A one-step synthesis of cadmium selenide quantum dots from a novel single source precursor.

A new approach to the one-step synthesis of cadmium selenide (CdSe) quantum dots is reported using the air stable complex cadmium imino-bis(diisopropylphosphine selenide); the ligand is readily prepared from elemental selenium and the precursor, quantum dots of comparable quality to those prepared by conventional methods are obtained.

Quantitative analysis of capsaicinoids in fresh peppers, oleoresin capsicum and pepper spray products.

Liquid chromatography-mass spectrometry was used to identify and quantify the predominant capsaicinoid analogues in extracts of fresh peppers, in oleoresin capsicum, and pepper sprays. The concentration of capsaicinoids in fresh peppers was variable. Variability was dependent upon the relative pungency of the pepper type and geographical origin of the pepper. Nonivamide was conclusively identified in the extracts of fresh peppers, despite numerous reports that nonivamide was not a natural product. In the oleoresin capsicum samples, the pungency was proportional to the total concentration of capsaicinoids and was related by a factor of approximately 15,000 Scoville Heat Units (SHU)/microg of total capsaicinoids. The principle analogues detected in oleoresin capsicum were capsaicin and dihydrocapsaicin and appeared to be the analogues primarily responsible for the pungency of the sample. The analysis of selected samples of commercially available pepper spray products also demonstrated variability in the capsaicinoid concentrations. Variability was observed among products obtained from different manufacturers as well as from different product lots from the same manufacturer. These data indicate that commercial pepper products are not standardized for capsaicinoid content even though they are classified by SHU. Variability in the capsaicinoid concentrations in oleoresin capsicum-based self-defense weapons could alter potency and ultimately jeopardize the safety and health of users and assailants.

Effects of intrathecal morphine on the ventilatory response to hypoxia.

BACKGROUND: Intrathecal administration of morphine produces intense analgesia, but it depresses respiration, an effect that can be life-threatening. Whether intrathecal morphine affects the ventilatory response to hypoxia, however, is not known. METHODS: We randomly assigned 30 men to receive one of three study treatments in a double-blind fashion: intravenous morphine (0.14 mg per kilogram of body weight) with intrathecal placebo; intrathecal morphine (0.3 mg) with intravenous placebo; or intravenous and intrathecal placebo. The selected doses of intravenous and intrathecal morphine produce similar degrees of analgesia. The ventilatory response to hypercapnia, the subsequent response to acute hypoxia during hypercapnic breathing (targeted end-tidal partial pressures of expired oxygen and carbon dioxide, 45 mm Hg), and the plasma levels of morphine and morphine metabolites were measured at base line (before drug administration) and 1, 2, 4, 6, 8, 10, and 12 hours after drug administration. RESULTS: At base line, the mean (+/-SD) values for the ventilatory response to hypoxia (calculated as the difference between the minute ventilation during the second full minute of hypoxia and the fifth minute of hypercapnic ventilation) were similar in the three groups: 38.3+/-23.2 liters per minute in the placebo group, 33.5+/-16.4 liters per minute in the intravenous-morphine group, and 30.2+/-11.6 liters per minute in the intrathecal-morphine group (P=0.61). The overall ventilatory response to hypoxia (the area under the curve) was significantly lower after either intravenous morphine (20.2+/-10.8 liters per minute) or intrathecal morphine (14.5+/-6.4 liters per minute) than after placebo (36.8+/-19.2 liters per minute) (P=O.003). Twelve hours after treatment, the ventilatory response to hypoxia in the intrathecal-morphine group (19.9+/-8.9 liters per minute), but not in the intravenous-morphine group (30+/-15.8 liters per minute), remained significantly depressed as compared with the response in the placebo group (40.9+/-19.0 liters per minute) (P= 0.02 for intrathecal morphine vs. placebo). Plasma concentrations of morphine and morphine metabolites either were not detectable after intrathecal morphine or were much lower after intrathecal morphine than after intravenous morphine. CONCLUSIONS: Depression of the ventilatory response to hypoxia after the administration of intrathecal morphine is similar in magnitude to, but longer-lasting than, that after the administration of an equianalgesic dose of intravenous morphine.

The effects of collection methods on oral fluid codeine concentrations.

The use of a variety of alternative biological specimens such as oral fluid for the detection and quantitation of drugs has recently been the focus of considerable scientific research and evaluation. A disadvantage of drug testing using alternative specimens is the lack of scientific literature describing the collection and analyses of these specimens and the limited literature about the pharmacokinetics and disposition of drugs in the specimen. Common methods of oral fluid collection are spitting, draining, suction, and collection on various types of absorbent swabs. The effect(s) of collection techniques on the resultant oral fluid drug concentration has not been thoroughly evaluated. Reported is a controlled clinical study (using codeine) that was designed to determine the effects of five collection techniques and devices on oral fluid codeine concentrations. The collection techniques were control (spitting), acidic stimulation, nonacidic stimulation, and use of either the Salivette or the Finger Collector (containing Accu-Sorb) oral fluid collection devices. Preliminary data were collected from two subjects using the Orasure device. The in vitro drug recovery was also evaluated for the Salivette and the Finger Collector devices. With the exception of a single time point, codeine concentrations in specimens collected by the control method (spitting) were consistently higher than concentrations in specimens collected by the other methods. The control collection concentrations averaged 3.6 times higher than concentrations in specimens collected by acidic stimulation and 1.3 to 2.0 higher than concentrations in specimens collected by nonacidic stimulation or collection using either the Salivette or the Finger Collector devices. When calculated using oral fluid codeine concentrations from the clinical study, the elimination rate constant, t(1/2), AUC and the peak oral fluid concentrations demonstrated device differences. The slope of the elimination curve for codeine using the acidic collection method exceeded that of the other four methods. As a result, the t(1/2) for the acidic method was significantly less than that of the control method (1.8 vs. 3.0 h, respectively). Oral contamination contributed to the control method having higher AUC than that calculated using the other methods. There was considerable variation in peak codeine concentrations between devices and between individuals within each collection method. When samples were collected simultaneously with the Salivette and the Finger Collector, the mean codeine concentrations were similar. We were able to recover > or = 500 microL of oral fluid from 81.8% of the clinical samples collected with the Salivette. However, we were able to recover this volume from only 25.5% of the samples collected with the Finger Collector. In addition, the in vitro drug recoveries were lower using the Finger Collector. When oral fluid was collected nearly simultaneously by the control method and by use of the Salivette, mean control codeine concentrations were 2.3 times higher, but the duration of detection was similar for both methods.

Validation of twelve chemical spot tests for the detection of drugs of abuse.

Validation procedures are described for 12 chemical spot tests including cobalt thiocyanate, Dille-Koppanyi, Duquenois-Levine, Mandelin, Marquis, nitric acid, para-dimethylaminobenzaldehyde, ferric chloride, Froehde, Mecke, Zwikker and Simon's (nitroprusside). The validation procedures include specificity and limit of detection. Depending on the specificity of each color test, between 28 to 45 drugs or chemicals were tested in triplicate with each of the 12 chemical spot tests. For each chemical test, the final colors resulting from positive reactions with known amounts of analytes were compared to two reference color charts. For the identification of unknown drugs, reference colors from the Inter-Society Color Council and the National Bureau of Standards (ISCC-NBS) and Munsell charts are included along with a description of each final color. These chemical spot tests were found to be very sensitive with limits of detection typically 1 to 50 microg depending on the test and the analyte.

Correlation of saliva codeine concentrations with plasma concentrations after oral codeine administration.

A clinical study was designed to determine if there was a predictable relationship between saliva and plasma codeine concentrations. Drug-free volunteers (n = 17) were administered a 30-mg dose of liquid codeine phosphate. Plasma and saliva specimens were collected at various times for 24 h after administration. Plasma and saliva were analyzed for codeine and morphine by positive-ion chemical ionization gas chromatography-mass spectrometry. The plasma codeine concentrations peaked between 30 min and 2 h after administration and ranged from 19 to 74 ng/mL with a mean of 46 ng/mL. Despite decontamination procedures, elevated saliva codeine concentrations were detected at the early collection times because of contamination of the oral cavity from the liquid codeine. Codeine concentrations in the 15 min specimens ranged from 690 ng/mL to over 15,000 ng/mL. After the initial 2-h period, the mean codeine saliva concentrations declined at a rate similar to that observed in the plasma, but remained 3 to 4 times greater than the plasma concentrations. During the elimination phase, half-life estimates for codeine in plasma and saliva were found to be equivalent, 2.6 and 2.9 h, respectively. However, the area under the curve (AUC) estimate for codeine in saliva was 13 times greater than the plasma AUC. Contamination of the saliva resulted in elevated saliva/plasma (S/P) concentration ratios for the first 1 to 2 h after drug administration. Consequently, S/P ratios in specimens collected in the first 15 to 30 min ranged from 75 to 2580. However, after the absorption phase, a significant correlation between saliva and plasma concentrations was observed (r = 0.809, p < 0.05) and mean S/P ratios remained constant (mean = 3.7). Although small changes in saliva pH were predicted to produce profound changes in the S/P ratios for codeine, this was not observed in the current study. Therefore, saliva codeine concentrations could be used to estimate plasma concentrations through the use of the S/P ratio once the oral contamination has been eliminated. However, these estimates should be made cautiously. One must ensure that oral contamination is not a factor. Also, as with blood-drug concentrations, considerable intersubject variability was observed.

Determination of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in plasma after intravenous and intrathecal morphine administration using HPLC with electrospray ionization and tandem mass spectrometry.

High-performance liquid chromatography (HPLC) coupled to atmospheric pressure ionization (API) mass spectrometry (MS) has become a useful technique in the direct analysis of low concentrations of conjugated opiate metabolites. Previous methods using HPLC with traditional detection methods do not have the sensitivity to detect low concentrations of most conjugated drug metabolites. Methods using gas chromatography-mass spectrometry (GC-MS) require hydrolysis and derivatization of the sample followed by an indirect quantitation of conjugated metabolites. Recently, several reports have described direct analysis of opiates and their glucuronide conjugates by HPLC and API-MS. These methods report lower limits of detection than GC-MS methods and quantitation in the low nanogram-per-milliliter range for the glucuronide metabolites of morphine. This report describes an HPLC-electrospray-MS-MS method capable of detecting subnanogram concentrations of morphine (MOR) and its 3- and 6-glucuronide metabolites (M3G and M6G, respectively). The assay has a dynamic range of 250-10,000 pg/mL for M3G and M6G and 500-10,000 pg/mL for MOR. Inter- and intra-assay precision and accuracy varied by less than 8% for all analytes at 750-, 2500-, and 7500-pg/mL concentrations. This assay was used for the determination of MOR, M3G, and M6G in human plasma after intravenous (i.v.) and intrathecal (i.t.) administration of MOR and its effects on the ventilatory response to hypoxia. Peak plasma concentrations of MOR and M6G were measured 1 h after i.v. administration of MOR. Peak concentrations of M3G were measured 2 h after i.v. administration of MOR. After i.t. administration of MOR, peak concentrations of M3G were measured 8 h postdose. MOR was not detected in plasma of patients administered MOR i.t.. Subnanogram concentrations of M6G were measured in the plasma of five of nine patients administered MOR i.t..

Quantitation of alprazolam and alpha-hydroxyalprazolam in human plasma using liquid chromatography electrospray ionization MS-MS.

A sensitive and specific electrospray ionization high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed for the quantitative determination of alprazolam (AL) and alpha-hydroxyalprazolam (OH-AL) in plasma. After the addition of deuterium labeled internal standards of AL and OH-AL, plasma samples were buffered to alkaline pH and extracted with toluene/methylene chloride (7:3). Dried extract residues were reconstituted in HPLC mobile phase and injected onto a reversed-phase C18 HPLC column. The analytes were eluted isocratically at a flow rate of 250 microL/min using a solvent composed of methanol and water (60:40) containing 0.1% formic acid. The analyses were performed using selected reaction monitoring. The assay was sensitive to 0.05 ng/mL for both the parent drug and metabolite and linear to 50 ng/mL. The intra-assay percent coefficients of variation (%CV) for AL at 2, 5, and 20 ng/mL were all < or = 5.6. At these concentrations, and all OH-AL intra-assay %CVs were < or = 8.4. The interassay variabilities for AL were 11.8%CV, 8.7%CV, and 8.7%CV at 2.0, 5.0, and 20.0 ng/mL, respectively. The OH-AL interassay variabilities were 9.6%CV, 9.2%CV, and 7.8%CV at the same concentrations, respectively. The assay accuracy was less than or equal to +/- 6.6% for both analytes at the three concentrations. The method was used to quantitate AL and OH-AL in plasma samples collected from 10 subjects who were administered a 1-mg oral dose of AL. The mean AL concentration peaked at 11.5 ng/mL 1 h after the dose and AL was detectable for 48 h. The mean OH-AL concentration peaked at 0.18 ng/mL 4 h after the dose and was undetectable by 36 h. Hydrolysis of the plasma samples had little effect on the detected AL concentrations but increased OH-AL concentrations substantially. Plasma/blood ratios for AL and OH-AL exceeded 1 in the study samples.

Benzonatate overdose associated with seizures and arrhythmias.

BACKGROUND: Benzonatate is an antitussive with a unique chemical structure. It can contain as many as 8 structural analogs. Therefore, laboratory analysis of benzonatate is difficult. We report 2 cases of benzonatate poisoning with seizures and cardiac arrest and an analytical method to identify and quantify benzonatate in human plasma. CASE REPORTS: Case 1: A 12-month-old male presented to the emergency department of a rural hospital following ingestion of an unknown amount of benzonatate. Upon arrival, the child was seizing and in full cardiac arrest. Resuscitative measures were unsuccessful and the child died shortly after arriving at the emergency department. Case 2: A 39-year-old male ingested 36 benzonatate capsules in a suicide attempt. Enroute to the health care facility, the patient experienced a seizure, had a cardiac arrest, and was cardioverted. Upon arrival at the emergency department, the patient was acidotic with a pH of 6.8. Gastric lavage was performed followed by the administration of activated charcoal. Six hours after arrival at the emergency department, the patient was alert, oriented, and hemodynamically stable. The patient was observed for 24 hours and subsequently discharged. Laboratory confirmation of benzonatate in the plasma of the patient was performed using high-pressure liquid chromatography with tandem mass spectrometry (MS/MS). The benzonatate concentration was estimated to be 2.5 micrograms/mL. CONCLUSION: Seizures and cardiac arrest are possible following an acute ingestion. Quantitative analysis of benzonatate is possible using high-pressure liquid chromatography with tandem mass spectrometry. Routine analysis for benzonatate is not common.

A multiple-site laboratory evaluation of three on-site urinalysis drug-testing devices.

Presented are findings from a multisite laboratory evaluation comparing on-site urinalysis drug-test results to results from Syva EMIT immunoassay and gas chromatography-mass spectrometry (GC-MS). Three laboratories participated in the NHTSA-funded project. Specimens were tested for amphetamines, benzodiazepines, cocaine, cannabinoids, and opiates. Each laboratory selected 20 urines that tested positive for a single drug/drug class and 20 that tested negative to challenge the on-site drug-testing devices. Qualitative and quantitative GC-MS confirmations were performed to ensure that all positive samples contained the target drug(s)/metabolite(s) and that all negative samples did not contain the target analytes. EZ-SCREEN, ONTRAK, and TRIAGE on-site test kits were selected for evaluation. On-site false-positive results, in which GC-MS-verified negative urine samples gave positive on-site results, were rare. Two such errors were recorded with both EZ-SCREEN and TRIAGE. Cross-reactivity from samples containing GC-MS-verified high concentrations of alternate drugs was also rare. One cross-reactive error was recorded while testing for cocaine with EZ-SCREEN, a second while testing for benzodiazepines with ONTRAK, and a third while testing for cocaine with ONTRAK. The EZ-SCREEN kit did not appear to adhere to a cutoff concentration as demonstrated by the number of samples that contained low concentrations of the target drugs that tested positive with this device. A significant finding of this study was that comparing on-site test device results with those of EMIT for samples with drug concentrations near the reporting cutoff was very complex. It required a thorough knowledge of the performance of each device, EMIT, and GC-MS. It also required an investigation of each discrepant result-a consideration not taken in many previous evaluations of on-site testing devices. Compared with current federal guidelines for workplace urinalysis testing, more donor samples would screen positive for cannabinoids and cocaine by the on-site devices than by EMIT immunoassay. However, fewer would be reported as positive because most contained GC-MS-determined drug concentrations lower than the federal confirmation and reporting limits.

Laboratory validation study of drug evaluation and classification program: alprazolam, d-amphetamine, codeine, and marijuana.

The Drug Evaluation and Classification (DEC) program is used by police agencies to identify drivers impaired because of drug use and to determine the class(es) of drug causing the impairment. The primary goal of this study was to determine the validity of the DEC evaluation in predicting whether research volunteers were administered alprazolam, d-amphetamine, codeine, or marijuana. A secondary goal was to determine the accuracy of Drug Recognition Examiners (DREs) in detecting if subjects were dosed with these drugs. Community volunteers (n = 48) were administered alprazolam (0, 1, 2 mg), d-amphetamine (0, 12.5, 25 mg), codeine (0, 60, 120 mg), or marijuana (0, 3.58% THC) in a double-blind, randomized, between-subject design. A single drug dose or placebo was administered at each experimental session, and blood samples were obtained before and after dosing. With the exception of marijuana, plasma drug concentration was at or near maximum during the DEC evaluation. The ability of the DEC evaluation to predict the intake of alprazolam, d-amphetamine, codeine, or marijuana was optimal when using 2-7 variables from the evaluation. DREs' decisions of impairment were consistent with the administration of any active drug in 76% of cases, and their drug class decisions were consistent with toxicology in 32% of cases, according to standards of the International Association of Chiefs of Police. These findings suggest that the DEC evaluation can be used to predict accurately acute administration of alprazolam, d-amphetamine, codeine, and marijuana and that predictions of drug use may be improved by focusing on a subset of variables.

A comparison of ONTRAK TESTCUP, abuscreen ONTRAK, abuscreen ONLINE, and GC/MS urinalysis test results.

This study was designed to compare results obtained from two separate on-site drug testing kits (ONTRAK TESTCUP and Abuscreen ONTRAK) with those obtained from laboratory based immunoassay and GC/MS. Abuscreen ONLINE immunoassay was used to select 250 negative samples and 100 presumptive-positive samples each for cocaine/metabolites, opiates and cannabinoids. Presumptive-positive samples were selected if the immunoassay response was > or = 300 ng/mL for cocaine/metabolites (BZE), > or = 300 ng/mL for opiates or > or = 50 ng/mL for cannabinoids (THC-COOH). GC/MS was used to confirm that each selected sample contained > or = 150 ng/mL BZE, > or = 300 ng/mL morphine/codeine or > or = ng/mL THC-COOH. TESTCUP results had a 100% agreement with GC/MS and a > 99% agreement with ONLINE when testing negative samples. The agreement between TESTCUP and ONLINE results for samples containing opiates was 100%. Results of testing samples containing BZE with TESTCUP demonstrated a 98% agreement with both GC/MS and ONLINE. Both discrepant samples contained BZE at concentrations < or = 300 ng/mL. The least agreement between TESTCUP and ONLINE results was found when testing samples containing THC-COOH. The agreement with ONLINE and GC/MS was 92% and all discrepant samples had GC/MS determined THC-COOH concentrations less than 50 ng/mL. A 100% agreement was obtained between expected and recorded TESTCUP results for QC samples fortified to contained BZE, morphine or THC-COOH at concentrations within 120% of the screening cutoffs. ONTRAK had a 100% agreement with both GC/MS and ONLINE when testing negative samples and samples that contained opiates. ONTRAK had a 91% agreement with GC/MS and ONLINE for testing of samples that contained BZE. The least agreement between ONTRAK and ONLINE results was found when testing samples that contained THC-COOH. The agreement was 89%, however, all discrepant samples contained GC/MS concentrations of THC-COOH less that the 50 ng/mL cutoff. With ONTRAK, a 100% agreement was obtained between expected and recorded results QC samples that contained morphine or THC-COOH and a 97.7% agreement was obtained between expected and recorded results on QC samples that contained BZE.

Determination of buprenorphine in human plasma by gas chromatography-positive ion chemical ionization mass spectrometry and liquid chromatography-tandem mass spectrometry.

Buprenorphine is used for the management of pain and has been advocated for the treatment of opioid addiction. Therapeutic doses result in low plasma concentrations of buprenorphine. In order to assess the safety and efficacy of buprenorphine, sensitive analytical methods are needed. Until recently, gas chromatography-positive ion chemical ionization mass spectrometry (GC-PCI-MS) offered the most sensitive method to selectively quantitate buprenorphine. We have developed and validated a sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) method for buprenorphine. The method is described and compared with a GC-PCI-MS method validated in this laboratory. One-milliliter aliquots of plasma are required for the LC-ESI-MS-MS method and 2-mL aliquots for the GC-PCI-MS method. Buprenorphine-d4 is used as internal standard for both methods. Derivatization with pentafluoropropionic acid anhydride is used for the GC-PCI-MS method, in which the derivatized protonated molecular ions after loss of water are monitored at m/z 596 and 600. For LC-ESI-MS-MS, the parent protonated molecule ions are monitored at m/z 468 and 472. A single-step extraction of basic plasma with n-butyl chloride provided recoveries of 70-87%. Although a limit of quantitation (LOQ) of 0.1 ng/mL could be established for LC-ESI-MS-MS, we could only achieve an LOQ of 0.5 ng/mL with the GC-PCI-MS assay. The GC-PCI-MS method has a linear range of 0.5 to 40 ng/mL (mean r2 = 0.998, n = 7). For quality control samples at 1.0, 2.5, and 12.5 ng/mL, the intra- and interassay coefficients of variation (CV) did not exceed 14%, and percent of targets were within 16%. The LC-ESI-MS-MS method had a linear range of 0.1 to 10 ng/mL (mean r2 = 0.999, n = 7). For quality control samples at 0.25, 2.5 and 7.5 ng/mL, the intra- and interassay CVs did not exceed 4%, and percent of targets were within 12%. Stability studies demonstrated buprenorphine was stable for up to 24 h, 125 days, and 55 days when stored at room temperature, 4 degrees C, and -20 degrees C, respectively. The utility of the lower LOQ was demonstrated in 40 plasma samples collected up to 96 h after a sublingual dose of buprenorphine; 10 were quantitatable using GC-PCI-MS and 38 using LC-ESI-MS-MS.

Detection of alprazolam in hair by negative ion chemical ionization mass spectrometry.

A sensitive and specific method for the quantitative determination of alprazolam (AL) in hair has been developed. After the addition of deuterium labeled triazolam as an internal standard, hair samples (20 mg) were digested with 1 N NaOH at 40 degrees C overnight. Calibrators containing known concentrations of AL dried onto drug-free hair were also prepared and digested. After digestion, the solution was cooled, adjusted to pH 9 with 6 N HCl and 1 ml of saturated sodium borate buffer was added. The digested solutions were extracted with toluene:methylene chloride (7:3) and the organic phase was evaporated to dryness. Extract residues were treated with BSTFA and 1% TMCS and analyzed on a Finnigan-MAT mass spectrometer in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was capable of detecting 25 pg/mg of AL and was linear to 250 pg/mg. Intra-assay precision was 11.1% at 25 pg/mg and 5.4% at 150 pg/mg. Inter-assay precision was 11.2% at 25 pg/mg and 5.3% at 150 pg/mg. This method has been used to study the hair incorporation of AL into Long-Evans rats who received 5.0 mg/kg or 7.5 mg/kg i.p. twice a day for 5 days. Preliminary results indicate that the AL concentration in the pigmented and non-pigmented hair on day 14 ranged from 60 to 100 pg/mg.

Detection of stanozolol in hair by negative ion chemical ionization mass spectrometry.

Stanozolol is an anabolic androgenic steroid occasionally abused by athletes. A sensitive, specific, and reproducible method for the quantitative determination of stanozolol in hair has been developed. After the addition of stanozolol-d3 as the internal standard, hair samples (10-25 mg) were digested with 2 mL of 1N NaOH at 65 degrees C for at least 2 h. Digest solutions were then extracted using solid-phase extraction. The eluents were evaporated, a mixture of N-methyl-N-trimethylsilylhepta-fluorobutryamide (MSHFBA) and trimethylsilylimidazole (TSIM) (1000:20, v/v) was added, and the mixture heated at 80 degrees C for 5 minutes. After cooling to room temperature, N-methyl-bisheptafluorobutyramide (MBHFBA) was added and the mixture heated at 80 degrees C for 30 min. The derivatized extracts were analyzed on a Finnigan MATTM 4500 mass spectrometer in the negative chemical ionization mode. Chromatographic separation was achieved with helium carrier gas on a HP-1 capillary column (15 m x 0.2-mm i.d.; 33-microns film thickness). The assay was capable of reliably quantitating 50 pg/mg of stanozolol and was linear to 2500 pg/mg. Intra-assay precision was 13.2% at 50 pg/mg and 6.6% at 2500 pg/mg. Interassay precision was 13.7% at 50 pg/mg and 6.1% at 2500 pg/mg. This method has been applied to the analysis of stanozolol incorporated into rat hair. Male Long-Evans rats were given stanozolol 20 mg/kg intraperitoneally once daily for 3 days. The mean concentrations of stanozolol in the rat hair collected on day 14 were 362.4 +/- 332.4 pg/mg in pigmented hair and 90.0 +/- 46.9 pg/mg in nonpigmented hair. These data demonstrate that stanozolol is incorporated preferentially into pigmented hair.

Laboratory validation study of drug evaluation and classification program: ethanol, cocaine, and marijuana.

The Drug Evaluation and Classification (DEC) program is used by police agencies to determine if individuals are behaviorally impaired because of drug use, and, if impaired, to determine the class of drug(s) causing the impairment. Although widely used, the validity of the DEC evaluation has not been rigorously tested. The primary goal of this study was to determine the validity of the variables of the DEC evaluation in predicting whether research volunteers had been administered ethanol, cocaine, or marijuana; a secondary goal was to determine the accuracy of trained police officers (Drug Recognition Examiner, DRE) in detecting whether subjects had been dosed with ethanol, cocaine, or marijuana. Community volunteers (n = 18) with histories of drug use received ethanol (0, 0.28, 0.52 g/kg), cocaine (4, 48, 96 mg/70 kg), and marijuana (0, 1.75, 3.55% THC) in a double-blind, randomized, within-subjects design. A single drug dose or placebo was administered during each of nine experimental sessions, and blood samples were obtained before and periodically after dosing. With the exception of marijuana, plasma drug concentration was at or near the observed maximum during the DEC evaluation. The ability of the DEC evaluation to predict the intake of ethanol, cocaine, or marijuana was optimal when using 17-28 variables from the evaluation. When DREs concluded impairment was due to drugs other than ethanol, their opinions were consistent with toxicology in 44% of cases. These findings suggest that the DEC evaluation can be used to predict accurately acute administration of ethanol, cocaine, or marijuana, and that predictions of drug use may be improved if DREs focused on a subset of variables.

Determination of alprazolam and alpha-hydroxyalprazolam in human plasma by gas chromatography/negative-ion chemical ionization mass spectrometry.

A sensitive and specific method was developed for the determination of alprazolam and its major metabolite alpha-hydroxyalprazolam in plasma. After the addition of deuterium-labeled internal standards, plasma samples were buffered to pH 9 with 1 ml of saturated sodium borate buffer, extracted with toluene-methylene chloride (7:3) and evaporated to dryness. The residues were treated with N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane and analyzed on a Finnigan-MAT mass spectrometer operated in the negative-ion chemical ionization mode with methane as the reagent gas. Chromatographic separation was achieved on a Restek-200 capillary column using hydrogen as the carrier gas. The assay was linear from 0.25 to 50 ng ml-1 for both compounds. The intra-assay precision for alprazolam was 16.1% at 0.5 ng ml-1 and 4.6% at 50 ng ml-1 and that for alpha-hydroxyalprazolam was 15.8% at 0.5 ng ml-1 and 4.2% at 50 ng ml-1. The method was used to determine alprazolam and alpha-hydroxyalprazolam in human plasma samples collected after a single 2 mg oral does of alprazolam. A peak concentration of 32.9 ng ml-1 of alprazolam was detected at 1 h following the dose.

Lack of long-term changes in cocaine and monoamine concentrations in rat CNS following chronic administration of cocaine.

In previous studies, we reported time-dependent and dose-dependent changes in the rat dopaminergic receptor system following chronic administration of cocaine. The aim of the present investigation was to monitor the concentration of monoamines (using HPLC-ECD) and cocaine (using GC-PCI/MS) in rat CNS following a dose schedule of 5, 10, 15, 20 and 25 mg/kg, i.p., b.i.d. for 21 days. 12 h after the last cocaine injection, cortical and striatal concentrations of monoamines and their metabolites were not significantly different in saline vs cocaine treated animals. In addition, the cocaine concentration in the brain regions examined did not change with the different doses used. Accumulation of a metabolite of cocaine (ecgonine methyl ester) was the only alteration found. These results indicate that alterations in the dopaminergic receptor system following chronic cocaine administration are not due to changes in neurotransmitter concentration or accumulation of cocaine in the brain.

A gas chromatographic-positive ion chemical ionization-mass spectrometric method for the determination of I-alpha-acetylmethadol (LAAM), norLAAM, and dinorLAAM in plasma, urine, and tissue.

l-alpha-Acetylmethadol (LAAM) is approved as a substitute for methadone for the treatment of opiate addiction. Analytical methods are needed to quantitate LAAM and its two psychoactive metabolites, norLAAM and dinorLAAM, to support pharmacokinetic and other studies. We developed a gas chromatographic-positive ion chemical ionization-mass spectrometric method for these analyses. The method uses 0.5 mL urine or 1.0 mL plasma or tissue homogenate, deuterated (d3) isotopomers as internal standards, methanolic denaturation of protein (for plasma and tissue), and extraction of the buffered sample with n-butyl chloride. For tissue homogenates, an acidic back extraction is included. norLAAM and dinorLAAM were derivatized with trifluoroacetic anhydride. Chromatographic separation of LAAM and derivatized norLAAM and dinorLAAM is achieved with a 5% phenyl methylsilicone capillary column. Positive ion chemical ionization detection using methane-ammonia as the reagent gas produces abundant protonated ions (MH+) for LAAM (m/z 354) and LAAM-d3 (m/z 357) and ammonia adduct ions (MNH4+) for the derivatized norLAAM (m/z 453), norLAAM-d3 (m/z 45 6), dinorLAAM (m/z 439), and dinorLAAM-d3 (m/z 442). The linear range of the calibration curves were matrix dependent: 5-300 ng/mL for plasma, 10-1000 ng/mL for urine, and 10-600 ng/g for tissue homogenates. The low calibrator was the validated limit of quantitation for that matrix. The method is precise and accurate with percent coefficients of variation and percent of targets within 13%. The method was applied to the analysis of human urine and plasma samples; rat plasma, liver, and brain samples; and human liver microsomes following incubation with LAAM.