Simultaneous determination of ketamine, tramadol, methadone, and their metabolites in urine by gas chromatography-mass spectrometry.

An automated solid-phase extraction procedure combined with gas chromatography-mass spectrometry methodology, without derivatization, has been developed for the identification and quantitation of ketamine, norketamine, tramadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and 2-ethyl-5-methyl-3,3-diphenylpyrroline in urine. The analytical approach is simple and rapid, yet reliable. Good linearity (r(2) > 0.995 over the concentration range of 30 to 600 ng/mL), sensitivity (limits of quantitation 15-30 ng/mL), accuracy (81.0-109.9%), precision (RSD < 13.8%), and recovery (> 79.6% in average) were achieved for all analytes. Ninety-one urine specimens from suspected drug users and 21 clinical urine specimens from methadone substitution therapy patients were analyzed to validate the method compatibility and stability. Results have demonstrated that this GC-MS method is a good confirmation and quantitation test scheme for the six target compounds in urine.

GC-MS quantification of ketamine, norketamine, and dehydronorketamine in urine specimens and comparative study using ELISA as the preliminary test methodology.

An automated solid-phase extraction procedure combined with the gas chromatography-mass spectrometry (GC-MS) methodology, without derivatization, has been developed for the determination of ketamine (K), norketamine (NK), and dehydronorketamine (DHNK) in urine. The analytical approach is simple and rapid, yet reliable, achieving good linearity (r(2)>0.999 over the concentration range of 30 to 1000 ng/mL), sensitivity (limits of quantification = 15, 10, and 20 ng/mL for K, NK, and DHNK, respectively), accuracy (90-104%), and precision (RSD<8.1%) for all analytes. Two hundred and six urine specimens collected from suspected drug users were analyzed by this protocol and also screened by Neogen ELISA method to evaluate the efficiency as well as the compatibility of these two methods. Neogen ELISA showed high efficiency (98.1%), high sensitivity (90.9%), high specificity (98.9%), low false-positive rate (1.1%), and moderate false-negative rate (9.1%), adopting 10 ng/mL K as the cutoff. Neogen ELISA screening followed by GC-MS analysis appeared to be a good screening-confirmation test scheme for the analysis of K in urine. Twenty of the 22 positive urine specimens contained all three analytes simultaneously, with DHNK showing the highest and K the lowest concentrations.

Characteristics and trends of 3,4-methylenedioxymethamphetamine (MDMA) tablets found in Taiwan from 2002 to February 2005.

One hundred and eighty-one 3,4-methylenedioxymethamphetamine (MDMA) containing tablets were sampled from confiscated drugs received by the Taiwan National Bureau of Controlled Drugs for testing from 2002 to February 2005. Sample tablets demonstrated various colors and logos. The appearances, contents of MDMA and other components in these tablets were analyzed in order to understand the characteristics and trends of MDMA use. Samples were analyzed using GC-MS methodology. Deuterated internal standards were used for drug quantification. The MDMA contents varied from 16 to 193 mg/tablet. 66-71% of the tablets seized each year contained only MDMA, and the content of MDMA in MDMA only tablets varied from 89 to 133 mg/tablet. There was a decreasing trend in MDMA content in these tablets over time. Other components commonly found besides MDMA included caffeine (18%), methamphetamine (7%), 3,4-methylenedioxyethylamphetamine (MDEA) (7%) and amphetamine (4%). 3,4-Methylenedioxyamphetamine (MDA), ketamine, ephedrine, diazepam, chlorzoxazone and nicotinamide were also detected. During the study period, the number of other drugs found as well as the combinations of different drugs detected in these tablets increased.

Performance characteristics of DRI, CEDIA, and REMEDi systems for preliminary tests of amphetamines and opiates in human urine.

Arrestee urine specimens (930) were tested with DRI, CEDIA, and REMEDi; those that tested positive for amphetamines and opiates (616 and 414, respectively) were then confirmed by gas chromatography-mass spectrometry. The performance characteristics of these three preliminary systems were evaluated using the following commonly used parameters: true positive, true negative, false positive, and false negative. The sensitivity, specificity, and efficiency of these methods were also calculated. Data derived from this study indicated DRI and CEDIA adapted by this study generated acceptable preliminary test results for amphetamine/methamphetamine and morphine/codeine, but not for MDA/MDMA and REMEDi has lower sensitivity than DRI and CEDIA, but with better specificity and efficiency, supporting its use under emergency room settings where drug concentrations in overdose cases are expectedly at high levels.

Quantitative detection of ketamine, norketamine, and dehydronorketamine in urine using chemical derivatization followed by gas chromatography-mass spectrometry.

A repeatable and highly sensitive analytical method using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM) is developed for the simultaneous detection of ketamine (KT), norketamine (NK), and newly introduced dehydronorketamine (DHNK) in urine. The test specimen along with the deuterium analogues as internal standards (IS): d4-KT for KT and d4-NK for NK/DHNK, was extracted on an automatic solid-phase extraction (SPE) apparatus. The extracted eluate then was dried and derivatized with N-methyl-bis(trifluoroacetamide) (CF3CONCH3COCF3, MBTFA). Finally, the cooled derivatized solution was directly injected into the GC-MS system for analysis. The proposed process achieves high sensitivity for the detection of KT, NK, and DHNK. Correlation coefficients derived from typical calibration curves in the range of 20-2000 ng/mL are 1.000 for KT and NK, 0.999 for DHNK. The limits of detection (LODs) and limits of quantitation (LOQs) are 0.5-1.0 and 1.5-3.0, respectively. The overall method recoveries of KT, NK, and DHNK are 82.2-93.4. The intra- and inter-day run deviations are smaller than 5.0%. The analytical scheme was also applied to the determination of KT, NK, and DHNK in 20 KT suspected urine specimens, and the results reconfirm that DHNK is a main metabolite of KT.

Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens.

Eight commercially available immunoassays for amphetamines (DRI Amphetamines, CEDIA DAU Amphetamines-Semiquantitative, EMIT d.a.u. Monoclonal Amphetamine/Methamphetamine, Synchron CX Systems AMPH, TDx/TDxFLx Amphetamine/Methamphetamine II, CEDIA Amphetamines/Ecstasy, COBAS INTEGRA Amphetamines, and Abuscreen((R)) OnLine HS Amphetamine/MDMA) are evaluated for their effectiveness in serving as the preliminary test methodology for the analysis of 3,4-methylenedioxymethamphetamine/3,4-methylenedioxyamphetamine (MDMA/MDA) and methamphetamine/amphetamine (MA/AM). Standard solutions (in urine matrix) of MDMA, MDA, MA, and AM are used to determine these immunoassays' reactivities (or cross-reactivities) toward these compounds of interest. Case specimens containing MDMA/MDA and MA/AM are also used to study the correlations of the apparent immunoassay MDMA (or MA) concentrations and the gas chromatographic-mass spectrometric concentrations of these compounds. Data resulting from this study suggest that CEDIA Amphetamines/Ecstasy can best predict the concentrations of MDMA and MA in case specimens and can also detect the presence of MDMA at low levels, whereas Abuscreen OnLine HS Amphetamine/MDMA can detect both MDMA and MA at low concentrations.

Improved screen and confirmation test of 7-aminoflunitrazepam in urine specimens for monitoring flunitrazepam (Rohypnol) exposure.

Confirmed and alleged misuses of flunitrazepam (FM2, Rohypnol) have brought about serious interest in the development of an analytical methodology that can be effectively used for preliminary screen and confirmatory test of FM2 (or its metabolites) in urine specimens under high-volume settings. Reported methods do not serve this need well for the following reasons: (1) common benzodiazepine (BZ) immunoassays (IAs) have broad cross-reactivities toward widely prescribed BZs (and their metabolites) and are therefore likely to generate an unacceptable number of false positives and (2) because FM2 is typically used at low doses (1-4 mg), IAs with low cross-reactivities toward FM2 (and its metabolites) are likely to generate false-negative results. In this current study, a familiar and effective two-step IA/gas chromatography-mass spectrometry (GC-MS) approach is successfully developed and applied to clinical specimens. Cross-reacting characteristics of the following BZ IAs toward various BZs (and their metabolites) are evaluated focusing on their effectiveness in serving as the preliminary test reagent in a two-step testing protocol: TDx, Beckman, CEDIA, Roche Cobas Integra, Emit II Plus, and Cozart ELISA. Although other IAs show broad cross-reactivities toward various BZs and their metabolites, diazepam is the only non-FM2 derived compound that exhibits noticeable cross-reactivity toward Cozart ELISA reagent. Cross-reactivity data and data derived from studies conducted on a limited number of clinical specimens demonstrate that, when used to monitor FM2 exposure in a large population group (including those exposed to other BZs), Cozart ELISA has the potential of being as effective as (or better than) those currently used in various workplace drug-testing programs for monitoring respectively targeted drugs. Data derived from this study further suggest that 50 ng/mL apparent 7-aminoflunitrazepam (Cozart ELISA) and 30 ng/mL free 7-aminoflunitrazepam (GC-MS) are potentially effective preliminary test and confirmation test cut-offs. To maximize efficiency, it is further suggested that urine specimens are first diluted by a factor of 5 for the preliminary test in which a 10-ng/mL 7-aminoflunitrazepam standard is used as the assay's cut-off standard.

Estimation of the measurement uncertainty in quantitative determination of ketamine and norketamine in urine using a one-point calibration method.

An approach was proposed for the estimation of measurement uncertainty for analytical methods based on one-point calibration. The proposed approach is similar to the popular multiple-point calibration approach. However, the standard deviation of calibration was estimated externally. The approach was applied to the estimation of measurement uncertainty for the quantitative determination of ketamine (K) and norketamine (NK) at a 100 ng/mL threshold concentration in urine. In addition to uncertainty due to calibration, sample analysis was the other major source of uncertainty. To include the variation due to matrix effect and temporal effect in sample analysis, different blank urines were spiked with K and NK and analyzed at equal time intervals within and between batches. The expanded uncertainties (k = 2) were estimated to be 10 and 8 ng/mL for K and NK, respectively.

A fast screening procedure for ketamine and metabolites in urine samples with tandem mass spectrometry.

A screening method based on electrospray tandem mass spectrometry (MS-MS) was developed. Urine samples were spiked with ketamine-d(4) and norketamine-d(4) as internal standard and extracted with 0.5 mL ethyl acetate. Extracted samples were monitored with triple-quadrupole MS-MS. Total analysis time was 1.5 min/sample. Limit of detection was 0.1 ng/mL for ketamine, norketamine, and dehydronorketamine (DHNK). Carryover rate was less than 0.06%. Within-run and between-run precision for ketamine, norketamine, and DHNK at three different concentrations (40, 75, and 125 ng/mL) was between 2.1 and 8.2%. Within-run and between-run accuracy, presented as % bias, was between -5.9 and 2.7%. A group of 76 urine samples were screened with ELISA and gas chromatography-mass spectrometry (GC-MS). With GC-MS as the reference method, when ketamine, norketamine, and DHNK were monitored at cutoff concentration of 100 ng/mL, there were 21 positive, 45 negative, 7 false-negative, and 3 false-positive results. A group of 243 samples was screened with MS-MS and analyzed with GC-MS; there were 74 positive, 163 negative, 6 false-positive, and no false-negative results. In conclusion, the MS-MS procedure is accurate, efficient, and suitable for use as a high-throughput screening method for ketamine and metabolites.

Issues pertaining to the analysis of buprenorphine and its metabolites by gas chromatography-mass spectrometry.

"Substitution therapy" and the use of buprenorphine (B) as an agent for treating heroin addiction continue to gain acceptance and have recently been implemented in Taiwan. Mature and widely utilized gas chromatography-mass spectrometry (GC-MS) technology can complement the low cost and highly sensitive immunoassay (IA) approach to facilitate the implementation of analytical tasks supporting compliance monitoring and pharmacokinetic/pharmacogenetic studies. Issues critical to GC-MS analysis of B and norbuprenorphine (NB) (free and as glucuronides), including extraction, hydrolysis, derivatization, and quantitation approaches were studied, followed by comparing the resulting data against those derived from IA and two types of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Commercial solid-phase extraction devices, highly effective for recovering all metabolites, may not be suitable for the analysis of free B and NB; acetyl-derivatization products exhibit the most favorable chromatographic, ion intensity, and cross-contribution characteristics for GC-MS analysis. Evaluation of IA, GC-MS, and LC-MS/MS data obtained in three laboratories has proven the 2-aliquot GC-MS protocol effective for the determination of free B and NB and their glucuronides.

Simultaneous determination of morphine, codeine, 6-acetylmorphine, cocaine and benzoylecgonine in hair by liquid chromatography/electrospray ionization tandem mass spectrometry.

A fast and sensitive liquid chromatography/triple quadrupole tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of morphine, codeine, 6-acetylmorphine (6-AM), cocaine and benzoylecgonine (BE) in hair. Pulverized hair samples were extracted with methanol, and a 50 microL supernatant aliquot was injected into the LC/MS/MS system. Chromatography was performed with an XBridge phenyl column (3.5 microm particle size, 4.6 x 150 mm), and the mobile phase was composed of methanol and 10 mM ammonium acetate adjusted to pH 4.00 with 99% formic acid (95:5, v/v). A separation run with isocratic elution was completed in 10 min at a flow rate of 500 microL/min. Positive electrospray ionization and multiple reaction monitoring (MRM) with one precursor ion/product ion transition were used for the identification of each analyte. Deuterated analogues as internal standards were used for quantification and qualification. Linearity was established in the concentration range of 100-3000 pg/mg. The limits of detection were 10 pg/mg for morphine, codeine and 6-AM; and 1 pg/mg for cocaine and BE. The precision and accuracy were determined by spiking hair samples at six concentration levels. For all analytes, the relative standard deviations of intra- and inter-day precision were 0.1-6.3% and 1.5-10.6%, respectively. The accuracy ranged from 92.7 to 109.7%. The validated LC/MS/MS method was successfully applied to the analysis of 79 authentic hair samples.

One step and highly sensitive headspace solid-phase microextraction sample preparation approach for the analysis of methamphetamine and amphetamine in human urine.

A fiber-stable, repeatable and highly sensitive headspace solid-phase microextraction (HS-SPME) method was developed for the analysis of methamphetamine (MA) and amphetamine (AM) in urine using gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode. For sample preparation, the test specimen was placed in a 7 ml vial along with the additives (KOH and NaCl) and the internal standards (d8-MA and d8-AM), a glass insert containing heptafluorobutyric anhydride (HFBA) and heptafluorobutyric chloride (HFBCl) as derivatizing reagents was inserted into the vial, the vial was then sealed tightly. A SPME device with a 100 microm polydimethylsiloxane fiber was inserted into the vial and the fiber was exposed to the headspace in the insert, then the vial was heated and stirred at 100 degrees C and 600 rpm for 20 min for evaporation/adsorption/derivatization. The vaporized analytes (AM and MA) in the vial diffused into the glass insert though the holes on the insert, they absorbed onto the fiber, and then interacted with the vapor of the derivatizing reagent. Some of the analytes in the headspace of the glass insert may react with the vapor of the derivatizing reagent first, and then adsorb onto the fiber. The needle was finally removed and inserted into the injection port to desorb the analytes with the fiber exposed to the liner of the GC-MS system for analysis. By combining HFBCl and HFBA as derivatizing reagents and placing them in an insert, the HS-SPME method achieves high sensitivity for the analysis of AM and MA. Correlation coefficients derived from typical calibration curves in the 1.0-1700 ng ml(-1) range are 0.998 for MA and 0.994 for AM. The limits of detection and the limits of quantitation using a sample size of 1 ml are 0.3 and 1.0 ng ml(-1), respectively, for both MA and AM in urine specimens. Because the water hydrolysis of derivatizing reagent is much faster than the acylation reaction of the primary and secondary amines with the derivatizing reagent, the amphetamines cannot be acylated effectively over heated aqueous solution, and therefore this study provides a new acylation design in moisture surroundings. The proposed process also simplifies the procedure for urine sample preparation, and makes the automation of SPME possible.