Analysis of hydroxylated phenylalkylamine stimulants in urine by GC-APPI-HRMS.

A gas chromatography-atmospheric pressure photoionization-high-resolution mass spectrometry (GC-APPI-HRMS) method was developed for the determination of eight phenylalkylamine stimulants in urine samples. Spiked urine samples were hydrolyzed, processed by solid-phase extraction, and derivatized before analysis. Two derivatization reactions were studied: the formation of trimethylsilyl (TMS) derivatives with N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) and trimethylsilyl/trifluoroacetyl (TMS/TFA) derivatives with MSTFA and N-methyl-bis (trifluoroacetamide) (MBTFA) as derivatization reagents. Gas chromatography of both derivatives was performed with a 100% dimethylsiloxane column and a good separation of all isomeric compounds was achieved. To maximize the signal of the protonated molecule [M+H]+, the APPI most critical parameters were optimized. Three solvents were tested as dopant agents, with acetone yielding the lower in-source collision-induced dissociation (CID) fragmentation. The acquisition was performed in full scan and product ion scan (parallel reaction monitoring, PRM) using a quadrupole-Orbitrap mass analyzer (35,000 FWHM at m/z 200) in positive ion detection mode. At the optimal working conditions, the full scan method was evaluated for the fulfillment of identification requirements in doping analysis. Selectivity, limits of detection, matrix effect, and precision were estimated to validate the method for confirmation purposes and its applicability was tested by the analysis of spiked samples as well as by the analysis of samples obtained after the administration of some of the compounds to healthy volunteers. Results were compared with those obtained by GC-electron ionization-MS, demonstrating that the GC-APPI-HRMS method improved selectivity and sensibility, achieving lower limits of detection and satisfactory reproducibility.

Direct and rapid quantitation of ephedrines in human urine by paper spray ionization/high resolution mass spectrometry.

A rapid and direct paper spray ionization/mass spectrometry (PSI/MS) method was developed for quantitative analysis of ephedrine, pseudoephedrine, norpseudoephedrine, and methylephedrine in human urine. This method involves the use of a triangular filter paper and high-resolution mass spectrometry, where the molecular ions of ephedrines were generated by applying high voltage after loading the spray solvent to the paper which urine sample was pre-loaded. Small amounts (2µL) of urine spiked with an internal standard were directly analyzed for ephedrines. The PSI/MS method was validated for linearity, within- and between-run precision, accuracy, and limit of detection. The results showed good linearity (R(2)≥0.9928) and acceptable precision and accuracy. Furthermore, the accuracy of the method was assessed by analyzing a blind urine sample from World Anti-Doping Agency and comparing the measured concentrations with the nominal concentrations. This test resulted in accuracies ranging from 96.4 to 106.1%, indicating that the PSI/MS method has the potential to be an alternative technique for the fast quantitation of ephedrines in doping control analysis.

Enantioselective separation and determination of adrafinil and modafinil on Chiralcel OJ-H column in rat serum and urine using solid-phase extraction followed by HPLC.

A simple and rapid normal-phase HPLC method for enantiospecific separation of a psychostimulant, adrafinil (ADL), and its metabolite modafinil (MDL) in rat serum and urine was developed. The separation was accomplished on a normal-phase polysaccharide stationary phase Chiralcel OJ-H using n-hexane-ethanol (62:38 v/v) as a mobile phase at a flow rate of 1.0 mL/min. Detection was carried out at 225 nm using a photo diode array (PDA) detector. The elution order of the enantiomers was determined by a polarimeter connected in series with the PDA. ADL and its metabolite were recovered from rat serum and urine by solid phase extraction using Oasis HLB cartridges and the mean recoveries were >or=80%. The enantiomers were eluted within 15 min without any interference from endogenous substances. The calibration curves were linear (r(2) > 0.998) in the concentration range of 1.20-500 microg/mL for ADL and MDL. The assay was specific, accurate, precise and reproducible (intra- and inter-day precisions RSDs <7.2%). ADL in rat serum was stable over three freeze-thaw cycles at ambient temperature for 4 h. The method was successfully applied to pharmacokinetic studies of adrafinil after an oral administration to rats.

Methamphetamine-related burns in the cornbelt.

Methamphetamine (MA) is a highly addictive drug that is easily manufactured from everyday household products and chemicals found at local farm stores. The proliferation of small MA labs has led to a dramatic increase in patients sustaining thermal injury while making and/or using MA. We hypothesized that these patients have larger injuries with longer hospital stays, and larger, nonreimbursed hospital bills compared with burn patients not manufacturing or using MA. In a retrospective case-control study, all burn patients >or=16 years of age admitted to our burn center from January 2002 to December 2005 were stratified into two groups based on urine MA status. Of the 660 burn patients >or=16 years of age admitted during this 4 year period, urine drug screens were obtained at admission on 410 patients (62%); 10% of urine drug screens were MA (+). MA (+) patients have larger burns compared with MA (-) patients (9.3 vs 8.6% body surface area burns), have higher rates of inhalation injuries (20.4 vs 9.3%, P = .015), and more nonthermal trauma (13.0 vs 3.1%, P = .001). When compared with MA (-) patients, MA (+) patients require longer hospital stays (median 9.5 vs 7.0 days, P = .036), accrue greater hospital bills per day (dollars 4292 vs dollars 2797, P = .01), and lack medical insurance (66.7 vs 17.7%, P < .0001). The epidemic of MA use and its manufacture mandates that burn centers monitor patients for MA use and develop and institute protocols to ensure proper care of this increasingly costly population.

Effects of lower-cost incentives on stimulant abstinence in methadone maintenance treatment: a National Drug Abuse Treatment Clinical Trials Network study.

BACKGROUND: Contingency management interventions that provide tangible incentives based on objective indicators of drug abstinence have improved treatment outcomes of substance abusers, but have not been widely implemented in community drug abuse treatment settings. OBJECTIVE: To compare outcomes achieved when a lower-cost prize-based contingency management treatment is added to usual care in community methadone hydrochloride maintenance treatment settings. DESIGN: Random assignment to usual care with (n = 198) or without (n = 190) abstinence incentives during a 12-week trial. SETTING: Six community-based methadone maintenance drug abuse treatment clinics in locations across the United States. PARTICIPANTS: Three hundred eighty-eight stimulant-abusing patients enrolled in methadone maintenance programs for at least 1 month and no more than 3 years. INTERVENTION: Participants submitting stimulant- and alcohol-negative samples earned draws for a chance to win prizes; the number of draws earned increased with continuous abstinence time. MAIN OUTCOME MEASURES: Total number of stimulant- and alcohol-negative samples provided, percentage of stimulant- and alcohol-negative samples provided, longest duration of abstinence, retention, and counseling attendance. RESULTS: Submission of stimulant- and alcohol-negative samples was twice as likely for incentive as for usual care group participants (odds ratio, 1.98; 95% confidence interval, 1.42-2.77). Achieving 4 or more, 8 or more, and 12 weeks of continuous abstinence was approximately 3, 9, and 11 times more likely, respectively, for incentive vs usual care participants. Groups did not differ on study retention or counseling attendance. The average cost of prizes was 120 dollars per participant. CONCLUSION: An abstinence incentive approach that paid 120 dollars in prizes per participant effectively increased stimulant abstinence in community-based methadone maintenance treatment clinics.

Simple and rapid determination of amphetamine, methamphetamine, and their methylenedioxy derivatives in urine by automated in-tube solid-phase microextraction coupled with liquid chromatography-electrospray ionization mass spectrometry.

A simple and rapid method for the determination of amphetamine, methamphetamine, and their 3,4-methylenedioxy derivatives in urine samples was developed using automated in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS). In-tube SPME is an extraction technique for organic compounds in aqueous samples in which analytes are extracted from the sample directly into an open tubular capillary by repeated draw/eject cycles of sample solution. LC-MS analyses of stimulants were initially performed by liquid injection onto an LC column to determine spectra. Five stimulants tested in this study gave very simple ESI mass spectra, and strong signals corresponding to [M+H]+ were observed for all stimulants. The stimulants were well separated with a Supelcosil LC-CN column using acetonitrile/50mM ammonium acetate (15:85) as a mobile phase. In order to optimize the extraction of stimulants, several in-tube SPME parameters were examined. The optimum extraction conditions were 15 draw/eject cycles of 35 microL of sample in 50mM Tris-HCI (pH 8.5) at a flow rate of 100 microL/min using an Omegawax 250 capillary column. The stimulants extracted by the capillary were easily desorbed by mobile phase flow, and carryover of stimulants was not observed. Using in-tube SPME-LC-ESI-MS with selected ion monitoring, the calibration curves of stimulants were linear in the range from 2 to 100 ng/mL with correlation coefficients above 0.9985 (n = 18) and detection limits (S/N = 3) of 0.38-0.82 ng/mL. This method was successfully applied to the analysis of human urine samples without interference peaks. The recoveries of stimulants spiked into urine samples were above 81%.

Robotic solid-phase extraction of amphetamines from urine for analysis by gas chromatography-mass spectrometry.

We have evaluated the use of the Hamilton Microlab 2200 robotic pipetting system modified to conduct solid-phase extractions of amphetamines from urine. The Hamilton system is a programmable XYZ robotic sample handling instrument compatible with commercial solid-phase extraction (SPE) columns in the most commonly available sizes. During the extraction and elution steps, the system delivers programmable positive pressure with pressure controlled feedback so as to ensure consistent recovery. The system increases sample throughput while reducing technician hands-on time and improving sample-to-sample and batch-to-batch consistency. In comparison with the manual SPE method, the automated scheme provides similar analyte recovery, accuracy, and precision and a reduced potential for laboratory errors. The method's upper limits of linearity, detection, and lquantitation were, respectively, 10,000, 100, and 100 ng/mL for amphetamine and 25,000, 50, and 50 ng/mL for methamphetamine. Extraction recoveries for the compounds ranged from 88 to 101%. Carryover amounted to less than 0.02% even at 50,000 ng/mL concentrations of analyte. A typical automated run required 20 min of technician time versus 90 min for a corresponding manual SPE procedure. The automated procedure proved to be a reliable and labor-efficient addition to the laboratory.

Determination of urinary metabolites of caffeine for the assessment of cytochrome P4501A2, xanthine oxidase, and N-acetyltransferase activity in humans.

Caffeine metabolism via the 3-demethylation pathway is sequentially catalyzed by cytochrome P4501A2 (CYP1A2), xanthine oxidase, and N-acetyltransferase. The activities of the three enzymes can be estimated from urinary metabolic ratios of four caffeine metabolites, 5-acetylamino-6-formylamino-3-methyluracil (AFMU), 1-methyluric acid (1MU), 1-methylxanthine (1MX), and 1,7-dimethyluric acid (17DMU), after the ingestion of caffeine. A method for quantitation of the four metabolites in human urine has been developed. The method is based on a one-step extraction with ethyl acetate/2-propanol followed by high-performance liquid chromatography with UV detection. The detection limit was 1 microM for AFMU, 1MU, and 1MX and 2 microM for 17DMU. The intraday and interday coefficients of variation were < 3% and < 7%, respectively, and the accuracy was within +/- 3%. The method was employed in a population study of 277 healthy volunteers, each of whom ingested 200 mg caffeine and provided a urine sample approximately 6 h later. The metabolite concentration ranges in the urines were 2.1-327 microM, 4.0-744 microM, 4.9-598 microM, and 6.4-260 microM for AFMU, 1MU, 1MX, and 17DMU, respectively. The CYP1A2 ratio (AFMU + 1MU + 1MX/17DMU) was significantly lower in women than in men, excluding smokers and oral contraceptive users. The CYP1A2 ratio was higher in smokers than in nonsmokers, confirming the induction of CYP1A2 by smoking. In women using oral contraceptives, the CYP1A2 ratio was, as expected, significantly lower than in women not using oral contraceptives. For the N-acetyltransferase ratio (AFMU/1MX) and the xanthine oxidase ratio (1MU/1MX), no differences were seen in terms of sex, smoking habits, or the use of oral contraceptives. All results are in agreement with previous reports on CYP1A2, N-acetyltransferase, and xanthine oxidase activities in humans. Thus, the method is both analytically and biologically reliable for the assessment of CYP1A2, N-acetyltransferase, and xanthine oxidase in humans.