John Boutagy

Simultaneous Analysis of Cimetidine and Ranitidine in Human Plasma by HPLC

Abstract A rapid method for the simultaneous quantitation of the H2-receptor antagonist drugs cimetidine and ranitidine in human plasma by isocratic ion-pair reverse-phase HPLC is described. The method involves a simple organic extraction step of the alkalinized plasma containing added internal standard followed by back extraction of the extract with dilute acetic acid and subsequent analysis of the aqueous acidic phase on a reverse-phase (C18) column. The eluting solvent was acetonitrile-water (20:80 v/v) containing 0.005 mole/litre octanesulphonic acid and was monitored at 229 nm. The run time for the assay was 12.5 minutes, with a detection limit for cimetidine of 50 ng/m1/(0.2 μmole/1) and that for ranitidine was 20 ng/ml (0.06 umole/1).

A screening test for detecting sulfonylureas in plasma

Hypoglycemia induced by surreptitious sulfonylurea ingestion can be difficult to distinguish from an insulin-secreting tumor. We describe a technique for detecting most of the common sulfonylurea drugs in plasma. After preliminary acidification, and extraction in ether, the residue is reconstituted and injected onto a Versapack high-performance liquid chromatography column. Detection is at 230 nm. This procedure gives good separation of chlor-propamide, glibenclamide, gliclazide, glipizide, and tolbutamide. Results are semiquantitative but the sensitivity of the assay is sufficient to detect and identify clinically active concentrations of all five drugs. It is a rapid and reliable screening test. Four representative case histories are reported in which the screen proved to be of diagnostic value.

The metabolism of glyburide in subjects of known debrisoquin phenotype

Ten normal subjects of known debrisoquin phenotype (six extensive (EM) and four poor (PM) metabolizers) were given of 5 mg glyburide (glibenclamide) suspension orally. Plasma glyburide and urinary cis‐3‐hydroxy‐(30H) and trans‐4‐hydroxyglyburide (40H) were measured by a sensitive HPLC assay. No unchanged glyburide was detected in urine but both metabolites were identified in urine in all subjects. There were no significant differences in any respect with regard to glyburide metabolism or pharmacokinetics between EM and PM of debrisoquin. Estimated mean elimination half‐life of glyburide was 3.3 ± 1.1 hours for EM and 2.5 ± 0.4 hours for PM. In one subject (EM), with reduced excretion of 30H, glyburide was detected in plasma at 24 and 30 hours and the apparent elimination half‐life was 9.3 hours. There was no significant difference for total metabolite recovery between EM and PM. Eight of the subjects (six EM and two PM) had previously taken part in a study of tolbutamide metabolism, and a comparison of metabolic clearances by hydroxylation for the two sulfonylureal drugs showed no significant correlation. Glyburide is therefore unlikely to be metabolized by the enzymes that metabolize either debrisoquin or tolbutamide.

Determination of cytosine arabinoside in human plasma by gas chromatography with a nitrogen-sensitive detector and by gas chromatography--mass spectrometry.

A method for the determination of cytosine arabinoside in the plasma of leukemic patients being treated with this drug is described using either gas--liquid chromatography with a nitrogen-sensitive flame ionization detector or gas chromatography--mass spectrometry (GC-MS). To increase volatility, a double derivative of cytosine arabinoside was used, prepared by acetylation and subsequent methylation. Cytidine was used as internal standard for the GC procedure. GC--MS was performed with either cytidine as internal standard and detection by single-ion monitoring or by the use of [2H3] acetate-methyl derivative of cytosine arabinoside as internal standard and subsequent multiple-ion monitoring. Attempted extraction of cytosine arabinoside from plasma with various organic solvents was unsuccessful, but protein precipitation with ethanol or trichloroacetic acid followed by washing of the aqueous residue with organic solvents to remove as many of the interfering substances as possible gave satisfactory results. The minimum detectable quantity of pure cytosine arabinoside was similar for both techniques (approximately 500 pg). However, with GC using a nitrogen-sensitive detector, the lower limit of detection from plasma was found to be approximately 40--70 ng per ml plasma whilst GC--MS showed greater analytical selectivity with a detection limit in some cases as low as 1 ng per ml plasma.

Analysis of cytosine arabinoside and related pyrimidine nucleosides by gas chromatography and gas chromatography-mass spectrometry

Abstract Acetyl methyl derivatives of cytosine arabinoside and six other pyrimidine nucleosides were prepared and examined by gas-liquid chromatography (GLC) using a nitrogen-sensitive flame ionization detector and by gas chromatography-mass spectrometry (GC-MS). The derivatives gave symmetrical GC peaks and could be separated on SE-30 or OV-17. Detection limit for cytosine arabinoside was approximately 500 pg using both the nitrogen-sensitive flame ionization detector and single-ion monitoring [(M − 15) + ion]. Methyl trimethylsilyl and three homologous alkyl oxime trimethylsilyl derivatives of cytosine arabinoside and cytidine were also prepared and examined by GC-MS. Retention indices are reported on four phases and the mass spectra of these derivatives discussed. The methyl trimethylsilyl derivatives gave a lower detection limit than the methyl acetyl derivatives (approximately 50 pg), but the oxime derivatives were less sensitive. Allyl and propyl dimethylsilyl derivatives of cytosine arabinoside were also examined.

Interactions between ethanol and oral contraceptive steroids

We investigated the effect of oral contraceptive steroids (OCSs) on plasma ethanol disposition and tolerance to ethanol. Fifty‐four healthy women between 18 and 40 years old were classified as light (31) or moderate (23) drinkers. Each group was further subdivided into controls (no OCS; 10 light, seven moderate drinkers), 30 or 35 μg estrogen OCS (14 fight, 11 moderate drinkers), and 50 μg estrogen OCS (seven light, five moderate drinkers). Four of the subjects were studied on a second occasion, thus acting as their own controls with and without OCS use. All women were studied between days 14 and 21 of their pill/menstrual cycle. Plasma ethanol concentrations and two simple tests of motor function were measured for 6 hours after ethanol, 0.9 gm/kg in orange juice drank over a 30‐minute period. The groups were well matched for age and weight. There were no significant differences between any of the six subgroups in mean peak plasma ethanol concentration, mean time to peak, mean AUC, or mean rate of ethanol disappearance. This was also the case for the four women who acted as their own controls. Analyses between those receiving high and low progestogen OCSs and between smokers and nonsmokers showed no significant differences. There was acute deterioration in functional performance as measured by two motor function tests in all subjects, regardless of OCS use. Moderate drinkers were significantly less functionally impaired than fight drinkers whether with or without OCS use, indicating acquired tolerance. The mean degree of impairment and mean recovery time for both tests were significantly less in the OCS groups than in the control groups. The same trend was seen in the four women who were their own controls. Our results suggest that OCS use may induce some form of “tolerance” to ethanol. However, because there is no evidence of any change in ethanol disposition even at high plasma ethanol concentrations (>100 mg/dl), women taking OCSs should not attempt to drink more than usual.

Simplified procedure for the determination of sotalol in plasma by high-performance liquid chromatography

A simple, specific and rapid reversed-phase high-performance liquid chromatographic (HPLC) procedure for sotalol determination is described requiring small plasma volumes. The high recovery of sotalol from plasma and the high precision of measurement obviate the need for an internal standard. Plasma samples (300 microliters) were deproteinised with 50 microliters of 70% (w/w) perchloric acid in disposable glass tubes. After vortex-mixing and centrifugation, 30 microliters of 4 M K2HPO4 were added followed by gentle shaking. A 20-microliters aliquot was then injected (by autosampler) for HPLC analysis. Chromatography was performed on a glass-lined 250 mm x 4 mm 5-micron C18 steel column. The mobile phase was 6% (v/v) acetonitrile in 0.08 M KH2PO4 buffer (pH 4.6). The flow-rate was 0.8 ml/min. Detection was by fluorescence with excitation and emission wavelengths at 235 and 310 nm, respectively. The retention time for sotalol was 7.1 min. Calibration was linear from 0.16 to 10 micrograms/ml in plasma (r greater than 0.999 for detector response to sotalol). The minimum concentration for quantitation was 0.08 micrograms/ml [within assay coefficient of variation (C.V.) less than 5%]. Recovery was near quantitative (greater than 98%) and replicate (intra-assay precision was less than 5% C.V.). Analysis of samples (n = 10) at concentrations of 0.42 and 4.2 micrograms/ml gave mean values of 0.44 and 4.3 micrograms/ml, respectively. The inter-assay C.V. values were 4.5 and 2.2%, respectively. Other clinically used antiarrhythmic drugs did not interfere. This assay can be performed using other commercial C18 analytical columns by suitable adjustment of mobile phase flow-rate and acetonitrile composition.

A rapid liquid chromatographic method for determination of flecainide in human blood plasma using ultraviolet detection

Abstract A liquid chromatographic method for the assay of the antiarrhythmic drug flecainide in plasma has been developed. The method is rapid, simple and with sufficient detection sensitivity to render it suitable for therapeutic drug monitoring. Flecainide and added internal standard, a non-fluorinated analogue, were extracted by a single ether extraction from alkalinized plasma followed by a back-extraction of the ether with dilute phosphoric acid. A portion of the acid extract was then applied directly to a 30 cm ODS column eluting isocratically with 30% acetonitrile in water containing 0.01M dibutylamine phosphate. Monitoring was by ultraviolet detection at 214 nm and the total run time was 8 min. This method is specific and can quantitate plasma levels to less than 30 ng/ml (free base) from 0.5 ml of plasma without interference from antiarrhythmic drugs commonly used in therapy.

Determination of dexfenfluramine and nordexfenfluramine in urine by high-performance liquid chromatography using ultraviolet detection.

A method to determine the concentration of dexfenfluramine and its active metabolite nordexfenfluramine in human urine from healthy volunteers is described utilising a high-performance liquid chromatographic procedure with liquid-liquid extraction and ultraviolet detection. Analytes are measured after extraction of alkalinised urine with diethyl ether and subsequent back extraction with 0.5 M H2SO4 and with chromatography performed on a reversed-phase C18 column, using a mobile phase of acetonitrile-50 mM K2HPO4 (25:75, v/v) (flow-rate 1.3 ml/min) and ultraviolet detection at 210 nm. The sensitivity of the technique (10 ng/ml) is appropriate to measure both parent drug and metabolite in urine in humans for up to 5 days after a single 30-mg dose. The method is selective, reproducible (within- and between-day coefficient of variation ranged from 4.2 to 15%) and accurate (bias less than 8%) and thus suitable for dexfenfluramine pharmacokinetic investigations.

The pharmokinetics of isradipine in hypertensive subjects

SummaryIn conjunction with a multicentre clinical trial of the calcium antagonist isradipine in hypertension, pharmacokinetic and pharmacodynamic studies were conducted in 9 subjects. An initial dose of 5 mg (capsule formulation) of isradipine was given orally. The mean Cmax, tmax and AUC(0–8) were 6.0 ng · ml−1, 1.5 h and 15.1 h · ng · ml−1 respectively. Seven subjects repeated the study at steady state after 10 weeks dose titration with isradipine. Cmax, tmax and AUC(0–8) were 3.7 ng · ml−1, 1.2 h and 12.2 h · ng · ml−1 respectively indicating that the drug does not accumulate over time.Control of blood pressure paralleled plasma isradipine concentrations which suggested that the drug should be given at least twice daily. Pharmacokinetic studies performed in conjunction with clinical trials can provide valuable information about the patterns of drug response.