Performance analysis of a rapid HPLC determination with the solvent demixing extraction of HIV antiproteases and efavirenz in plasma.

A rapid and simple high-performance liquid chromatographic method without internal standardization is evaluated for the drug-level monitoring of most marketed antiproteases and efavirenz. Following plasma deproteinization with acetonitrile, the analytes are extracted into the solvent while it is demixed by the addition of a saturating amount of neutral salt. The organic supernatant is diluted by half with water up to the polarity of the mobile phase before being injected. The isocratic mobile phase is unbuffered water-acetonitrile (52:48), and the stationary phase is LiChrospher 100 RP-8 (5 microm). Analytes are eluted between 4 min (amprenavir and indinavir) and 20 min (nelfinavir). A spreadsheet program including analysis of variance (ANOVA) and regression is used for both the overall validation of milligrams-per-liter determinations and the performance evaluation of analytical steps from chromatographic raw data. Extraction shows acceptable 5% repeatability and nearly 100% recovery, although it is somewhat concentration-dependent. The calibration function is better fitted by bilogarithmic than arithmetical regression, and the ANOVA of raw data is found quite predictive of the quality of the final determinations.

Therapeutic isoniazid monitoring using a simple high-performance liquid chromatographic method with ultraviolet detection.

Simultaneous measurement of isoniazid and its main acetylated metabolite acetylisoniazid in human plasma is realized by high-performance liquid chromatography. The technique used is evaluated by a factorial design of validation that proved to be convenient for routine drug monitoring. Plasma samples are deproteinized by trichloroacetic acid and then the analytes are separated on a microBondapak C18 column (Waters). Nicotinamide is used as an internal standard. The mobile phase is 0.05 M ammonium acetate buffer (pH 6)-acetonitrile (99:1, v/v). The detection is by ultraviolet absorbance at 275 nm. The validation, using the factorial design allows one to: (a) test the systematic factors of bias (linearity and matrix effect); (b) estimate the relative standard deviations (RSDs) related to extraction, measure and sessions assay. The linearity is confirmed to be within a range of 0.5 to 8 microg/ml of isoniazid and 1 to 16 microg/ml of acetylisoniazid. This method shows a good repeatability for both extraction and measurement (RSD INH=3.54% and 3.32%; RSD Ac.INH=0.00% and 5.97%), as well as a good intermediate precision (RSD INH=7.96%; RSD Ac.INH=15.86%). The method is also selective in cases of polytherapy as many drugs are associated (rifampicin, ethambutol, pyrazinamide, streptomycin). The matrix effect (plasma vs. water) is negligible for INH (3%), but statistically significant for Ac.INH (11%). The application of this validation design gave us the possibility to set up an easy and suitable method for INH therapeutic monitoring.

Concurrent high-performance liquid chromatographic measurement of loxapine and amoxapine and of their hydroxylated metabolites in plasma.

The dibenzoxazepine neuroleptic loxapine, its N-demethylated metabolite amoxapine, and their 7- and 8-hydroxymetabolites were measured simultaneously in plasma by reversed-phase high-performance chromatographic method. An original liquid-liquid extraction procedure was performed, consisting in coextraction of the substances together with a water-miscible solvent (acetonitrile) by a non-water-miscible solvent (toluene). The substances were separated on a 5-microm CN 25-cm column, and eluted with a mobile phase consisting of acetonitrile-acetic acid 0.5 N (30:70) and hexylamine (0.05%). They were detected by ultraviolet spectrophotometry at 310 nm. Clozapine was used as internal standard. Linearity was demonstrated in the range of 10 to 250 microg/l, and detection limits were found to be 3.5 to 6.3 microg/l according to the substance. Within-day repeatability ranged from 2.7% to 6.5%, and between-day reproducibility ranged from 0.9% to 20.2%. The extraction procedure provided a mean absolute recovery of 51.1% (range, 40.7% to 58.6%) with a mean coefficient of variation of 4.2%. This technique was applied to the concurrent determination of plasma concentrations of the compounds in 10 patients administered loxapine 75 to 600 mg daily. Steady state plasma levels of loxapine were significantly correlated with oral doses (n = 10, r = 0.858, p < 0.002). In conclusion, the method proved to be a convenient and reproducible procedure allowing the simultaneous measurement of loxapine, amoxapine, and their metabolites in patients.

Application of a standardized coextractive cleanup procedure to routine high-performance liquid chromatography assays of teicoplanin and ganciclovir in plasma.

Deproteinization of plasma samples with acetonitrile followed by coextracting acetonitrile and lipophilic solutes with chloroform, as already proposed for methotrexate, is stressed as a general sample cleanup procedure for liquid chromatography of highly polar drugs, and was validated for two more applications: teicoplanin and ganciclovir. A dedicated "prevalidation" experimental design was used to assess performances of both assays, including sample preparation. Deviations from linearity were less than 10% over the ranges of 3.1 to 50 mg/l (teicoplanin) and 0.2 to 15 mg/l (ganciclovir), respectively, and limits of quantitation were 0.09 and 0.01 mg/l, respectively. Mean chromatographic measurement R.S.D.s were 4.6% and 1.9%, respectively, with an additional mean cleanup R.S.D. of 2% for both. Mean analyte losses ascribable to cleanup were 6% and 2.5%, respectively from water, and 18% and 12%, respectively from the plasma matrix.

Prevalidation statistical design to assess analytical methods. Example of a quick liquid chromatographic assay of itraconazole in serum.

(A) An analysis-of-variance spreadsheet program is presented which allows to readily test and/or quantitate in a single run analytical linearity, matrix effect on recovery, repeatability of measurement and of extraction and the ruggedness of these features for up to three sessions. Owing to napierian logarithmic transformation, ANOVA mean squares directly read as relative standard deviations and checking linearity is straightforward. (B) A quick assay for therapeutic drug monitoring of itraconazole and its main metabolite was devised with the help of the program, and subsequently validated according to current quality control recommendations. The assay involves acetonitrile demixing extraction, reversed-phase HPLC and UV detection and shows acceptable performance from 0.06 to 5.0 mg/l (limit of detection about 0.03 mg/l). The prevalidation design fairly predicted precision and accuracy, was more informative about matrix effect and was even more demanding about analytical linearity.

High-performance liquid chromatographic determination of fluconazole in plasma.

A high-performance liquid chromatographic method with ultraviolet absorbance detection at 260 nm was developed for the analysis of fluconazole in plasma. The method involves sample clean-up by liquid-liquid extraction. The proposed technique is reproducible, selective, reliable and sensitive. Calibration standards were prepared in the range 1.25-20 mg/l. The limit of quantitation was 0.4 mg/l. The coefficients of variation were 5% between measurements of a single extract injected in duplicate, and 7% between two extractions of spiked samples at the same concentrations. The separation between fluconazole and endogenous substances was satisfactory. This method was designed in order to minimise the risk of interference from substances that could be co-administered to critically ill patients undergoing hemodiafiltration. With a run time below 5 min, the present method is rapid and easy to use for later clinical studies, as well as for routine monitoring.

Clonazepam in acute mania: time-blind evaluation of clinical response and concentrations in plasma.

This study was performed to evaluate the optimal doses and clinical efficacy of clonazepam as a first-line drug in acute mania, as well as to determine its effective plasma concentrations. Clonazepam was administered orally to 11 newly admitted inpatients. On day 0, the loading dose was titrated upward according to the clinical global impression; the maintenance dose was calculated with intent to maintain the plasma level that had been achieved after initial dose escalation. Clinically based dose adjustments were allowed on days 4 and 7. Manic symptoms were scored on days 0, 4 and 14 according to a time-blind procedure; clonazepam plasma levels were measured by HPLC. On day 14, there was a significant decrease in manic symptoms and 66.7% of the patients who completed the trial were markedly improved. Steady-state plasma levels of clonazepam were significantly correlated with daily doses (rs = 0.795, P < 0.005) and therapeutic concentrations ranged between 6.5-83.9 micrograms/l. At the onset of therapy, the clinically titrated loading dose resulted in plasma concentrations within the narrow range of 18.9-34.0 micrograms/l. These results support the potential value of clonazepam in the short-term management of acute mania; the initial control of agitation was achieved with plasma drug levels in a remarkably narrow range as compared with the further control of mania.

Validation of a quick modeling program generating clearance estimates at steady state for routine therapeutic drug monitoring.

Therapeutic drug monitoring (TDM) of chronic treatments is justified for several reasons, including relative over- or underdosage due to variable individual elimination, pharmacokinetic interactions in drug combinations, and noncompliance. In all these circumstances, the prescribing physician is interested in having an estimation of the patient's clearance of the drug, even from one measurement. We compare a validated bayesian program, USC*Pack of Jelliffe, found difficult to use in daily routine, with a "home-made" program. The latter, which is capable of taking data from a clinical database, will generate a graphic simulation of daily plasma drug concentrations together with an estimation of steady-state clearance more rapidly than does USC*Pack. Both programs were run with only one measured plasma level. The patients were 83 children or young adults treated with phenobarbital (PB), carbamazepine (CBZ), and/or Valproic acid (VPA) who were resistant to monotherapy and who were to be sampled two to four times between doses. Drugs were routinely assayed by high-performance liquid chromatography (HPLC). Despite the rough character of Phacile (numeric integration and adjustment of only two of three parameters, without an acknowledged minimization algorithm), the results are comparable to those obtained with USC*Pack for estimating clearance and predicting plasma drug concentrations. Phacile algorithm, although simple, has proven of interest in routine TDM and as an introduction for medical students to the bayesian approach of population pharmacokinetics.

Correlation between methotrexate pharmacokinetic parameters, and clinical and biological status in rheumatoid arthritis patients.

OBJECTIVE: To determine the correlation between the pharmacokinetic (PK) parameters of methotrexate (MTX), clinical status and laboratory test results in rheumatoid arthritis (RA) patients. METHODS: 22 patients (4 M/18F, mean age: 50 +/- 12 years, mean duration of RA: 8.5 +/- 6.5 years, mean duration on MTX: 8 +/- 10 months) were included in a prospective study. The mean dose of MTX administered was 6 +/- 0.7 mg/m2 of body area/week. No patient received any nonsteroidal antiinflammatory drug (NSAID). Blood and urine samples were collected over 24 hours (9 blood samples). The MTX concentrations were assayed by fluorescence polarization immunoassay. Clinical parameters (Ritchie articular index, morning stiffness, joint pain count, joint swelling count), hematological, liver and renal function tests, and ESR were recorded. Correlations between the patients' PK parameters, laboratory tests and clinical status were carried out using Pearson's correlation coefficient test. RESULTS: A significant correlation was observed between the Ritchie articular index, morning stiffness and the area under the curve (p = 0.009 and p = 0.026, respectively). No correlation was found with the other parameters. CONCLUSION: These results suggest that when the patient's disease activity is higher, the AUC becomes more important, reflecting a greater body exposure to MTX.

A double Weibull input function describes the complex absorption of sustained-release oral sodium valproate.

The pharmacokinetics of valproic acid after oral administration of sustained-release formulations were studied in 12 healthy volunteers. The objective of the present study was to find an appropriate mathematical model to describe the complex drug intake process. The concentration of valproic acid in plasma was measured by HPLC. For each subject, during the input process a double peak phenomenon was observed, the plasma concentrations were fitted according to a single or a double Weibull input function, and then a first-order elimination rate was used to describe the observed data. The Weibull model was considered as an approximation of the overall process. The mean peak plasma concentration, 34.6 +/- 8.9 mg/L, was reached after 8.6 +/- 2.7 h. A single Weibull function adequately described the observed data for three subjects; the mean Weibull parameters were td (the time necessary to transfer 63% of the administered drug into the systemic circulation) of 7.87 +/- 3.53 h and gamma (shape) of 1.16 +/- 0.66. A double Weibull input function was used for nine subjects; the mean Weibull parameters were td1 = 2.35 +/- 1.18 h and td2 = 9.36 +/- 4.47 h and gamma 1 = 1.77 +/- 2.27 and gamma 2 = 3.68 +/- 3.26. The mean half-life value of the elimination phase was 14.4 +/- 4.6 h.

A double-blind comparison of clonazepam and placebo in the treatment of neuroleptic-induced akathisia.

The present study was designed to investigate the efficacy of clonazepam in neuroleptic-induced akathisia. Twelve patients were treated during 2 weeks with clonazepam or placebo in a double-blind randomized design. Akathisia was scored by an independent rater before and after treatment, as well as 1 week after medication withdrawal. Clonazepam (0.5-2.5 mg/day) induced a significantly higher reduction in the akathisia scores than placebo (p < 0.05). One week after stopping the drug, there was a partial but significant relapse in the treated group as compared with controls, in whom the symptoms remained stable. In addition, the clinical improvement was significantly correlated with the daily dose of clonazepam (rs = 0.827; p < 0.002). These results support the potential usefulness of clonazepam in the treatment of neuroleptic-induced akathisia and suggest an optimal daily dose in the range of 10-40 micrograms/kg.

Effect of etodolac on methotrexate pharmacokinetics in patients with rheumatoid arthritis.

OBJECTIVE: To determine if the pharmacokinetics of methotrexate (MTX) is modified by the coadministration of etodolac. METHODS: MTX (10 mg) was administered intramuscularly in the absence and presence of steady state levels of etodolac in 19 patients with rheumatoid arthritis. MTX levels were assayed by fluorescence polarization immunoassay. Concentrations of 7-hydroxymethotrexate (7-OH-MTX) were assayed by a reverse phase high performance liquid chromatography method. The unbound fraction was determined by ultrafiltration. RESULTS: When etodolac was coadministered with MTX, the highest observed concentration decreased and the mean residence time increased to become statistically significant. However, these differences are not of great clinical importance especially given that the area under the curve and clearances were unchanged. There were no significant differences in binding protein and 7-OH-MTX concentrations between MTX with and without etodolac administration. CONCLUSION: The pharmacokinetics of MTX is slightly modified by coadministration of etodolac. Moreover, no clinical toxicity was observed.

Performance analysis of a reversed-phase liquid chromatographic assay of lamotrigine in plasma using solvent-demixing extraction.

A reversed-phase column liquid chromatographic assay is described and validated for lamotrigine, a new anticonvulsant drug. The drug and its internal standard were extracted from plasma into acetonitrile according to a previously described solvent-demixing procedure, separated on LiChrospher 100CN, and measured by ultraviolet absorption at 280 nm. The assay performance was evaluated through analysis of variance and of regression with our usual validation design. The method detects ca. 2 ng (55 micrograms/l x 30 microliters) and shows a linear response with a constant 5% coefficient of variation from 1 to 10 mg/l. It is easy and robust, and seems well suited to therapeutic drug monitoring.

Pharmacokinetics of an indomethacin pro-drug: apyramide after intravenous administration in dog.

The pharmacokinetics of apyramide, an ester of indomethacin and acetaminophen (paracetamol), were determined after intravenous administration to nine beagle dogs. Indomethacin and its pro-drug, apyramide, were extracted from acetonitrile-precipitated plasma by a solvent-demixing procedure and the concentration of these two drugs was measured by a reversed-phase liquid chromatographic assay. The kinetic evolution with time of plasma levels of apyramide and of indomethacin resulting from enzymatic hydrolysis was compared with values obtained for indomethacin injected in equimolar dose. Plasma levels of apyramide quickly decreased and the mean (+/- SD) half life was 0.15 +/- 0.08 h. For metabolic indomethacin, the mean (+/- SD) area under curve was 12.36 +/- 4.80 mg.h/l and the mean (+/- SD) half life of terminal phase was 16.71 +/- 9.46 h. After administration of indomethacin, these values were 17.60 +/- 4.12 mg.h/l and 7.89 +/- 2.20 h, respectively.

Carbamazepine and its 10,11-epoxide metabolite in acute mania: clinical and pharmacokinetic correlates.

The study was designed to investigate the antimanic profile of carbamazepine as a first-line drug in affective or schizoaffective disorders, to correlate the clinical efficacy with the plasma level of carbamazepine and its 10,11-epoxide metabolite, and to test the potential value of monitoring the salivary level. It was administered alone for 3 weeks to 21 acute manic inpatients. During the first week, the dosage was rapidly increased to 800 mg/day in order to produce steady-state plasma levels of carbamazepine on Day 7. The individual dose was then adjusted to maintain the therapeutic range of 8-12 mg/l. Plasma and saliva levels of the drug and its metabolite, as well as clinical status were assessed weekly. Overall, there was 62% globally improved patients and 77% in affective disorders. The improvement of manic symptoms was significantly lower in schizoaffective than in affective disorders, whereas the dropout rate and the need for antipsychotic medication was higher in the former group. The antimanic response was significantly correlated with the plasma levels both of carbamazepine and its epoxide metabolite, with a time-lag consistent with a delayed drug effect. Drug and metabolite concentrations in saliva were close to their plasma free fraction and were strongly correlated with their plasma levels, suggesting the potential value of salivary drug monitoring.

[Use of a volatile substrate to explore oxidative metabolism of xenobiotics in the rabbit lung]. / Utilisation d'un substrat volatil pour explorer le métabolisme oxydatif des xénobiotiques par le poumon chez le lapin.

A demethylation breath test was modeled in rabbits receiving 14C-anisole either by the intravenous route or by inhalation. The exhalation rate of 14CO2 was measured in a metabolic cage. The modeling hypothesis involved two metabolic compartments: a "central" compartment receiving the infusion, and a "pulmonary" compartment receiving the inhalation. The modeling succeeded in identifying a highly significant metabolic contribution of the lungs following inhalation of the substrate.

[Experimental validation of a procedure for modulated perfusion of thiopental]. / Validation expérimentale d'une procédure de perfusion modulée du thiopental.

The pharmacokinetics of sodium thiopental was studied in 9 normal dogs of various ages by the usual bolus method. Data were fittable by a two compartment open model in 8, three compartments were needed in one. Constant-rate infusions, as used in cerebral resuscitation, were performed in 6 of them. As expected, constant blood levels could not be obtained during the distribution phase which lasted about 6 hours. From the individual pharmacokinetic constants, the time equation of infusion rate intended to exactly compensate for distribution was calculated. The resulting "modulated infusions" succeeded in reducing the blood level trough from 50% to 20%. After the end of infusions, the decrease of plasma levels exhibited a non linear pattern which had not been noticeable in the elimination phase after boluses. Such unknown non linearity may lead to systematic errors when calculating pharmacokinetic parameters: this could explain why a complete correction was not obtained with modulated infusions. A better parametrization method in the case of non-linear elimination is under study.

Statistical analysis of performance of a liquid chromatographic assay of anticonvulsants involving solvent-demixing extraction and reciprocal internal standardization.

Analysis of variance both factorial and nested was used to validate a HPLC method intended for routine clinical assay of ethosuximide, phenobarbital, phenytoin and carbamazepine. Drugs were salted out, together with the solvent, from 0.5 ml acetonitrile-deproteinized plasma samples with 80-90% recovery. The acetonitrile extraction solution contained a known amount of all four drugs. This added amount of any drug was used when absent from plasma as an internal standard for those present and when present as a calibrator. Results showed that assay precision was acceptable (CV 6%) over and above the therapeutic range when additions did not exceed the lower therapeutic plasma level and if as many replications were made as there were drugs to assay. In return for some loss of sensitivity, reciprocal internal standardization provides increased assay reliability owing to the usual availability of more than one internal standard and to easier identification of interfering chromatographic peaks.