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.

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.

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.