Determination of lamotrigine and its metabolites in human plasma by liquid chromatography-mass spectrometry.

A method based on electrospray ionization liquid chromatography-mass spectrometry was developed for the quantitative determination of lamotrigine and three of its reported metabolites, lamotrigine-2-N-glucuronide, lamotrigine-2-N-methyl, and lamotrigine-2-N-oxide in human blood plasma. The method utilized sample preparation by precipitation of proteins with acetonitrile, chromatographic separation on a reversed-phase system by gradient elution, and monitoring of the protonated molecular ions. Two internal standards, 3,5-diamino-6-(2-methoxyphenyl)-1,2,4-triazine and morphine-3-glucuronide-D3, were utilized to achieve precise quantification. The method validation comprised a demonstration of an agreement in the quantification of lamotrigine with that of a routine HPLC-UV method. The limits of detection were between 0.05 and 0.16 micromol/L. The method was employed for the measurement of clinical samples collected from 55 patients in steady-state prior to the dose intake (trough level). Lamotrigine and the 2-N-glucuronide were typically detected, while the N-methyl and N-oxide metabolites were detected only rarely. The median lamotrigine plasma level was 24.0 micromol/L (range, 4.3 to 64 micromol/L), the median 2-N-glucuronide level was 2.4 micromol/L (range, <0.05 to 24 micromol/L), and the median lamotrigine 2-N-glucuronide/lamotrigine ratio was 0.11 (range, <0.01 to 0.64). In conclusion, this liquid chromatographic-mass spectrometric method is suitable for simultaneous determination of lamotrigine and its metabolites in human plasma.

Application of direct urine LC-MS-MS analysis for screening of novel substances in drug abusers.

A newly developed liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was used to study 3000 human urine samples from 3 different populations for 23 analytes covering phenylethylamines, benzylpiperazine, and non-benzodiazepine hypnotics. Direct injection of urine and LC-MS-MS with rapid chromatography and atmospheric pressure chemical ionization was used in the screening step. The cutoff levels were chosen to be at the limit of detection for most analytes to identify as many positive samples as possible. Typically one ion transition was monitored from the pseudo-molecular ions in the multiple reaction monitoring mode. Of the 797 positive screening findings, 518 (65%) were confirmed by a second LC-MS-MS analysis including solid-phase extraction. Confirmed analytical findings included 22 cases positive for N-benzylpiperazine, 88 for 3,4-methylenedioxy-N-methylamphetamine and metabolites, 4 for 1-phenyl-2-butylamine, 24 for zolpidem and metabolites, 118 for zopiclone and metabolites, and 1 for zaleplon. In conclusion, LC-MS-MS was found to be a robust alternative for drugs of abuse screening, offering high sensitivity compared with immunochemical screening methodology.

Multicomponent screening for drugs of abuse: direct analysis of urine by LC-MS-MS.

By allowing for direct injection of urine, liquid chromatography-tandem mass spectrometry (LC-MS-MS) with atmospheric-pressure chemical ionization has proven to be useful for analysis of drugs of abuse in human urine. The purpose of this study was to evaluate this technique by direct screening for 23 different substances (phenylethylamines, hypnotics, and N-benzylpiperazine) in urine samples. It was possible to achieve lower detection limits compared with commercial immunochemical methods. There was a linear response for all analytes with an intraday coefficient of variation of about 16%, and the gradient elution gave a variability in relative retention time of about 1%. Positive results were confirmed by reanalysis including sample preparation by solid-phase extraction. Among the 529 authentic urine samples analyzed, 35 samples were screened positive for phenylethylamines, and 20 for hypnotics. Of these, 23 (66%) samples were confirmed to be positive for phenylethylamines, and 11 (55%) for hypnotics. This study demonstrates that LC-MS-MS is a valuable complement to immunochemical screening analysis, especially for substances for which immunochemical methods are not yet available or when an increased sensitivity is needed.

Direct screening of urine for MDMA and MDA by liquid chromatography-tandem mass spectrometry.

There is a limit in the number of substances detected by commercially available reagents. It is therefore important to have other, complementary techniques. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) may offer one possibility. An LC-MS-MS procedure based on the detection of positive ions after atmospheric pressure chemical ionization (APCI) was developed for direct measurement of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine in urine. It was compared with Online Amphetamines. The LC-MS-MS methodology showed improved sensitivity (1 00-ng/mL cutoff and specificity with a coefficient of variation of 10%. It was compared to the immunochemical analysis using 1000 clinical patient urine samples. The positive samples were confirmed by gas chromatography-mass spectrometry. The LC-MS-MS method detected almost four times as many samples positive for MDMA compared to the immunochemical method, with no false positives and one false negative. Our study suggests that LC-MS-MS offers an alternative to immunochemical methods in drug of abuse screening.

Chromatographic screening for zopiclone and metabolites in urine using liquid chromatography and liquid chromatography-mass spectrometry techniques.

Zopiclone is a benzodiazepine-like hypnotic that was believed not to have any abuse potential. Nevertheless, during the past few years there have been an increasing number of reports on the abuse and misuse of zopiclone. Despite this, methods for screening analysis in urine are lacking. To investigate whether UV detection would be possible to use for this purpose, a liquid chromatography method with ultraviolet detection for analyzing zopiclone and its urinary metabolites was developed, with liquid chromatography-mass spectrometry for confirmation. The method was used for analyzing samples from subjects receiving methadone. The limits of detection were approximately 100 ng/mL in control urine samples and 500 ng/mL in urine samples from subjects receiving methadone. However, due to the high background in these patients' urine, a single therapeutic dose was impossible to detect.