Jhg Jonkman

CYP2D6 and CYP2C19 activity in a large population of Dutch healthy volunteers: indications for oral contraceptive-related gender differences.

AbstractObjective: We examined a large database containing results on CYP2D6 and CYP2C19 activity of 4301 Dutch volunteers phenotyped in the context of various clinical pharmacology studies. Methods: The subjects were given 22 mg dextromethorphan, 100 mg mephenytoin and 200 mg caffeine. For CYP2D6, the dextromethorphan/dextrorphan metabolic ratios in urine samples taken for a subsequent 8 h were used. Dextromethorphan and dextrorphan were quantified by reversed-phase high performance liquid chromatography. For CYP2C19 similarly obtained (R)-mephenytoin and (S)-mephenytoin ratios were used. (S)-mephenytoin and (R)-mephenytoin were analysed and quantified by enantioselective capillary gas chromatography. In addition, CYP2C19 poor metabolizer (PM) subjects were reanalysed after acidic pre-treatment of urine samples to confirm the PM status. Results: The investigated population mainly comprised Caucasian (98.9%) males (68%). The age ranged from 18 to 82 years. For CYP2D6, it was found that 8.0% of the subjects were PMs. The average metabolic ratio was 0.014 (0.033) for subjects who showed extensive metabolizing activity (EM) and 5.4 (7.6) for PM subjects. For CYP2C19, it was found that 1.8% of the subjects were PMs. The metabolic ratio was 0.162 (0.124) for EM subjects and 1.076 (0.040) for PM subjects. Within the EM group the metabolic ratio in females was significantly lower for CYP2D6 (−20%) and significantly higher for CYP2C19 (+40%) compared with males. For PMs there was no such difference for CYP2D6 (P = 0.79) or CYP2C19 (P = 0.20). Oral contraceptive (OC) use significantly decreased the CYP2C19 activity by 68% for mephenytoin as compared to non-OC using females. Conclusions: For CYP2D6, the PM incidence (8.0%) is in accordance with literature data. The CYP2C19, PM incidence (1.8%) is low compared to reports from other European countries. For mephenytoin, the acidification procedure has been shown to be very important for the confirmation of CYP2C19 PMs. In EM females compared to EM males, CYP2D6 activity is increased and CYP2C19 activity is reduced. For CYP2C19 in particular this reduction is substantial and most pronounced in the age range from 18 to 40 years. For CYP2C19, the reduced activity is associated with the use of oral contraceptives.

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.

Chronopharmacokinetics of theophylline after sustained release and intravenous administration to adults

SummaryThe influence of time of drug administration on pharmacokinetics of theophylline was studied both after ingestion of a sustained-release tablet, containing choline theophyllinate (Zy 15061-S. R.; Teovent®; Sabidal®; ZYMA S.A.) and after intravenous infusion of aminophylline to eight healthy volunteers. Both drugs were administered in the morning (10 a.m.) and on a separate occasion in the evening (10 p.m.) after a 12 h period of fasting.After oral administration of a dose of 540 mg theophylline, the drug was steadily absorbed, both during day-time and during night-time. In some subjects absorption was slower in the evening. Maximum theophylline plasma concentrations were reached after 3.3±0.4 h (mean±SD) and 3.9±1.4 h respectively (not significantly differentp>0.05).The maximum plasma concentrations were almost identical after administration in the morning and in the evening (12.6±3.3 mg·l−1 and 13.1±1.4 mg·l−1 respectively).There was also no significant difference (p>0.05) between the areas under the plasma concentration-time curves after oral and intravenous administration, both at day-time and at night-time. This finding indicates complete bioavailability of the sustained release tablets on both occasions.After administration of the tablets in the morning the plasma concentration 12 h post dosing was significantly lower than after administration in the evening: c121 accounted for 6.0±2.0 mg·l−1 after intake at 10 a.m. and for 7.9±2.1 mg·l−1 after ingestion at 10 p.m. (p<0.01).A similar observation was done after intravenous administration of the drug: c12 was 6.6±1.6 mg·l−1 after starting the infusion in the morning and 8.0±1.8 mg·l−1 after infusing the drug in the evening (p<0.01). This phenomenon could be explained by the finding of a significantly prolonged half-life of theophylline during night-time, provided that the plasma concentrations were in the range of 5 to 15 mg·l−1 (which coincides approximately with the therapeutic range of the drug). For day-time elimination the half-life of theophylline was found to be 6.2±0.9 h and for night-time elimination 8.0±2.0 h (p<0.01), which means an increase of 29.6±20.9% during the night. The prolonged half-life of theophylline at night-time might be of therapeutic benefit in preventing bronchus obstruction in the morning.

'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.

DETERMINATION OF CARBOXYLIC-ACIDS IN PICOMOLE RANGE AFTER DERIVATIZATION WITH PENTAFLUOROBENZYL BROMIDE AND ELECTRON-CAPTURE GAS-CHROMATOGRAPHY

Abstract A method is presented for the determination of picomole quantities of carboxylic acids by gas chromatography in combination with electron capture detection. The acids are extracted from aqueous media into dichloromethane by ion-pair extraction with tetrapentylammonium ions, and derivatized as their pentafluorobenzyl esters. These derivatives have good chomatographic properties with minimum detectable amounts of ca . 0.15 pg 250 pg or greater quantities of the acids can be used. Recoveries are ca . 90% with a precision of ca . 6% at the 10-ng level.

Rapid and selective theophylline serum and saliva assay by means of high pressure liquid chromatography

A rapid, selective and sensitive high pressure liquid Chromatographic (HPLC) method for the determination of theophylline in human serum (or plasma) and saliva was developed.When using 0.5 ml of serum, concentrations down to 0.4 mg.l−1 could be accurately measured. Each sample requires only about 15 minutes for the completion of the assay, including sample preparation. The actual chromatography time is about 8 minutes. The theophylline metabolites and other xanthines, as theobromine, caffeine (and its metabolite paraxanthine) are well separated. The sensitivity, precision and accuracy are sufficient for routine monitoring of therapeutic theophylline serum levels in patients (approximately 10 to 20 mg.l−1). Reliability of the method was demonstrated during analysis of about 3000 samples on the same column.

Disposition and clinical pharmacokinetics of microcrystalline theophylline.

SummaryVariation in the systemic disposition of theophylline after ingestion of a new microcrystalline product (Theolair®) has been investigated in 7 hospitalized patients with generalized obstructive lung disease. Disposition (absolute bioavailability) was determined by comparing in the same patients the areas under the serum concentration-time curves after a single oral dose of microcrystalline theophylline and after an intravenous infusion of aminophylline. Oral absorption appeared to be fast. The half-life of absorption was 19±9 min (mean±SD). Maximal serum concentrations reached after 100±30 min were found to be in a rather narrow range: 9.8±2.5 mg · 1−1. The absolute bioavailability of the microcrystalline preparation was high and it showed only small variation: 102.7±10.2% of the dose. Relevant pharmacokinetic parameters (half-life of elimination, volume of distribution and total body clearance) were determined after both routes of administration. Individual dosage regimens required to obtain a therapeutic serum concentration were calculated for each individual patient on the basis of the observed pharmacokinetic parameters.

Optimization of chromatographic selectivity of twelve sulphonamides in reversed-phase high-performance liquid chromatography using mixture designs and multi-criteria decision making

Abstract For the optimization of the reversed-phase high-performance liquid chromatographic separation of twelve sulphonamides using a quaternary mobile phase, quadratic regression models were calculated. The three pseudo-components were buffer-methanol, buffer-acetonitrile and buffer-tetrahydrofuran and had identical solvent strengths. The capacity factors of the sulphonamides were determined at ten mobile phase compositions. The calculated regression models were used to optimize the resolution of the mobile phase and to simulate a chromatogram under optimum mobile phase conditions. The simulated optimum separation showed great similarity with a chromatogram measured under optimum conditions.

DETERMINATION OF LOW CONCENTRATIONS OF QUATERNARY AMMONIUM COMPOUND THIAZINAMIUM METHYLSULFATE IN PLASMA AND URINE

A sensitive and selective method for the quantitative determination of the quaternary ammonium antiacetylcholine‐compound thiazinamium methylsulphate (Multergan) in plasma and urine is described. The procedure is based on ion pair extraction of the compound with iodide as the counter ion. This is followed by gas chromatography using an alkali flame ionization detector. The detection limit is 2 ng ml−1 with a recovery of 88·0 ± 6·2% from plasma, 91·4 ± 4·6% from urine. The described method can also be applied to other quaternary ammonium compounds.

Determination of amoxycillin in plasma by ion pair column extraction and reversed-phase ion pair high-performance liquid chromatography

A quantitative method is described for the determination of amoxycillin in plasma. The method utilizes ion pair extraction of amoxycillin on disposable columns packed with Baker-10 SPE octadecyl, with tetrabutylammonium ion as the counter ion and methanol as the eluent. Separation and quantitation is performed by reversed-phase ion pair high-performance liquid chromatography (Nucleosil C-18) using the same counter ion and a mobile phase of methanol-phosphate buffer (pH 6.0) (31:69 v/v) with detection at 229 nm.