Mephenytoin as a probe for CYP2C19 phenotyping:effect of sample storage, intra-individual reproducibility and occurrence of adverse events.

AIMS: To further evaluate mephenytoin as a probe for CYP2C19 phenotyping. METHODS: Healthy subjects (n = 2638) were phenotyped using the urinary (S)-mephenytoin to (R)-mephenytoin ratio. This method was evaluated for (a) the stability of the S/R-ratio following sample storage, (b) the intraindividual reproducibility of the ratio, and (c) the occurrence of adverse events. RESULTS: After prolonged storage, the S/R-ratio of samples from extensive metabolisers (EM) increased up to 85%. In 1.5% of the cases (1 out 66), this led to incorrect classification of phenotype. In EMs, but not in poor metabolisers (PMs), the S/R-ratio increased after acid treatment. The intraindividual reproducibility of the mephenytoin phenotyping procedure was 28%. No major side-effects were observed and there was no relationship between the incidence of side-effects and the phenotype of the subject. CONCLUSIONS: After prolonged storage the S/R-ratio significantly increased in EMs and, although low, the risk of incorrect classification should not be ignored. Our data support the use of mephenytoin as a safe drug for CYP2C19 phenotyping.

Evaluation of analytical and clinical performance of a dual-probe phenotyping method for CYP2D6 polymorphism and CYP3A4 activity screening.

A bioanalytical method for the determination of dextromethorphan (DEX) and its metabolites dextrorphan (DTX), 3-methoxymorphinan (3MM), and 3-hydroxymorphinan (3HM) in human urine was developed for CYP2D6 phenotyping and CYP3A4 activity measurements in clinical pharmacology studies using dextromethorphan administered in a drinking solution as substrate. The method was evaluated by thorough conventional validation and by a cross-validation of the method with a previously applied method for dextromethorphan and dextrorphan only (CYP2D6 phenotyping). Cross-validation with the former method showed no significant differences in measured concentrations of volunteer samples. This guaranteed the consistency of epidemiologic data in the database collected from two methods. For the CYP2D6 and CYP3A4 evaluations, the clinical parameters are ratios of concentrations. It appeared that severe variance in individual concentrations generally did not influence the variance of ratios significantly, because experimental errors in concentrations of two analytes proved to correlate considerably. For CYP2D6 values around the antimodes, the chance of a misclassification is very small. The chance of classifying an extensive metabolizer as a poor metabolizer or vice versa is negligible. For CYP3A4 activity determinations it was concluded that in general a change in dextromethorphan/3-methoxymorphinan (DEX/3MM) ratios of 10% or more as detected with the current method, is a significant increase or decrease in the activity of CYP3A4. The authors concluded that they had obtained an analytically valid and clinically reliable bioanalytical method for the determination of dextromethorphan and its metabolites dextrorphan, 3-methoxymorphinan, and 3-hydroxymorphinan in human urine for CYP2D6 phenotyping and CYP3A4 activity measurements for clinical pharmacology studies.

'High throughput' solid-phase extraction technology and turbo ionspray LC-MS-MS applied to the determination of haloperidol in human plasma.

A quantitative method for the analysis of haloperidol in human plasma is described. Sample clean-up was performed by means of solid-phase extraction using 3M Empore extraction disk plates in the 96-well format, automated with a Canberra Packard pipetting robot. Separation was performed by reversed phase high performance liquid chromatography with turbo ionspray tandem mass spectrometric detection by monitoring the decay of protonated haloperidol of m/z 376 to its fragment at m/z 165, versus the decay of protonated haloperidol-D4 at m/z 380 to its fragment at m/z 169. The validated concentration range was from 0.100 to 50.0 ng ml(-1), with an inaccuracy and overall imprecision below 10% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. The average sample preparation time for a batch of 96 samples is approximately 50 min. The chromatographic run time is 3 min. A sample throughput of at least 240 samples per day can be achieved.

High-throughput solid-phase extraction for the determination of cimetidine in human plasma.

For the implementation and validation of an automated 'high-throughput' solid-phase extraction (SPE) system, using microtiter solid-phase technology and a pipetting robot, a SPE method previously validated manually for cimetidine in human plasma was adapted. Sample cleanup was performed by means of SPE using Microlute extraction plates in the 96-well format, each well filled with 50 mg of Varian C18 sorbent. Separation was performed by reversed-phase high-performance liquid chromatography (HPLC) with UV detection at 234 nm. The validated calibration range was from 0.100 to 5.00 mg/l, with an inaccuracy and imprecision below 20% at all concentration levels. Validation results on linearity, specificity, precision, accuracy and stability are shown and are found to be adequate. Cross-check analysis of samples from a clinical trial showed that there is a good correlation between results obtained by the automated method and results obtained by the manual method. The average sample preparation time for a technician decreased from approximately 4 min per sample to 0.6 min. A sample throughput of at least 160 samples per day can be achieved, the HPLC analysis time being the rate-limiting step.

Pharmacokinetics of [14C]-labelled Losigamone and enantiomers after oral administration to healthy subjects.

Losigamone ((+/-)-(R*,S*)-5-(2-chlorophenylhydroxymethyl)-4-methoxy-2 (5H)-furanone; AO-33) is a new potential antiepileptic drug undergoing clinical development. In a crossover study, 200 mg [14C]-labelled Losigamone, as well as 100 mg of each of the unlabelled enantiomers, was administered to 5 healthy volunteers as an oral suspension. The objectives of the study were to determine the mode of elimination, the excretion balance, metabolic profile, the in vitro and in vivo binding to plasma proteins and the pharmacokinetics of both enantiomers in plasma. From the plasma concentration-time profiles of [14C]-radioactivity and unchanged Losigamone it can be concluded that the absorption of Losigamone occurs very rapidly and the plasma concentration of the parent compound versus total radioactivity was consistently about 40%. An overall recovery of total radioactivity of about 97% with 85% in urine and 12% in faeces was found. Protein binding was 50%. Losigamone was extensively metabolized, with only traces of unchanged drug found in urine. The predominant metabolic pathways are hydroxylation and conjugation. After administration of the pure enantiomers, significant differences in pharmacokinetics were observed. The mean oral clearance of the (-)-enantiomer was 1863 ml/min and of the (+)-enantiomer was 171 ml/min. There was no chiral inversion after administration of the enantiomers.

Rational experimental design for bioanalytical methods validation. Illustration using an assay method for total captopril in plasma.

Generally, bioanalytical chromographic methods are validated according to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper, a rational, total performance evaluation programme for chromatographic methods is presented. The design was developed in particular for the pre-validation and main validation phases. The entire experimental design can be performed within six analytical runs. The first run (pre-validation phase) is used to assess the validity of the expected concentration-response relationship (lack of fit, goodness of fit), to assess specificity of the method and to assess the stability of processed samples in the autosampler for 30 h (benchtop stability). The latter experiment is performed to justify overnight analyses. Following approval of the method after the pre-validation phase, the next five runs (main validation phase) are performed to evaluate method precision and accuracy, recovery, freezing and thawing stability and over-curve control/dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. Analysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stability and over-curve control/dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/dilution, additional to ANOVA, also a paired comparison is applied. As a consequence, the recommended design combines the performance of as few independent validation experiments as possible with modern statistical methods, resulting in optimum use of information. A demonstration of the entire validation programme is given for an HPLC method for the determination of total captopril in human plasma.

Chemometrics in bioanalytical sample preparation. A fractionated combined mixture and factorial design for the modelling of the recovery of five tricyclic amines from plasma after liquid-liquid extraction prior to high-performance liquid chromatography.

A general systematic approach is described for the chemometric modelling of liquid-liquid extraction data of drugs from biological fluids. Extraction solvents were selected from Snyder's solvent selectivity triangle: methyl tert.-butyl ether, methylene chloride and chloroform. The composition of a mixture of the three extraction solvents was varied and the extraction yield (recovery) of a group of tricyclic amines was measured at all compositions selected. Two process variables, the extraction time and the extraction intensity, were varied simultaneously with the mixture variables to study their influence and their interaction with the mixture composition. The combined mixture and factorial design statistical techniques obtained in this way enabled the recovery to be modelled as a function of both the composition of the extraction liquid and the process variables. The models were assessed with regard to both descriptive and predictive capacities. The results showed that structurally related compounds may demonstrate different partitioning behaviour with regard to both mixture variables and process variables. It was concluded that mixtures of solvents result in higher extraction efficiencies for the amines. A positive effect on the extraction efficiency was demonstrated by the extraction intensity process variable and extraction time. A positive effect on the extraction efficiency was demonstrated by an interaction between extraction intensity and time. Mixture models in which process variables were introduced were recognized as being very suitable for modelling liquid-liquid extraction systems.

Development of a laboratory robotic system for automated bioanalytical methods--I. The determination of theophylline in human plasma: a comparison between the robotized and manual method.

The quality of bioanalytical methods is often determined by the quality of sample preparation. Using a robot for sample treatment may give better results than manual sample preparation, since the robot lacks human behaviour and incidental errors that are part of it. The use of a laboratory robot has the additional advantage of giving each sample the same analytical history, resulting in better reproducibility. An automated method has been developed for the analysis of drugs in plasma using a laboratory robot. Theophylline was used as a probe drug. Sample preparation was automated with a Zymate II robot, followed by separation and quantitation on an HPLC-system. The robotic method showed a good correlation with the manual method, while sample throughput was doubled.

Development of a laboratory robotic system for automated bioanalytical methods--II. A robot computer program for guarding totally automated bioanalytical methods.

The application of fully automated, unattended sample preparation performed by a laboratory robot for the analysis of drugs in biological samples requires the prevention of system failures which may arise in the on-line coupled chromatographic system or in other components of the robotic system. A computer program has been developed which can help to detect such problems. The control program for the robotic sample preparation contains a number of safety measures to intercept robotic or human errors. A routine is implemented, guarding for chromatographic malfunctions and errors in dispensing liquids by the robot. After detection of trouble, sample preparation is interrupted.