Robert Dugal

Studies on anabolic steroids : I. Integrated methodological approach to the gas chromatographic-mass spectrometric analysis of anabolic steroid metabolites in urine

The analytical and methodological imperatives for large-scale and routine gas chromatographic-mass spectrometric screening of anabolic steroid urinary metabolites are described. Several aspects of their isolation, enzymatic hydrolysis, derivatization and metabolism in humans are discussed. Gas chromatographic-mass spectrometric data illustrating artifacts arising from enzymatic hydrolysis of 3 beta-ol-5-en steroids, and describing new metabolites of boldenone, methanedienone and stanozolol, as well as the conversion of norethisterone into 19-nortestosterone metabolites through de-ethylation at C-17, are presented. The analytical approach developed for gas chromatographic-mass spectrometric screening of anabolic steroids is based on the sequential selection-ion monitoring of specific and discrete ion groups characteristic to the steroids of interest under high-resolution chromatographic conditions. The major analytical and methodological requirements necessary to provide irrefutable evidence, in the case where the presence of a synthetic anabolic steroid or a testosterone to epitestosterone ratio higher than 6:1 is suspected in a given urine specimen, are also discussed.

Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography

A rapid and reliable screening procedure using high-performance liquid chromatography for the detection of 23 diuretics (belonging to five different pharmacological groups) in urine has been developed. Two aliquots of 2-ml urine samples were extracted separately under acidic and basic conditions. The acidic and basic extracts were pooled, evaporated to dryness and reconstituted in methanol. The methanolic extract was injected onto a Hewlett-Packard Hypersil ODS C18 (5 microns) column (column I) and a Hewlett-Packard LiChrosorb RP-18 (5 microns) column (column II; an alternative column). The same gradient mobile phase was used for both columns. A diode array ultraviolet detector was set to monitor the signal to the integrator (Chem Station) at 230 and 275 nm. Recovery studies of the 23 diuretics were performed under acidic and basic conditions. The overall lower limits for detection on column I using both extraction procedures ranged from 0.5 to 1.5 micrograms/ml of urine (average 1.0 micrograms/ml). Amiloride, ethacrynic acid and probenecid could not be detected below 5 micrograms/ml of urine. No interference from the biological matrix was apparent. Amiloride could be detected in urine 4 h after oral administration of 15 mg of amiloride to a healthy volunteer, when the sample was extracted under alkaline conditions. The suitability of the screening method for the analysis of urine samples was tested by studying the variation with time of chlorthalidone, furosemide, probenecid, acetazolamide, quinethazone, spironolactone, bendroflumethiazide, bumetanide, triameterene and hydrochlorothiazide concentrations in the urine of normal human volunteers after minimum single or multiple (probenecid) doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on anabolic steroids: V. Sequential reduction of methandienone and structurally related steroid A-ring substituents in humans: gas chromatographic—mass spectrometric study of the corresponding urinary metabolites

The biotransformation of methandienone (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one) in human adults, more particularly the sequential reduction of its A-ring substituents, was investigated by gas chromatography-mass spectrometry. Two pairs of 17-epimeric tetrahydro diols resulting from the stereoselective reduction of the delta 4- and 3-oxo groups and of the delta 1-function were characterized. The major diols were 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol, which were both excreted in the conjugate fraction in a 1:3.8 ratio. The immediate metabolic precursors of the 5 beta-diol, namely 17 beta-hydroxy-17 alpha-methyl-5 beta-androsta-1-en-3-one and 17 alpha-methyl-5 beta-androsta-1-en-3 alpha,17 beta diol and their corresponding 17-epimers, were also identified in post-administration urine samples. These data indicated that reduction of methandienone A-ring substituents proceeds according to the sequence. delta 4-, 3-oxo- and delta 1-. The A-ring reduction products of the structurally related steroids mestanolone, 17 alpha-methyltestosterone and oxymethone were also characterized and provided further analytical and metabolic evidence supporting the proposed route of methandienone A-ring reduction. It was also demonstrated using synthetic 17 beta-sulfate conjugates of methandienone and 17 alpha-methyltestosterone that their corresponding 17-epimers are formed by nucleophilic substitution by water of the labile sulfate moiety. The steroidal metabolites were identified on the basis of their characteristic mass spectral features and by comparison with authentic reference standards. Metabolic pathways accounting for the occurrence of the metabolites of interest in post-administration urine samples are proposed.

Pharmacokinetics of Netilmicin in Renal Insufficiency and Haemodialysis

The pharmacokinetics of intravenously administered netilmicin, an investigational aminoglycoside antibiotic, were studied in 38 patients with Creatinine clearance ranging from 150 to 0ml/min/1.73m2 in order to determine the influence of kidney function status on the disposition of the antibiotic. The serum disappearance of netilmicin followed first order kinetics and the elimination rate constant decreased proportionally with decreasing renal function. Half-lives averaged 2.2 hours in normal individuals (creatinine clearance > 80ml/min/1.73 m2) and reached 42 ± 10 hours(mean ± SD) in virtually anephric patients. The elimination rate constant lowered proportionally with decreasing renal function.Several linear relationships between pharmacokinetic parameters and renal function indicators were defined. A clinically useful correlation indicates that the half-life may be approximated as 3 times the serum Creatinine concentration and may be used for adjustment of dosage of netilmicin in the treatment of patients with impaired renal function. During haemodialysis, netilmicin extraction from the blood reaches 75 ± 14% (mean ± 95% confidence interval) of that of Creatinine and 88 ± 19% of that of blood urea nitrogen.

Nanogram-range determination of plasma imipramine by gas-liquid chromatography using a selective nitrogen/phosphorus detector.

A sensitive and specific gas chromatographic method for the quantitative determination of plasma concentrations resulting from a single normal therapeutic dose of imipramine has been developed and is descriged. After extraction into a mixture of n-heptane/isoamyl alcohol (98.5:1.5), imipramine is well separated from its main metabolite, desmethylimipramine, and both compounds are detected using a selective detector operating in the N/P mode. The procedure permits the rapid routine quantitative analysis of relatively small plasma volumes (1-2 ml) containing as little as 1-2 ng of imipramine. No interference from the biological matrix is apparent. The suitability of the method for the analysis of biological samples was tested by studying the time of course of imipramine plasma concentrations in normal human volunteers, after administration of a single therapeutic dose.

Determination of loxapine in human plasma and urine and identification of three urinary metabolites

1. A g.l.c. method for quantitative determination of loxapine (2-chloro-11-(4-methyl-1-piperazinyl)dibenz(b,f)(1,4)oxazepine), in human plasma and urine is described. 2. Preliminary pharmacokinetic data on plasma concn of loxapine over 12 h from five psychiatric patients who received a total average dose of 80 mg of loxapine succinate per day orally for twelve weeks are presented. 3. In addition to unchanged loxapine, three urinary metabolic products, namely aromatic ring-hydroxy loxapine, desmethyl loxapine and loxapine-N-oxide, were identified using g.l.c.--mass spectrometry.

Clinical Pharmacokinetics of Sisomicin: Dosage Schedules in Renal-Impaired Patients

The pharmacokinetics of intravenously administered sisomicin were studied in 33 patients with normal renal function and different degrees of renal impairment. In all patients, the serum disappearance of sisomicin, once distribution equilibrium had been achieved, followed first-order kinetics and percentage of hourly loss from serum decreased proportionally with decreasing renal function. Half-lives averaged 2.06 h in normal subjects (endogenous creatinine clearance greater than 80 ml/min per 1.73 m2) and reached 35.3 h in a virtually anephric subject. Linear relationships were defined between sisomicin serum half-life and the reciprocal of the endogenous creatinine clearance and serum creatinine concentration. The latter relationship indicates that the half-life of sisomicin may be approximated as twice the serum creatinine concentration and may be used for dosage adjustment in renal-impaired patients. Prediction of the extent of sisomicin removal by hemodialysis may be made from the relationship between the dialyzate of sisomicin and that of creatinine and blood urea nitrogen. Dosage schedules and methods of administration compatible with the pharmacokinetic properties of the antibiotic are finally proposed.

Metabolic interaction between amitriptyline and perphenazine in psychiatric patients

1. Steady-state plasma level samples of sixty-five schizophrenic patients from two psychiatric hospitals assigned to three treatment groups (amitriptyline 150 mg/day, perphenazine 20 mg/day and a combination of amitriptyline and perphenazine at 150 mg and 20 mg/day) were assayed for amitriptyline (AT), endogenous nortriptyline (NT) and perphenazine (PPZ) using gas-liquid chromatography. 2. Results reveal that AT and NT levels are independent of sex and hospital environment. 3. PPZ significantly increased the steady-state NT plasma levels, probably through inhibition of the hydroxylation biotransformation pathway, but had no effect on AT levels, thus indicating that PPZ has no influence on the desmethylation pathway, or alternatively, the hydroxylation of AT.

Penfluridol steady‐state kinetics in psychiatric patients

Twenty‐two hospitalized schizophrenic patients, participating in a large‐scale phase II double‐blind dose‐effect study (30, 60, and 120 mg weekly) of penfluridol, a new diphenylbutylpiperidine neuroleptic, were maintained on a regular dosage regimen for 13 wk. Several blood samples were taken during the last dosage interval. Results show that the peak concentration develops within 12 hr after the last dose. A rapid decline, probably due to tissue re‐equilibration, then occurs and is followed by a much slower falloff. Detectable concentrations 168 hr after administration are consistent with the long duration of action of penfluridol. Statistically significant differences between doses were found in the analysis of variance of plasma concentrations at all sampling times and in mean steady‐state plasma concentrations. Wide differences in plasma concentrations were noted in patients receiving the same absolute dose, but a good relationship was defined between mean steady‐state concentration and the dose expressed as mg per either kg of body weight or square meter of body surface area.

Quantitative determination of plasma oxyphenbutazone by gas-liquid chromatography with selective nitrogen detection.

A sensitive and specific gas chromatographic method, using the nitrogen-phosphorus detector for the detection and determination of oxyphenbutazone extracted from plasma is described. The method involves extraction and back-extraction steps followed by derivatization of both oxyphenbutazone and the internal standard with trifluoroacetic anhydride. The procedure permits the rapid and specific routine determination of oxyphenbutazone in plasma with a detection limit of 0.5 microgram/ml. The procedure is linear over the range of concentrations encountered after administration of a single oral therapeutic dose. No interference from the biological matrix is apparent. The suitability of the method for the analysis of biological samples was tested by studying the variation with time of oxyphenbutazone plasma concentrations in normal human volunteers over a period of several biological half-lives.