1% for the high control. The method was used to analyze a variety of dietary supplements for ETH as well as PEA and its N, N-diethyl derivative (NDP).

Methylhexaneamine (MHA) has been marketed in dietary supplements based on arguments that it is a constituent of geranium (Pelargonium graveolens) leaves, stems, roots or oil, and therefore qualifies as a dietary ingredient. The purpose of this study is to determine whether P. graveolens plant material (authenticated) or its oil contains detectable quantities of MHA. Two analytical methods were developed for the analysis of MHA in P. graveolens using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. The results were further confirmed using liquid chromatography-high-resolution mass spectrometry. Twenty commercial volatile oils, three authenticated volatile oils and authenticated P. graveolens leaves and stems (young and mature, and fresh and dried) were analyzed for MHA content. In addition, three dietary supplements containing MHA that alleged P. graveolens as the source are analyzed for their MHA content. The data show that none of the authenticated P. graveolens essential oils or plant material, nor any commercial volatile oil of Pelargonium (geranium oil) contain MHA at detectable levels (limit of detection: 10 ppb). The dietary supplements that contained MHA as one of their ingredients (allegedly from geranium or geranium stems) contained large amounts of MHA. The amounts of MHA measured are incompatible with the use of reasonable amounts of P. graveolens extract or concentrate, suggesting that MHA was of synthetic origin.

Methods using liquid chromatography/mass spectrometry (LC/MS) and LC with diode array detection (DAD) in the UV range (LC/UV) were developed for the determination of low levels of the anthraquinones aloe-emodin and aloin-A (barbaloin) in aloe-based products. The methods were used to analyze several commercial products (liquids, semisolids, and solids) for the 2 anthraquinones. The wavelengths used for quantification of aloin-A, aloe-emodin, and emodin (internal standard) by DAD were 357, 257, and 289 nm, respectively. The on-column sensitivities were 0.25 and 0.05 ng by LC/UV and 0.01 and 0.025 ng by LC/MS for aloin-A and aloe-emodin, respectively. The methods are simple and sensitive and provide reproducible results; therefore, they are suitable for the determination of these anthraquinones in various aloe-based products.

Foods containing seeds or oil of the hemp plant (Cannabis sativa L.) are increasingly found in retail stores in the U.S. The presence of delta9-tetrahydrocannabinol (THC) in these foods has raised concern over their impact on the results of workplace drug tests for marijuana. Previous studies have shown that eating hemp foods can cause screening and confirmed positive results in urine specimens. This study evaluated the impact of extended daily ingestion of THC via hemp oil on urine levels of its metabolite 11-nor-9-carboxy-delta9-tetrahydrocannabinol (THC-COOH) for four distinct daily THC doses. Doses were representative of THC levels now commonly found in hemp seed products and a range of conceivable daily consumption rates. Fifteen THC-naïve adults ingested, over four successive 10-day periods, single daily THC doses ranging from 0.09 to 0.6 mg. Subjects self-administered THC in 15-mL aliquots (20 mL for the 0.6-mg dose) of four different blends of hemp and canola oils. Urine specimens were collected prior to the first ingestion of oil, on days 9 and 10 of each of the four study periods, and 1 and 3 days after the last ingestion. All specimens were screened for cannabinoids by radioimmunoassay (Immunalysis Direct RIA Kit), confirmed for THC-COOH by gas chromatography-mass spectrometry (GC-MS), and analyzed for creatinine to identify dilute specimens. None of the subjects who ingested daily doses of 0.45 mg of THC screened positive at the 50-ng/mL cutoff. At a daily THC dose of 0.6 mg, one specimen screened positive. The highest THC-COOH level found by GC-MS in any of the specimens was 5.2 ng/mL, well below the 15-ng/mL confirmation cutoff used in federal drug testing programs. A THC intake of 0.6 mg/day is equivalent to the consumption of approximately 125 mL of hemp oil containing 5 microg/g of THC or 300 g of hulled seeds at 2 microg/g. These THC concentrations are now typical in Canadian hemp seed products. Based on our findings, these concentrations appear to be sufficiently low to prevent confirmed positives from the extended and extensive consumption of hemp foods.

Buprenorphine, which is a powerful analgesic, a substitution drug for opioids widely used in Europe, and a promising new drug currently undergoing clinical trials in the treatment of opioid dependence in the U.S., is excreted in human urine mainly as glucuronide conjugates. In gas chromatographic-mass spectrometric analysis, the urine specimens must be first hydrolyzed to release buprenorphine from its glucuronide conjugates. In order to evaluate the existing hydrolysis methods and to find the optimal hydrolysis conditions, buprenorphine-3-beta-D-glucuronide (B3G) was synthesized. Urine fortified with synthetic B3G was hydrolyzed using acid, base, and beta-glucuronidases from different source species, including Helix pomatia, Escherichia coli, and Patella vulgata. Glusulase, a preparation containing both beta-glucuronidase (H. pomatia) and sulfatase, was also tested. Whereas both acidic and basic hydrolysis were ineffective, quantitative hydrolysis could be achieved by using beta-glucuronidases under appropriate conditions. However, we found that there was a marked difference in the reactivity of these enzymes (E. coli > H. pomatia >> P. vulgata). The optimal incubation conditions for enzymatic hydrolysis of B3G were 2 h at 37 degrees C for E coli and 4 h at 60 degrees C or 16 h at 37 degrees C for H. pomatia. Using 1000 Fishman units of either of these two enzymes, effective hydrolysis could be achieved even when the B3G concentration was as high as 2000 ng/mL. Glusulase was equally effective toward B3G if the fortified urine samples were incubated with 25 microL of this enzyme for 1 h at 60 degrees C.

A procedure for extraction of heroin and metabolites for gas chromatography-mass spectrometry (GC-MS) analysis of meconium specimens that would allow detection of these analytes at low levels was needed. Solid-phase extraction (SPE) cartridges were therefore evaluated for their effectiveness in sample preparation. Four different types of commercially available extraction cartridges were used. Heroin, 6-monoacetylmorphine (6-MAM), morphine, and codeine were extracted from meconium samples using these SPE cartridges and then simultaneously analyzed using GC-MS. In each case, the extraction efficiency, linearity range, limit of detection (LOD), limit of quantitation (LOQ), between-run precision, and within-run precision were determined. Although satisfactory results were obtained with the four different types of SPE cartridges, best overall performance was observed using Clean Screen columns following the procedures outlined here. LODs as low as 20 ng/g for codeine, 10 ng/g for morphine, and 2.5 ng/g for 6-MAM were obtained, and LOQs as low as 20 ng/g for codeine, 10 ng/g for morphine, and 5 ng/g for 6-MAM were obtained. In all cases linearities were observed (r =0.99) for codeine, morphine, and 6-MAM over a wide concentration range (100-2000, 100-2000, and 5-100, respectively). At 50 ng/g codeine and morphine and 10 ng/g 6-MAM, the precision of analysis using these cartridges showed coefficients of variation ranging from 4.75% to 15.5%.

An EMIT-ETS d.a.u. immunoassay screening method for methadone in meconium and a gas chromatography-mass spectrometry (GC-MS) method for methadone and its metabolites including 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) in meconium were described. The GC-MS method showed good linearity (r2 > or = 0.998) over a concentration range of 25-2000 ng/g with limits of detection of 10, 25, and 10 ng/g for methadone, EDDP, and EMDP, respectively, and a limit of quantitation of 25 ng/g for all three analytes. Fifty pooled meconium samples were screened using a cutoff of 200 ng/g, and all samples screened negative. GC-MS analysis of all samples showed four samples to contain methadone (35.2 to 79.9 ng/g), EDDP (28.5 to 557.2 ng/g), or both, with no detectable amount of EMDP. The negative results on the four specimens at the cutoff used may be explained by the fact that EMIT-ETS d.a.u. antibody for methadone was specific to the parent drug. The results point to the fact that immunoassays should be directed to EDDP for detection of prenatal exposure of methadone through analysis of meconium specimens.

A simple extraction procedure for delta9-tetrahydrocannabinol (delta9-THC) and its metabolites from various biological specimens was developed based on immunoaffinity chromatography. Using the affinity resin prepared by immobilization of THC antibody onto cyanogen bromide-activated Sepharose 4B, delta9-THC and its major metabolites including 11-nor-delta9-THC-9-carboxylic acid (delta9-THCCOOH), 11-hydroxy-delta9-THC (11-OH-delta9-THC), and 8beta,11-dihydroxy-delta9-THC (8beta,11-diOH-delta9-THC) were extracted simultaneously from plasma or urine after enzyme hydrolysis. The samples were derivatized as TMS derivatives and analyzed by gas chromatography-mass spectrometry in EI mode with SIM monitoring. Greater than 87% extraction recovery of the four analytes was obtained from both plasma and urine at 5 and 50 ng/mL concentration levels. The method was also used for meconium analysis with some modification. The extraction recovery from meconium, however, was lower than that of plasma and urine, ranging from 52 to 72% at the 10-ng/g level. All compounds showed good linearity within the tested ranges up to 100 ng/mL (g). The limits of detection ranged from 0.5 to 2.5 ng/mL in plasma and urine, and from 1.0 to 2.5 ng/g in meconium. Analysis of 24 meconium specimens showed that 11-OH-delta9-THC is indeed an important metabolite in meconium.

developed for the analy~s of t]unJtrazepam (FN) and its major metabolite, 7-amino-flunitrazepam (7-amino-FN), in both plasma and whole blood. The method was based on acid hydrolysis of the samples after dilution with HPLC water followed by extraction and derivatization (heptafluorobutyrate) of the resulting benzophenones. Analysis of plasma and whole blood samples from subjects administered 2-mg doses of FN showed that FN was only detected in whole blood (rOD 5 ng/mL) and not in plasma. However, 7-amino-FN was detected in both plasma and whole blood, although the levels were much higher in plasma. 7-Amino-FN was detected for the entire period of specimen collection (12 h), but FN was only detected in whole blood for 4 h after ingestion with peak levels after I the time course of detection of the parent drug versus its metabolite in both plasma and blood samples collected at the same time.

The analysis of meconium specimens for metabolites of substances of abuse is a relatively accurate method for the detection of fetal exposure to drugs. Most of the methods reported in the literature before the early 1990s relied on radioimmunoassays. The purpose of this study was to develop and validate methods for meconium sample preparation for the screening and gas chromatography-mass spectrometry (GC-MS) confirmation of meconium extracts for cannabinoids, cocaine, opiates, amphetamines, and phencyclidine. EMIT and TDx immunoassays were evaluated as screening methods. The sample preparation method developed for screening included extraction and purification prior to analysis. Cutoff levels were administratively set at 20 ng/g for 11-nor-delta9-THC-9-COOH (THCCOOH) and phencyclidine and at 200 ng/g for benzoylecgonine, morphine, and amphetamines, although lower levels could be detected in meconium using the EMIT-ETS system. Ninety-five meconium specimens were subjected to the screening procedure with GC-MS confirmation of presumptive positives. In addition, 30 (40 for cocaine) meconium specimens were subjected to GC-MS analysis for all analytes regardless of the screening results to determine the false-negative rate, if any, of the immunoassay. Although there were no false negatives detected, the GC-MS confirmation rate for the immunoassay-positive specimens was generally low, ranging from 0% for amphetamines to 75% for opiates. The lowest rate of confirmed positives was found with the cannabinoids, suggesting that tetrahydrocannabinol (THC) metabolites other than free 11-nor-9-carboxy-delta9-THC may be major contributors to the immunoassay response in meconium.

A simple method for the direct analysis of gamma-hydroxybutyrate (GHB) from human urine is described. The method uses solid-phase extraction, liquid-liquid extraction, and silyl-derivatization, then gas chromatographic-mass spectrometric analysis using GHB-d6 as the internal standard. The method was linear from 5 to 500 mg/L, and coefficients of variation were less than 10%. Twenty-six urine specimens previously analyzed by an existing method were analyzed and yielded GHB concentrations ranging from 0 to 6100 mg/L; the results correlated between the two methods. Compared with existing methods, the method described here is superior because it is specific to GHB and can discriminate between GHB and gamma-butyrolactone.

Gas chromatography-mass spectrometry (GC-MS) analysis of meconium specimens screening positive for cannabinoids by the EMIT 20 Assay showed a low confirmation rate for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (THCCOOH). A study was designed to investigate the possible contribution of other delta 9-tetrahydrocannabinol (THC) metabolites, including glucuronides, to the overall response of the EIA. delta 9-THC-glucuronide was synthesized in order to develop the most efficient procedure for hydrolysis of glucuronides in meconium. Procedures were developed for the extraction and GC-MS analysis of delta 9-THC, 11-OH-delta 9-THC, 8 alpha- and 8 beta-OH-delta 9-THC, 8 beta,11-diOH-delta 9-THC, and THCCOOH, after enzymatic hydrolysis of meconium extracts. It is concluded that enzymatic hydrolysis of meconium extracts is necessary for efficient recovery of delta 9-THC metabolites; delta 9-THC and its 8-OH metabolite(s) are basically absent in meconium specimens; and 11-OH-delta 9-THC and 8 beta,11-diOH-delta 9-THC contribute significantly to the immunoassay response of meconium extracts. Analysis of several meconium specimens that screened positive for cannabinoids but failed to confirm for THCCOOH showed significant amounts of 11-OH-delta 9-THC and 8 beta,11-diOH-delta 9-THC. Therefore, GC-MS confirmation of cannabinoids in meconium should include analysis for these two metabolites in addition to THCCOOH.

A study was conducted to compare the performance of the OnLine and OnTrak immunoassays for benzodiazepines with gas chromatographic-mass spectrometric (GC-MS) analysis in detecting flunitrazepam (FNP) and its metabolites in human urine. Urine was collected over a 72-h period from six individuals (four male and two female) who had taken a single oral dose of either 1 or 4 mg of FNP. The OnTrak assay was run at a 100-ng/mL cutoff of nordiazepam (NDP), and the OnLine assay was run with a standard curve from zero to 200 ng/mL of NDP with and without beta-glucuronidase treatment. Each sample was analyzed by GC-MS using FNP, 7-amino-FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP as standards with beta-glucuronidase treatment. The specimens from the 1-mg dose did not yield a positive result by immunoassay over the 72-h collection period. Specimens from the 4-mg dose did yield positive results in both immunoassays. The time of the first positive result ranged from 4 to 12 h, and the time to the last positive result ranged from 18 to 60 h. Treatment of the samples with beta-glucuronidase increased the OnLine values between 20 and 60%, but it did not appreciably increase the detection time. GC-MS analysis showed no detectable levels of FNP, 3-hydroxy-FNP, desmethyl-FNP, 7-amino-3-hydroxy-FNP, and desmethyl-3-hydroxy-FNP. However, all samples collected past time zero showed detectable levels of 7-amino-FNP (2 ng/mL) with peak concentrations at 12-36 h. The peak levels of 7-amino-FNP by GC-MS paralleled the peak levels of the immunoassay response. The amount of 7-amino-FNP metabolite quantitated by GC-MS, however, accounted for only 15-20% of the total immunoassay crossreactive FNP metabolites.

A quantitative method using size exclusion chromatographic (SEC) analysis was performed to determine the concentration of Aloe vera mucilaginous polysaccharide (AVMP) in 18 selected commercial preparations. Of the 18 preparations studied, 9 contained quantitatable amounts of AVMP at 0.22 to 1.30 mg/mL, and 2 showed trace amounts (

This manuscript reviews data available in the scientific literature relative to drug testing for the five mandated drug classes and circumstances other than abuse of the drug itself that could result in a positive test. For marijuana, passive inhalation, unknowing oral ingestion, and the use of Marinol are discussed. Data are presented on the concentration of delta9-tetrahydrocannabinol (THC) and its precursors, acid-A and acid-B, in illicit marijuana and the extent of extraction of THC in boiled (tea) or cooked products. For cocaine, passive inhalation and passive exposure issues are reviewed. For opiates, poppy seed ingestion and guidelines for exclusion of poppy seeds as a cause for a positive test are discussed. For amphetamines, issues such as the presence of other phenethylamines, l-methamphetamine (Vicks' inhalers), and other prescription drugs are discussed. Although passive inhalation of methamphetamine and phencyclidine is theoretically possible, no data were available on these issues.

GC/MS analysis of biological specimens is believed to be the most forensically accepted method for confirming the presence of abused drugs. 11-Nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (delta 9-THC-COOH) is the major metabolite of delta 9-tetrahydrocannabinol (delta 9-THC) for which testing (including GC/MS) is directed as an indication of marijuana use. The currently available internal standard for delta 9-THC-COOH is d3-delta 9-THC-COOH, which has the deuterium atoms located on the side chain. In addition to the high cost of this compound, it suffers from a limited dynamic range of analysis, especially when the methyl derivative is used. This is because of a contribution to one of the internal standard ions (m/z 316) from a fragmentation of the natural drug which involves loss of the side chain. The new internal standard, d6-11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (d6-delta 9-THC-COOH), avoids these disadvantages. The six deuterium atoms are located on the two methyl groups of Carbon 6 in the dibenzopyran structure. The dynamic range of analysis with the new internal standard was tested between 6.25 to 1,000 ng/mL with a correlation coefficient of 0.998. Analysis of several urine specimens for delta 9-THC metabolite using both d3- and d6-internal standards showed a correlation coefficient of 0.9987.

There is controversy as to whether the mature fruit of Erythroxylum coca var. coca Lam. contains the cocaine alkaloid (benzoylmethylecgonine). In the present study, cocaine was monitored to determine if it was present in embryos and endosperms of mature fruit of E. coca var. coca Lam., and if present, the time required for de novo synthesis in imbibing seed. Seeds from mature fruit of E. coca were dissected to separate the embryos from the endosperms. The separated embryos and endosperms were analysed for cocaine. Subsequently, endosperms and embryos from seed imbibed under a light and dark treatment were separated on days 3, 6, 9, 12 and 15 and analysed for cocaine/Cocaine was present in embryos (0.005% of d. wt) and endosperms (0.001% of d. wt) of mature fruit of E. coca. De novo synthesis of cocaine occurred only in embryos of seed imbibed under light after day 9 of imbibition.

With the advent of drug testing in the workplace and the consequences of a positive drug test, several issues have arisen in defending or explaining a positive result for a given drug of abuse. Marijuana is the illicit drug with the highest percentage of positives in workplace drug testing. Consequently, marijuana was the first such drug for which excuses were provided to explain the positive test result. Passive inhalation was the first defense offered. This prompted several studies to ascertain whether passive inhalation of marijuana smoke could produce a positive drug test (1)(2)(3)(4). The majority of these studies showed that although passive inhalation of marijuana smoke under certain circumstances could produce detectable concentrations of Δ-9-tetrahydrocannabinol (THC) metabolite in urine, those concentrations were not above the cutoffs used under the federal workplace drug testing guidelines (50 μg/L for screening and 15 μg/L for confirmation). One study (5), however, showed that concentrations above these cutoffs could be attained when the exposure conditions were very severe and, hence, unrealistic. Today, passive inhalation is not nearly the issue that it was at the beginning of drug testing in the workplace. The second issue was passive ingestion. Unknowing ingestion of marijuana tea, brownies prepared with marijuana, or the like was the line of defense that followed passive inhalation. Although studies were carried out to investigate this issue (6)(7), there was no question that if someone orally ingested cooked marijuana, urinary excretion of Δ-9-THC …

Different methods, dichromate (iodometric and colorimetric), vanadate (spectrophotometric), critical miscibility temperature, and gas chromatography, were investigated for the determination of ethanol in various products. The results were compared with those from the official specific gravity method (USP and Egyptian Pharmacopoeia). The advantages and disadvantages, as well as the precision, are given for each method.

A high performance liquid chromatographic (HPLC) procedure was developed for the analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine. Hydrolyzed urine samples were cleaned up using Bond-Elut columns. 11-Nor-cannabinol-9-carboxylic acid was used as internal standard. The analysis was carried out using a C8 reversed-phase column with acetonitrile:50 mM phosphoric acid (65:35) as the solvent. Detection was done by ultraviolet detector at 214 nm. The minimum detectable level of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid was 25 ng/mL.

A gas chromatographic/electron capture detection procedure was developed for the analysis of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine. Hydrolyzed urine samples (1 mL or less) were extracted by a simple acid-base partitioning step. The extracted metabolite was derivatized with pentafluorobenzyl bromide in a biphasic system using benzyl tributylammonium hydroxide as a phase transfer catalyst. 11-Nor-cannabinol-9-carboxylic acid was used as internal standard. A linear relationship was observed between the peak height ratio of the metabolite/internal standard and the concentration of the metabolite (r = 0.9996), and between the peak height and concentration of the metabolite (r = 0.9995). Concentration of 5 ng/mL of metabolite in urine resulted in a peak-to-noise ratio of 4:1; however, concentrations down to 1 to 2 ng/mL could be determined. The procedure has many advantages over currently available methods, of which sensitivity and speed are the most important.

The potential for cross-reactivity of an antiserum used to detect 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in a urine sample by a radioimmunoassay procedure has been examined. The degrees of cross-reactivity of 41 cannabinoids and non-cannabinoid phenolic constituents of Cannabis have been determined. In general, only (delta 8- or delta 9-THC type) cannabinoids of the dibenzopyran type structure were found to appreciably cross-react with the antiserum. Cross-reactivity was not observed with any non-cannabinoid constituent.