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.

An automated analytical method for the determination of felbamate in human plasma by robotic sample preparation and reversed-phase high performance liquid chromatography.

An automated analytical method for the determination of felbamate in human plasma is described. Sample cleanup and preparation was performed by means of a Zymate II laboratory robot and consisted of a liquid-liquid extraction of felbamate and the internal standard, primidone, from human plasma to dichloromethane. The dichloromethane was evaporated and reconstituted in a phosphate buffer. Separation was performed by reversed-phase high performance liquid chromatography using a 5 microns Hypersil ODS column (150 x 4.6 mm) and a mobile phase consisting of a mixture of phosphate buffer (pH = 6.5, 0.015 M) and acetonitrile (79:21, v/v). Quantitation was performed by measurement of the UV absorbance at a wavelength of 210 nm. The lower limit of quantitation was 0.100 micrograms ml-1 using 200 microliters of plasma. The mean absolute analytical recovery of felbamate was 75.2% (n = 28). The recovery of the internal standard, primidone was 74.7% (n = 10). The within-day precision was below 3.8% at all concentration levels, except at the lower limit of quantitation (18.3%). The within-day accuracy varied between -3.7 and +7.4%. The between-day precision was below 5.0% at all concentration levels. The between-day accuracy of the method varied between -5.7 and +1.6%. The selectivity of the method towards several other anti-epileptic drugs has been demonstrated.

A comparative study of the steady-state pharmacokinetics of immediate-release and controlled-release diltiazem tablets.

We have studied the controlled-release properties and relative systemic availabilities of two dosages of the same controlled-release (CR) diltiazem tablet formulation by comparing them at steady state with those of an immediate-release formulation. We measured 24-hour plasma concentration profiles during 4-day treatments with diltiazem 90 mg CR tablet bd diltiazem 120 mg CR tablet bd, and conventional diltiazem 60 mg immediate-release (IR) tablet tid. The study had a randomized, three-way crossover design. Twelve healthy men (38-52 y) participated. Trough plasma concentrations were determined on days 3 and 4. The 24-h plasma concentration-time profiles were assessed after the last morning dose on day 4 of each period. The following steady-state pharmacokinetic values were calculated: the minimum plasma concentration (Cmin), the maximum plasma concentration (Cmax), the time interval during which the plasma concentration exceeded 75% of Cmax (t75), the area under the plasma concentration-time curve (AUC72-96), the peak-to-trough fluctuation (PTF), and the area-under-the-curve fluctuation (AUCF). Steady state was achieved on day 3. The pharmacokinetics were comparable. For diltiazem CR 90 mg and diltiazem CR 120 mg, AUC84-96 (night) was approximately 75% of AUC72-84 (daytime). The diltiazem plasma concentration increased slowly from about 6 h after the evening dose of both CR tablets, resulting in relatively high plasma concentrations in the early morning hours. Only during treatment with diltiazem CR 120 mg were the plasma concentrations of diltiazem maintained above the minimum therapeutic plasma concentration of 50 micrograms.l-1 throughout the full 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Electrophysiologic and pharmacokinetic profile of the new antiarrhythmic drug TYB-3823 in humans.

In an open-label study, electrophysiology and pharmacokinetics of TYB-3823, a new antiarrhythmic compound, were investigated. Sixteen patients underwent an electrophysiologic study before and after intravenous (i.v.) administration of TYB-3823. Two patients each received the following increasing doses: 0.2, 0.4, 0.8, and 1 mg/kg. Eight patients received 1.2-mg/kg. TYB-3823 concentrations followed a biexponential decrease with a terminal half-life (t1/2) of 3.88 +/- 0.87 h. Clearance was 47.2 +/- 18.5 L/h, and volume of distribution was 250 +/- 77 L. Dose-dependent pharmacokinetics were evident. Significant effects of TYB-3823 were apparent at doses > or = 0.8 mg/kg (n = 12), including increase in the AH and HV interval from 92 +/- 17 to 105 +/- 19 ms (p < 0.002) and 47 +/- 7 to 60 +/- 12 ms (p < 0.005), respectively. QRS duration was prolonged from 100 +/- 16 to 126 +/- 22 ms (p < 0.001), accompanied by an increase of the corrected QT interval from 425 +/- 28 to 465 +/- 37 ms (p < 0.002). The corrected JT interval remained unchanged, however, refractoriness did not change, but monophasic action potential duration (APD) tended to decrease. TYB-3823 appeared effective against reinduction of all arrhythmias observed during the control study [atrial fibrillation, atrioventricular (AV) nodal tachycardias]. TYB-3823 depresses conduction velocity significantly without prolonging refractoriness. Therefore, TYB-3823 may be classified as a class 1C antiarrhythmic. On the basis of the present results, additional class 1B activity cannot be excluded. TYB-3823 has antiarrhythmic properties, appears to be devoid of proarrhythmic effects, and is well tolerated.