Gas chromatography-mass spectrometry quantitative method was developed to monitor concentrations of methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in plasma and urine of patients. The developed method was simple, accurate and reproducible to quantify methadone and EDDP in plasma and urine samples in the concentration range of 15-1,000 and 50-2,000 ng/mL, respectively. The proposed analytical method was applied to plasma and urine samples obtained from 96 patients undergoing methadone maintenance treatment (MMT) with daily methadone doses of 2-120 mg/day. Urinary methadone excretion was observed to be significantly affected by pH, in which the ratio of methadone to EDDP was two times higher in acidic urine (P = 0.029). The findings of this study further enhance the guidelines for monitoring of methadone treatment among outpatients. Methadone-to-EDDP ratio in urine was found to be consistent at 24 and 4 h, hence suggesting the possibility that outpatients may be monitored with single urine sample in order to check for compliance. This study which provides data on peak concentrations of methadone and EDDP as well as the ratio of both compounds has added to the body of knowledge regarding pharmacokinetic properties of methadone among heroin-dependent patients under MMT.
Study site: University Malaya Medical Centre (UMMC), HKL, University Malaya Centre for Addiction Sciences (UMCAS) and Rehabilitation Centre of Al-Rahman Mosque, Kuala Lumpur, Malaysia
A novel mass spectrometry detection technique based on a multi-period and multi- experiment (MRM-EPI-MRM3) with library matching in a single run for fast and rapid screening and identification of amphetamine type stimulants (ATS) related drugs in whole blood, urine and dried blood stain was developed and validated. The ATS-related drugs analyzed in this study include ephedrine, pseudoephedrine, amphetamine, methamphetamine, MDMA (3,4-Methylenedioxymethamphetamine), MDA (3,4-Methylenedioxyamphetamine), MDEA (3,4-Methylenedioxy-N-ethylamphetamine) and phentermine. The relative standard deviation for inter and intraday was less than 15% while recoveries ranged from 80% to 120% for all three matrices, i.e., whole blood, urine and dried blood stain. All compounds gave library matching percentage of more than 85% based on the purity. This method was proven to be simple and robust, and provide high confident results complemented with library matching confirmation.
An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse.