Replacing immunoassays for mephedrone, ketamines and six amphetamine-type stimulants with flow injection analysis tandem mass spectrometry.

A screening procedure was developed for the simultaneous detection of mephedrone, six amphetamine-type stimulants (ATS), ketamine and its two metabolites with electrospray ionization flow injection analysis tandem mass spectrometry (FIA-MS-MS). Urine samples were fortified with deuterated analogues as internal standards, extracted with ethyl acetate and analyzed with FIA-MS-MS. The mass analyzer was operated in multiple reaction monitoring mode. Two product ions were monitored for each drug and internal standards. For each analyte, the limit of detection was less than 4 µg/L, within-day and between-day precisions (percent coefficient of variation) at three different concentrations were less than 7.3% and bias was between -17.3 and 11.8%. Total analysis time with FIA-MS-MS is 1.8 min per sample. A group of 215 urine samples were screened with immunoassay for ATS and analyzed with FIA-MS-MS and gas chromatography-mass spectrometry (GC-MS) for ketamines and ATS. The analysis of ATS by immunoassay and GC-MS was 96.7% concordant. The analysis of three ketamines and seven ATS by FIA-MS-MS and GC-MS was 97.2% concordant. The FIA-MS-MS procedure is efficient, accurate, flexible and capable of detecting analytes of different chemical groups. It can replace immunoassays for the screening of new designer drugs when commercial immunoassays are unavailable.

Detection of p-chloroamphetamine in urine samples with mass spectrometry.

Designer drugs are introduced periodically to avoid detection and to provide new drugs with different pharmacological activities. During our routine analysis of amphetamine in urine samples, we observed one sample that reacted with immunoassay with high activity. There is one prominent peak in the gas chromatography- mass spectrometry (GC-MS) chromatogram. However, no amphetamine, methamphetamine, MDA, MDMA, MDEA, or ephedrine was detected with GC-MS. Careful examination of the mass spectrum indicated the presence of one fragment ion (m/z 140), which is similar to the base peak of trifluoroacetic anhydride derivative of amphetamine. The characteristic ion cluster representing the presence of one chlorine atom was observed. Investigation with liquid chromatography (LC)-MS detected an unknown compound with molecular ion of m/z 170. This compound was tentatively identified as chloroamphetamine. Pure standard material of p-chloroamphetamine (PCA) was purchased and analyzed with both GC-MS and LC-MS. Identical GC-MS spectra and LC-MS-MS fragmentation patterns were obtained. A GC-MS procedure was developed for the quantitation of PCA. The limits of detection and quantification were 10 µg/L. Precision was between 1.26% and 4.26%, and bias was between -0.91% and 4.27%. The prevalence PCA positive rate is 0.35% of the samples screened positive for amphetamine.

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.

Enantiomeric quantification of (S)-(+)-methamphetamine in urine by an immunoaffinity column and liquid chromatography-electrospray-mass spectrometry.

A method using an immunoaffinity column (IAC) and liquid chromatography-electrospray ionization mass spectrometry (LC/MS) for on-line detecting the presence of MA in the effluent was developed for the quantitative and enantiomeric determination of (S)-(+)-methamphetamine (d-MA) in urine. The IAC was made in our laboratory and utilized in the LC/MS to simultaneously extract and separate enantiomers of MA from urine samples. An aqueous ammonium acetate buffer was used as the mobile phase. Urine samples were spiked with racemic deuterated methamphetamine (MA-d14) as internal standard (IS), filtered through a membrane, and injected into the LC/MS without any further pre-treatment. Protonated molecular ion of MA and MA-d(14) (m/z 150 and 164) were isolated and further fragmented, the respective product ions, m/z 119 and 130, were collected for quantitative determination. This is an improvement of our previous method (A.C. Lua, Tsong-Yung Chou, J. Chromatogr. A 967 (2002) 191). In the previous method, MA was separated with HPLC, the efflux was fractionated and each fraction was either determined with an immunoassay or GC/MS. Monitoring of MA in the efflux is tedious and time consuming. Urine samples spiked with different concentrations of d-MA were measured by this method. A linear relationship exists in the 150-1050 ng/mL range, and the detection limit (defined as signal-to-noise ratio 3) of d-MA was determined to be 18 ng/mL. The linearity of the method for d-MA can be described by the equation (Y=1.415 x 10(-3)X+0.034, correlation coefficient: r2=0.999). Within run, accuracy and precision (n=6, relative error: -7.2 to +4.0% and relative standard deviation: 3.8-9.3%) of the method are fairly good.

A rapid and sensitive ESI-MS screening procedure for ketamine and norketamine in urine samples.

Traditionally, ketamine was analyzed with gas chromatography (GC) equipped with nitrogen-phosphorus detection, flame-ionization detection, and mass selective detection (MSD) or with liquid chromatography-mass spectrometry (LC-MS). These procedures are sensitive but tedious and slow. There is no commercial immunoassay for ketamine. We have developed a simple and rapid electrospray ionization MS (ESI-MS) procedure to screen ketamine and norketamine (NK) in urine samples. Samples were spiked with ketamine-d(4) (K-d(4)) as internal standard and extracted with 0.2 mL of hexane. An experienced technician can prepare a batch of 60 samples in 1 h. An Agilent LC-MSD trap system with autosampler was employed to inject 10-microL extracted samples directly for mass analysis without chromatographic separation. Total analysis time was 1.3 min per sample. The ESI-MS was operated in scan mode. The ion pairs (m/z 238/242 for K/K-d(4) and m/z 224/242 for NK/K-d(4)) extracted from the full scan mass spectrum were used for quantification. Because of the nature of the ion trap mass detector employed, the presence of other compounds at high concentration could cause the suppression of target analyte ion intensity determined. Limits of detection were 3 ng/mL for ketamine and 15 ng/mL for NK. Carryover was 0.28% for ketamine and 0.39% for norketamine. Within-run precision (%CV) for K and NK at 3 different concentrations (80, 200, and 600 ng/mL) was 4.0% to 14.7%. A group of 168 urine samples collected from disco-dancing club participants were screened with ESI-MS and confirmed with GC-MS. The sensitivity was 97.1% and specificity was 85.7%. These results indicated that the ESI-MS screening procedure is rapid, sensitive, accurate, and reliable.

Detection of acid-labile conjugates of ketamine and its metabolites in urine samples collected from pub participants.

The rapid increase of ketamine (K) abuse worldwide has created a need for a sensitive and reliable detection procedure. Ketamine and its major metabolite, norketamine (NK), are usually determined with gas chromatography-mass spectrometry (GC-MS). Phase II metabolism of K has not been fully investigated. In this report, we studied the phase II biotransformation of ketamine. Urine samples were hydrolyzed with concentrated HCl and alkalinized and extracted with organic solvent. GC-MS (electron impact mode) was employed to determine K, NK, and dehydronorketamine (DHNK). Acidic hydrolysis of urine samples resulted in the detection of a significant increase of K, NK, and DHNK. This indicated the presence of acid-labile conjugates of K, NK, and DHNK in positive urine samples. Because we were unable to obtain DHNK reference materials, the determined value of DHNK was only presumptive. The limit of detection of the procedure was 1 ng/mL for K and 5 ng/mL for NK. The limit of quantitation was 5 ng/mL for K and 10 ng/mL for NK. The range of linearity was 5 micro g/mL for K and NK. The within-run precisions (%CV) for K at concentrations of 40, 120, and 360 ng/mL were 1.54%, 3.41%, and 2.08%, respectively. The within-run precisions for NK were 2.09%, 1.08%, and 1.16%, respectively. Between-day precisions for K were 5.04%, 1.99%, and 5.31%, respectively. Between-day precisions for NK were 3.93%, 2.37%, and 4.51%, respectively. The accuracy of the controls was between 93.7% and 102.5% of the target values. The effect of acidic hydrolysis was determined with a group of 50 samples. The median concentration ratios of hydrolyzed to unhydrolyzed K, NK, and DHNK were 1.15, 1.35, and 1.44, respectively.

Preparation of immunoaffinity columns for direct enantiomeric separation of amphetamine and/or methamphetamine.

Immunoaffinity chromatographic columns were prepared for direct enantiomeric determination of racemic methamphetamine and amphetamine in this study. The stationary phase was synthesized by covalently bonding an anti-D-methamphetamine monoclonal antibody onto a pre-activated support (e.g. silica, sepharose 4B). Chromatographic results revealed that the immunoaffinity columns achieved enantiomeric separation of racemic amphetamine and methamphetamine. The immunoaffinity columns also have the ability to directly extract D-methamphetamine from urine by changing the pH of the mobile phase, this ability making it practical for the columns to determine a very low concentration of D-methamphetamine in urine.

Detection of serologic responses to GB virus C/hepatitis G virus infection.

OBJECTIVES: To investigate the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) and compare the serologic responses to various GBV-C/HGV markers in eastern Taiwan aborigines. METHODS: We used RT-PCR and anti-HGenv u-plate to investigate the prevalence of GBV-C/HGV in eastern Taiwan aborigines. We also used ELISA, dot blot assay, and Western blot to detect the serologic responses to various GBV-C/HGV markers. RESULTS: The prevalence of GBV-C/HGV RNA in the general population of eastern Taiwan aborigines is about 5% (17/317), while 14% (43/317) have anti-E2 antibodies. There were no significant differences in antibody titer against one consensus core peptide (PPSSAAACSRGSPR) between GBV-C/HGV RNA-positive and -negative sera. Only 23 of 42 serum samples positive in the anti-HGenv u-plate EIA assay were positive (55%) in the dot blot assay. No positive signal was detected by Western blot using either recombinant NS3 or commercial E2 proteins. CONCLUSIONS: Antibodies against one consensus core peptide (PPSSAAACSRGSPR) may not constitute a good marker for the detection of GBV-C/HGV viremia. For the detection of anti-E2 antibodies, the anti-HGenv u-plate assay is more sensitive than the dot blot assay. Western blot assay is not a sensitive method for detecting GBV-C/HGV infection.