Quantitation and confirmation of the diazolo- and triazolobenzodiazepines in human urine by gas chromatography/mass spectrometry.

We report a reliable, rapid, sensitive, and quantitative method for the N- and O-trimethylsilyl (TMS) derivatives of the diazolo- and triazolobenzodiazepines after enzymatic incubation (2 h) and liquid-liquid extraction of 0.5-mL urine volumes. Each analyte was identified by gas chromatography/mass spectrometry through the retention time for the total ion current (TIC) and selected ion monitoring (SIM) of four ion currents. Quantitation of the diazolobenzodiazepines was obtained by the ratio of the base peak ion of the TMS analyte to that of the internal standard bromazepam in the concentration range 50-500 ng/mL (1-10 ng injected into the GC/MS). The limit of detection (LOD) at p less than 0.01 was 50 ng/mL for all the diazolobenzodiazepines. The assay was not quite as sensitive for triazolobenzodiazepines (5-20 ng injected in the GC/MS). The extraction efficiency of the assay ranged from 75 to 92% for all the analytes. The coefficient of variation (CV) for the diazolobenzodiazepines ranged from 5.4 to 9.4% for within-day runs and from 11.1 to 13.9% for between-day runs. For the triazolobenzodiazepines the values were 3.8 to 18.9% for a single day and 3.4 to 19.9% between days. The selected ion current ratio for each analyte was determined for a single day and over a one-week period. There was no statistical difference in the ratios during this time. The confirmation of diazolobenzodiazepines in urine by this method was relatively easy after screening by the immunoassay technique. Identification of triazolobenzodiazepines appeared to be more difficult by both the screening technique and the GC/EI/MS analysis.

Confirmation and quantitation of barbiturates in human urine by gas chromatography/mass spectrometry.

A sensitive, reliable, rapid quantitative method was developed for the N,N'-dimethyl derivatives of the 5,5'-disubstituted barbiturates (NNDM-barbiturates) after liquid-liquid extraction of 0.5-mL urine volumes. Each barbiturate was identified by GC/MS through the retention time for the total ion current and selected ion monitoring of four ion currents for each analyte. Quantitation was achieved through the base peak ion ratios for each NNDM-barbiturate/tolylbarbiturate (IS) over the concentration range 20-250 ng/mL (0.4 to 5 ng injected into the GC/MS). The limit of detection for all the barbiturates (p less than 0.01) was 20 ng/mL (0.4 ng total). The extraction efficiency ranged from 75 to 84% for all the barbiturates. The coefficient of variation of the barbiturates for the within-day run was 2.5 to 4.8% and between days was 6.7 to 8.6%. The percentage abundances of the ion current ratios for each NNDM-barbiturate was determined and found to be fully stable over a one-week period. This method is currently in routine use in our laboratory for the GC/MS confirmation of presumably positive barbiturate urine samples.

Rendering the "poppy-seed defense" defenseless: identification of 6-monoacetylmorphine in urine by gas chromatography/mass spectroscopy.

We report a sensitive, rapid, quantitative gas chromatographic/mass spectroscopic method for measuring the 6-monoacetylmorphine (6-MAM) metabolite of heroin in 0.5 mL of human urine. After a simple liquid-liquid extraction and derivatization, the trimethylsilyl derivative of 6-MAM is identified from its retention time (total ion current) and by selected ion monitoring. The limit of detection was 10 micrograms/L, corresponding to 0.2 ng of trimethylsilyl-6-MAM injected into the gas chromatograph/mass spectrometer. The presence of 6-MAM in urine is indicative of heroin. 6-MAM is not present in poppy seeds or in urine after the ingestion of products containing poppy seed.

Active and realistic passive marijuana exposure tested by three immunoassays and GC/MS in urine.

Human urine samples obtained before and after active and passive exposure to marijuana were analyzed by immune kits (Roche, Amersham, and Syva) and gas chromatography/mass spectrometry (GC/MS). Seven of eight subjects were positive for the entire five-day test period with one immune kit. The latter correlated with GC/MS in 98% of the samples. Passive inhalation experiments under conditions likely to reflect realistic exposure resulted consistently in less than 10 ng/mL of cannabinoids. The 10-100-ng/mL cannabinoid concentration range essential for detection of occasional and moderate marijuana users is thus unaffected by realistic passive inhalation.

Confirmation and quantitation of cocaine, benzoylecgonine, ecgonine methyl ester in human urine by GC/MS.

A rapid, sensitive, reliable quantitative GC/MS method using 0.2 mL of urine was developed for the confirmation of cocaine use. After a simple organic solvent extraction and derivatization with pentafluoropropionic anhydride, cocaine, benzoylecgonine, and ecgonine methyl ester were identified by GC/MS through the retention time for the total ion current and selected ion monitoring (SIM) for each analyte. Quantitation was achieved by obtaining the calibration curves for the molecular ion ratios of the analyte/ketamine (IS) over a range of 12.5-250 ng/mL (0.1-2 ng total). The extraction efficiency for these analytes ranged from 70 to 82%. The sensitivity limit of detection for each analyte was 12.5 ng/mL (0.1 ng) at p less than 0.01. Intra- and interday precision for these analytes ranged between 14.7 and 29.5% CV. This method is in routine use in our laboratory for the GC/MS confirmation of enzyme immunoassay cocaine-positive urine samples.

Confirmation of marijuana, cocaine, morphine, codeine, amphetamine, methamphetamine, phencyclidine by GC/MS in urine following immunoassay screening.

Rapid, reliable, sensitive, qualitative, and quantitative methods using small urine volumes (0.2-0.5 mL) were developed primarily for confirmation of marijuana, cocaine, benzoylecgonine, ecgonine methyl ester, morphine, codeine, amphetamine, methamphetamine, and phencyclidine. Using capillary gas chromatography/mass spectrometry (GC/MS) and selected ion monitoring (SIM), mass spectra were obtained for each analyte. Samples were prepared by hydrolysis where applicable, organic solvent extraction, and derivatization where necessary. Confirmation was achieved by comparing abundance of major ions and retention time of the total ion current (TIC) of an analyte with those of the appropriate analytical standard. Quantitation was achieved and calibration curves derived by obtaining the molecular ion ratios of that analyte/internal standard (IS) over a concentration range of 10-300 ng/mL (0.16-4.0 ng total injected into GC/MS). The overall extraction efficiency for these analytes ranged from 53% to 96%. Statistically significant cut-off values (p less than 0.01) were obtained for each analyte. The slope, y-intercept, and coefficient of determination (r2) were calculated for each analyte. All of the GC/MS methods were extensively tested against urine samples determined positive or negative by immunoassay (IA) and are now used in our laboratory.

Quantitation of l-alpha-acetylmethadol and its metabolites in human serum by capillary gas-liquid chromatography and nitrogen detection.

A procedure is described for the simultaneous measurement of l-alpha-acetylmethadol and its two pharmacologically active metabolites: noracetylmethadol and dinoracetylmethadol. In the method an intramolecular conversion reaction of the two metabolites to their amide configuration is utilized. The reaction is performed while the metabolites are still in the serum. Following solvent extraction the samples are analyzed by capillary gas-liquid chromatography coupled with nitrogen detection. Quantitation is achieved by internal standardization. The lower limit of sensitivity is 5 ng/ml in serum. Absolute sensitivity is 0.1 ng for all three compounds. The advantages over other procedures are: speed due to the single extraction step; increased recovery of noracetylmethadol and dinoracetylmethadol due to decreased polarity of the amides; greater stability of the metabolites in the amide configuration; better chromatographic quantitation and separation because detector response for the amides is greater than it is for the original configuration of the metabolites and the area of the chromatographic tracing is free of interfering substances.

Confirmation of EMIT benzodiazepine assay with GLC/NPD.

A simple, rapid, and reliable gas-liquid chromatographic (GLC) confirmation procedure was developed for urine samples found positive by the EMIT-dau benzodiazepine metabolite assay. The procedure involves acid hydrolysis, organic extraction, and identification using GLC-nitrogen-phosphorus detection (NPD). The method was validated in three subjects who took 10 mg diazepam daily for five days. Urinary excretion of the diazepam related substances was monitored quantitatively for 12 days. Considerable differences in diazepam metabolism was observed, despite the small number of subjects used. The data further indicated that both the physical characteristics and the metabolic profile of each individual may determine whether or not their occasional benzodiazepine use would be detected by the EMIT procedure. In some individuals taking daily diazepam, 48 to 72 hours may be required before sufficient metabolites accumulate in the urine to give a positive EMIT reaction.

A rapid, quantitative GLC method for the simultaneous determination of nicotine and cotinine.

Published gas-liquid chromatographic (GLC) methods for the determination of nicotine and cotinine have proved impractical for the analysis of a large number of clinical samples. Significant improvements over other methods have been achieved, being low sample volume (0.5 mL plasma), rapid two-step extraction from plasma, no evaporation step, and good separation. The lower limits of sensitivity for nicotine and cotinine were 1 and 5 ng/mL, respectively. The method was validated by the analysis of plasma samples from cigarette-smoking volunteers. The method described permits the quick, routine determination of nicotine and cotinine in a large number of samples.

Estimation of phencyclidine in leafy mixtures and in vapor phase after smoking.

Five street samples of leafy material coated with phencyclidine (PCP) were analyzed by a gas chromatographic nitrogen detection assay. The samples contained 15.6+1.8% PCP by weight or 32.2+ 13.8 mg PCP per "joint". An aliquot of a joint was smoked with a laboratory apparatus and the vaporized PCP was collected on a filter. Only 22.6+8.0% of the PCP or 6.7+2.1 mg PCP, reached the filter. This amount is in an approximation of the dose of PCP which becomes available to the oral and pulmonary mucosa following the smoking of a single PCP coated joint.

Simultaneous determination of nicotine and cotinine in human plasma by nitrogen detection gas-liquid chromatography.

Human plasma levels of nicotine and its principal metabolite, cotinine, were simultaneously quantitated by gas-liquid chromatography combined with nitrogen selective detection. Nicotine, cotinine, and the added internal standard ketamine are extracted from plasma at basic pH into methylene chloride, back-extracted into acid, and then re-extracted into methylene chloride. Analysis is carried out on a packed glass column of 3% SE-30 while column temperature is programmed from 150 to 200 degrees C. Detector response is linea over the range of 2 to 50 ng/mL nicotine and 50 to 500 ng/mL cotinine. The method was validated on 150 plasma samples obtained from habitual smokers. Mean levels of 19.5 and 219 ng/mL were found for nicotine and cotinine, respectively. Both the mean and the range of the levels were in agreement with previously reported plasma levels for nicotine and cotinine.

Evaluation of the TRI 'dipstick' test for the detection of drugs of abuse in urine.

An evaluation of Technology Resources Inc. (TRI) Amphetamine, Barbiturate, Narcotic (G) and Narcotic (S) "Dipsticks" for drugs of abuse in urine was made. The results obtained by six individuals reading the "Dipstick" papers was compared with the analysis of the same urine samples, by a combination of TLC, EMIT, RIA and GLC. The data obtained with "Dipstick" papers, regardless of the drug tested, were clearly unreliable (high percentage of false negatives, low percentage of true positives) and the assay was unsuitable as a technique for screening urines for drugs of abuse.

Phencyclidine-induced stereotype in rats: effects of methadone, apomorphine, and naloxone.

Phencyclidine (PCP) given to male Wistar rats produced hyperactivity and various stereotypic motor behaviors. Methadone, apomorphine, and naloxone were tested for their effects on PCP-induced stereotypy. Methadone (0.5 mg/kg) had no effect on the hyperactivity produced by PCP, but significantly attenuated PCP-induced stereotypy when given both before and after PCP. Low doses of apomorphine were equally effective as methadone in attenuating PCP-induced stereotypy. However, when naloxone was given after methadone or apomorphine to PCP-treated rats, the full PCP-induced stereotypy was again observed. Naloxone pretreatment on doses up to 20 mg/kg was not effective in antagonizing PCP-induced behavioral effects. Methadone and apomorphine antagonism of PCP-induced stereotypy may be mediated by opiate receptors. The results of this study and observations from human studies collectively suggest the possible effectiveness of opiates in treating PCP-induced and functional psychoses.