A. Sioufi

High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection

An analytical method for the determination of letrozole (CGS 20,267) in plasma and of letrozole and its metabolite, CGP 44,645, in urine is described. Automated liquid-solid extraction of compounds from plasma and urine was performed on disposable 100-mg C8 columns using the ASPEC system. The separation was achieved on an ODS Hypersil C18 column using acetonitrile-phosphate buffer, pH 7, as the mobile phase at a flow-rate of 1.5 ml/min. A fluorescence detector was used for the quantitation. The excitation and emission wavelengths were 230 and 295 nm, respectively. The limits of quantitation (LOQ) of letrozole in plasma and in urine were 1.40 nmol/l (0.4 ng/ml) and 2.80 nmol/l, respectively. The respective mean recoveries and coefficient of variation (C.V.) were 96.5% (9.8%) in plasma and 104% (7.7%) in urine. The LOQ of CGP 44645 in urine was 8.54 nmol/l (2 ng/ml). The mean recovery was 108% (6.3%). The compounds were well separated from co-extracted endogenous components and no interferences were observed at the retention times of compounds. The sensitivity of this method for letrozole in plasma should be sufficient for kinetic studies in humans with single doses of 0.5 mg and possibly less.

The effect of age on the pharmacokinetics of valsartan

Twelve young (mean age 23 years, range 18–28) and 12 elderly (mean age 76 years, range 65–89) volunteers were given a single oral dose of 80 mg valsartan after an overnight fast. Each group consisted of six male and six female subjects. Mean systemic exposure to valsartan was higher in the elderly when compared with the young (AUC(0–24 h), 52% increase and AUC(0–∞), 70% increase). Variability, as shown by the coefficient of variation (CV), was larger for the elderly subjects and ANOVA of the log transformed AUC showed a significant difference between the two groups. This difference was largely brought about by five elderly subjects (one male, four females), whose AUC was about 2‐fold higher than the rest of the group. For the remaining elderly subjects, plasma valsartan AUC was similar to that observed for the young volunteers. This higher systemic exposure in five of the elderly subjects is not thought to be of clinical relevance when data from the patient population are considered. Other covariates—such as body weight, comedication, creatinine clearance, valsartan kinetics (absorption rate, distribution, and elimination)—did not explain the higher AUC in this subset of the elderly group. Data from the present study were compared with population kinetic data obtained from larger clinical trials including hypertensive patients in all age groups. Using this population approach, there was no difference in the pharmacokinetics of valsartan between male and female patients. Also, a relationship between plasma clearance of valsartan and age was established. The median age of patients in the hypertensive pool was 55 years. For an average 70‐year‐old patient, plasma clearance of valsartan is predicted to fall by 22% compared with an average 55‐year‐old. For the population, this difference is not sufficient to warrant initial dose adjustment based on age per se. The covariate age, does not completely explain the variability in the pharmacokinetics of valsartan within the general population. The treatment was well tolerated.

Determination of artemether and its metabolite, dihydroartemisinin, in plasma by high-performance liquid chromatography and electrochemical detection in the reductive mode

An analytical method for the determination of artemether (A) and its metabolite dihydroartemisinin (DHA) in human plasma has been developed and validated. The method is based on high-performance liquid chromatography (HPLC) and electrochemical detection in the reductive mode. A, DHA and artemisinin, the internal standard (I.S.), were extracted from plasma (1 ml) with 1-chlorobutane-isooctane (55:45, v/v). The solvent was transferred, evaporated to dryness under nitrogen and the residue dissolved in 600 microliters of water-ethyl alcohol (50:50, v/v). Chromatography was performed on a Nova-Pak CN, 4 microns analytical column (150 mm x 3.9 mm I.D.) at 35 degrees C. The mobile phase consisted of pH 5 acetate-acetonitrile (85:15, v/v) at a flow-rate of 1 ml/min. The analytes were detected by electrochemical detection in the reductive mode at a potential of -1.0 V. Intra-day accuracy and precision were assessed from the relative recoveries (found concentration in % of the nominal value) of spiked samples analysed on the same day (concentration range 10.9 to 202 ng/ml of A and 11.2 to 206 ng/ml of DHA in plasma). The mean recoveries over the entire concentration range were from 96 to 100% for A with C.V. from 6 to 13%, from 92% to 100% for DHA (alpha-tautomer) with C.V. from 4 to 16%. For A, the mean recovery was 96% at the limit of quantitation (LOQ) of 10.9 ng/ml with a C.V. of 13%. For DHA, the mean recovery was 100% at the LOQ of 11.2 ng/ml with a C.V. of 16%.

Determination of the S(+)- and R(−)-enantiomers of baclofen in plasma and urine by gas chromatography using a chiral fused-silica capillary column and an electron-capture detector

A sensitive and enantiospecific gas chromatographic method for the determination of the S(+)- and R(-)-enantiomers of baclofen (I and II) in plasma and urine has been developed and validated. The method is based on the complete resolution of the derivatized enantiomers on a chiral fused-silica capillary column. The hydrochloride salt of a (-)-fluoro analogue of baclofen (III.HCl) was used as the internal standard in plasma, the hydrochloride salt of a (+)-fluoro analogue of baclofen (IV.HCl) as the internal standard in urine. Rapid and convenient isolation of the compounds was achieved using reversed-phase Bond-Elut C18 columns. After elution, the compounds were converted into isobutyl esters and purified by base-specific solvent extraction. The isobutyl esters were then N-acylated with heptafluorobutyric anhydride. The derivatives were quantitated after separation on the chiral column using electron-capture detection. The analysis of spiked plasma and urine samples demonstrated the good accuracy and precision of the method, with limits of quantitation of 25 nmol/l for I and II in plasma and of 2 mumol/l for I and II in urine. The method appears to be suitable for use in pharmacokinetic studies of the enantiomers in plasma and urine from animals and man after administration of the racemic baclofen.

Chromatography of benzodiazepines

An overview of methods for the determination of benzodiazepines in biological media, based on the application of chromatographic techniques, is presented. A general discussion of the techniques in terms of stability, selectivity, validation, standardization, detection and sensitivity is given. No single technique can be claimed as the method of choice for benzodiazepines. Gas chromatography with electron-capture detection has some strong claims and shows generally good sensitivity and reproducibility. High-performance liquid chromatographic equipment is readily available in most laboratories. The ultimate choice of an assay method for benzodiazepines will be determined by the clinical application (routine monitoring, pharmacokinetics, overdose, forensic medicine) and by the characteristics of the benzodiazepine, the expertise of the analyst, the equipment available, the desired sensitivity and specificity and the time involved in method development or adaptation and validation.

Automated determination of an angiotensin II receptor antagonist, CGP 48 933, in plasma by high-performance liquid chromatography

Abstract A fully automated high-performance liquid chromatography method with fluorimetric detection is described for the determination of CGP 48 933 in human plasma. Liquid-solid extraction was performed automatically on C8 reversed phase column using the Gilson ASPEC system. The on-line chromatography was performed on a ODS Hypersil C18 5 μm column. The mobile phase, acetonitrile- pH 2.8 phosphate buffer (50:50, v/v) was used at a flow rate of 1.3 ml/min. The fluorimetric excitation and emission wavelengths were set at 265 and 378 nm, respectively. The limit of quantitation of CGP 48 933 was 11.5 nmol per litre of plasma.

Gas chromatographic determination of amantadine hydrochloride (symmetrel) in human plasma and urine

A method for the determination of amantadine hydrochloride at concentrations down to 10 ng/ml in human plasma and urine is described. After addition of a known amount of amphetamine sulphate as internal standard to 1 ml of plasma or urine, amantadine is extracted at basic pH in toluene. Both compounds are derivatized with trichloroacetyl chloride. The derivatives are determined by gas chromatography using a 63Ni electron-capture detector. The technique was applied in a study of the elimination of amantadine after oral administration to man; plasma concentrations are reported.

Simultaneous determination of imipramine and its metabolite desipramine in human plasma by capillary gas chromatography with mass-selective detection

An analytical method for the simultaneous determination of imipramine (IMI) and its N-desmethyl metabolite, desipramine (DIMI) in human plasma by capillary gas chromatography-mass selective detection (GC-MS), with D4-imipramine (D4-IMI) and D4-desipramine (D4-DIMI) as internal standards, was developed and validated. After addition of the internal standards, the compounds were extracted from plasma at basic pH into n-heptane-isoamyl alcohol (99:1, v/v), back-extracted into acidic aqueous solution and re-extracted at basic pH into toluene. Desipramine and D4-desipramine were converted into their pentafluoropropionyl derivatives. The compounds were determined by gas chromatography using a mass selective detector at m/z 234 for IMI, m/z 238 for D4-IMI, m/z 412 for DIMI and m/z 416 for D4-DIMI. The method was applied to clinical samples.

Determination of diclofenac in plasma and urine by capillary gas chromatography—mass spectrometry with possible simultaneous determination of deuterium-labelled diclofenac

A specific and sensitive method for the determination of diclofenac at concentrations down to ca. 1 ng/ml, the limit of detection being 100 pg/ml, in human plasma and urine by gas chromatography-mass spectrometry with 2H4-labelled diclofenac as internal standard is described. The method is also suitable for the simultaneous assay of these two compounds when both are present in samples of human plasma or urine. In this case, 5-chlorodiclofenac is used as internal standard. After toluene extraction from plasma or without extraction for urine, the method involves the formation of a dimethylindolinone derivative by extractive alkylation. The technique was applied to determine low plasma concentrations and urinary excretion of labelled and unlabelled diclofenac after percutaneous applications of Voltaren Emulgel to humans applied simultaneously under occlusive dressing as deuterated diclofenac sodium, and without occlusive dressing as unlabelled diclofenac sodium.

Gas chromatographic determination of phentolamine (Regitine) in human plasma and urine.

A method for the determination of unconjugated phentolamine at concentrations down to 5 ng/ml in human plasma, and of free and total (free plus conjugated) phentolamine down to 25 ng/ml in urine is described. After addition of 2-[N-(p-chlorophenyl)-N-(m-hydroxyphenyl)-aminomethyl]-2-imidazoline as internal standard, both compounds are extracted into benzene-ethyl acetate (1:1, v/v) at pH 10, transferred into an acidic aqueous solution and back-extracted at pH 10 into benzene-ethyl acetate. They are then derivatized with N-heptafluorobutyrylimidazole. The derivatives are determined by gas chromatography using a 63Ni electron-capture detector. In urine, total (free plus conjugated) phentolamine is determined after enzymatic hydrolysis. The technique was applied for the study of the plasma concentrations and urinary elimination after oral administration to man.