Detection of mono-hydroxylated metabolites of stanozolol by HPLC-ESI (+) MS/MS in Indian sports persons.

The abuse of stanozolol is quite widespread in Indian sport. Its analysis is challenging and this has led to the development of new methods to improve its detection. A method was developed and validated for the detection of the three main monohydroxylated metabolites of stanozolol. The excretion profile of these metabolites was studied in four healthy male volunteers. The excretion study samples, after a single oral dose of drug, showed that 3'-OH-stanozolol was excreted at the highest concentration, followed by 16beta-OH stanozolol, with 4beta-OH stanozolol as the least excreted. Ninety-eight old doping samples with adverse analytical findings for 3'-OH-stanozolol were reanalysed using this method. This showed 3'-OH-stanozolol and 16beta-OH stanozolol in all the 98 samples whereas 4beta-OH-stanozolol was identified in 90 samples. The percentage of positive identifications of stanozolol in Indian sportspeople has increased markedly in the last five years, from 31.9% in 2004 to 81.8% in 2009; however, this may be due to the more effective detection of stanozolol metabolites. It can thus be concluded that the marked increase in percent positive of stanozolol in Indian sportspersons in 2009 may be due to the improved detection by a more effective LCMS/MS method.

A novel study of screening and confirmation of modafinil, adrafinil and their metabolite modafinilic acid under EI-GC-MS and ESI-LC-MS-MS ionization.

OBJECTIVE: Adrafinil and modafinil have received wide publicity and have become controversial in the sporting world when several athletes were discovered allegedly using these drugs as doping agents. By acknowledging the facts, the World Anti-Doping Agency (WADA) banned these drugs in sports since 2004. The present study explores the possibility of differentiating adrafinil and modafinil and their major metabolites under electron impact ionization in gas chromatograph-mass spectrometer (GC-MSD) and electrospray ionization in liquid chromatograph-mass spectrometer (LC-MS/MS) by studying the fragmentation pattern of these drugs. MATERIALS AND METHODS: Adrafinil, modafinil and their major metabolite, modafinilic acid were analyzed on EI-GC-MSD and ESI-LC-MS/MS using various individual parameters on both the instruments. The analytical technique and equipment used in the analysis were an Agilent 6890N GC with 5973 mass selective detector for the GC-MSD analysis and an Agilent 1100 HPLC with API-3200 Triple quadrupole mass spectrometer for the LC-MS/MS analysis. Validation of both methods was performed using six replicates at different concentrations. RESULT AND DISCUSSION: The results show that adrafinil, modafinil and their major metabolite modafinilic acid could be detected as a single artifact without differentiation under EI-GC-MSD analysis. However, all drugs could be detected and differentiated under ESI-LCMS/MS analysis without any artifaction. The GC-MSD analysis gives a single artifact for both the drugs without differentiation and thus can be used as a marker for screening purposes. Further, the Multiple Reaction Monitoring (MRM) method developed under LC-MS/MS is fit for the purpose for confirmation of suspicious samples in routine sports testing and in forensic and clinical analysis.

Sample purification procedure for confirmation of 3'-hydroxy stanozolol by gas chromatography/high resolution mass spectrometry.

OBJECTIVE: To improve the detection limit of 3'-hydroxy-stanozolol by using the double extraction procedure, specific for basic drugs. MATERIALS AND METHODS: The urine samples were spiked in four replicates with 3'-hydroxy-stanozolol at different concentrations of 1, 2, 5 and 10 ng/mL, processed by two different methods and injected into gas chromatography/high resolution mass spectrometry (GC-HRMS) instrument. The data was analyzed by comparing the recovery values and the ion match criterion of the two procedures. RESULTS: The results show that the recovery percentage and ion match criterion of 3'-hydroxy-stanozolol at lower concentrations has a significant improvement when Solid phase extraction was performed, instead of Liquid-liquid extraction in the second extraction procedure. CONCLUSION: The sample preparation procedure using Oasis-MCX cartridges allows confirmation of 3'-hydroxy-stanozolol at the minimum required performance limit (MRPL) decided by the World Anti Doping Agency. This procedure may be used for the confirmation of suspicious samples found in routine testing, as it efficiently fulfills the ion-matching criterion laid down by the World Anti Doping Agency.

Comparative bioavailability of two brands of norfloxacin.

Twelve adult healthy volunteers participated on two occasions in a cross-over study with an interval of 30 days. The bioavailability of norfloxacin 400 mg administered as single oral dose was compared in an Indian preparation A (Torrent) and imported preparation B (Merck Sharp and Dohme (MSD), USA). Plasma was separated from the blood and stored at -20 degrees C for analysis by High Performance Liquid Chromatography. Time taken to achieve mean peak plasma concentration (Tmax) was 2.00 +/- 0.74 hours in case of Torrent (A) and 1.70 +/- 0.49 hours in case of Merck Sharp and Dohme, USA (B). The maximum plasma concentration (Cmax) ranged from 1.60 to 2.87 ug/ml in Torrent (A) and 1.18 to 2.28 ug/ml in case of MSD (B). Area under plasma concentration curve (AUCO-12hr) was 12.70 +/- 3.2 ug/ml/hour for 'A' and 14.80 +/- 2.80 ug/ml/hr for 'B'. Elimination half life (t1/2) for Torrent (A) was 9.25 +/- 5.10 hours and for MSD (B) it was 12.05 +/- 1.05 hours. There was no significant difference in the pharmacokinetic parameters between the two brands (Student's 't' test). Increased elimination half life and large bioavailability (AUC) with both the preparations in the present study suggest the need to be cautious while treating patients with renal problems and to use lower doses in Indian population to achieve desirable kinetics of norfloxacin.

Comparative bioavailability of two brands of ciprofloxacin.

Twelve adult healthy volunteers participated on two occasions in a cross-over study with an interval of 30 days. The bioavailability of ciprofloxacin 500 mg administered as single oral dose was compared in preparation A (Indian, Torrent) and preparation B (Imported, Bayer). The drug was administered early morning on an empty stomach. Blood samples were collected at 1/2,1,2,4,6,8,12 and 24 hours. Plasma levels of ciprofloxacin were determined by HPLC. Time taken to achieve peak plasma concentration (Tmax) was 2 hours (A) and 1.67 +/- 0.49 hours (B). Maximum plasma concentration (Cmax) ranged from 1.8 to 5.1 ug/ml (mean 3.08 +/- 0.99 ug/ml) for 'A' and 2.08 to 5.5 mu g/ml (mean 3.58 +/- 1.37 mu g/me for 'B'. Area under plasma concentration time curve ranged from 30.91 to 118.27 ug/ml/hr (mean 59.97 +/- 26.68 ug/ml/hr) in 'A' and 36.52 to 108.05 (mean 62.80 +/- 22.33 ug/ml/hr) in 'B' which is more than reported in Western literature. Large bioavailability of ciprofloxacin in the present study suggests the need to be cautious while treating patients with renal problems and to use lower doses in Indian patients to achieve desirable results. However, there was no significant difference in the pharmacokinetic parameters between the two brands (Paired 't' test and Wilcoxon Sign Rank test). It is therefore, concluded that both the preparations are comparable in terms of bioavailability.

Isoniazid and acetylisoniazid kinetics in serum and urine in pulmonary primary complex with intermittent regimen.

Twenty patients, 1 through 13 years of age from Pediatric Tuberculosis Clinic of All India Institute of Medical Sciences, New Delhi, suffering from pulmonary primary complex (PPC) were investigated for serum and urine concentrations of isoniazid (INH) and acetylisoniazid (AcINH). Patients were put on an intermittent regimen - 2HR, 4H2R2, INH (H) was given in a dose of 10 mg/kg/day for first 2 months (the daily dose phase), followed by 20 mg/kg/dose in biweekly phase of regimen for rest of the 4 months, whereas, rifampicin (R) was given as 12 mg/kg in both daily as well as biweekly phases. In the biweekly phase of regimen, after 7 days of biweekly administration of drugs, INH and AcINH concentrations were estimated by HPLC at 0,1,3,5 and 7 hours in serum, and at 0-3, 3-6, 6-12 and 12-24 hour-intervals of drug administration in urine. Peak concentrations of INH and AcINH (Mean +/- SD) were 2.6 +/- 1.8 and 5.5 +/- 2.6 micrograms/ml in serum (Cmax), and 5.7 +/- 4.8 and 21.5 +/- 12.1 mg in urine, respectively. Time to achieve Cmax (Tmax), for INH and AcINH were 1 and 5 hours respectively while time of peak concentration in urine for INH was 3-6 hours and for AcINH 6-12 hours. The half-life (T1/2) of INH was 4.5 hours and area under serum-concentration time-curve (AUC0-7h) was 20.7 micrograms/ml/h (mean values). In biweekly phase (4H2R2) of regimen, just before administration of next dose, 0 hour (or 72 hours) concentration of INH was estimated at 0.47 +/- 0.3 micrograms/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Tuberculous meningitis in children: manifestation of an immune compromised state.

Thirty children in the age group 0-4 years with tuberculous meningitis (TBM) were investigated for their immune status (cell-mediated and humoral). Twenty age, sex and nutritional status matched children were investigated on the same lines, who served as controls. Absolute T cell counts were significantly increased (3126 +/- 1623) (p < 0.01) in TBM, though T cell percentages were comparable (59.8% in TBM versus 54.44% in controls). Leucocyte migration inhibition (LMI) test positivity was high (93%) in TBM patients. Mean LMI index showed a highly statistically significant difference (p < 0.001) between the 'Before-therapy' (0.62 +/- 0.16) and 'During-therapy' (0.77 +/- 0.23) groups in TBM patients. Mantoux test positivity with 1 TU of PPD was low (53.0%) in TBM in comparison to LMIT positivity. In humoral immune response, quantitative function measured by EAC rosettes was not altered. However, there was a significant decrease in the levels of IgA (79.48 +/- 33.78 IU) (p < 0.01), IgG (115.01 +/- 32.56 IU) (p < 0.01) and IgM (148.50 +/- 51.88 IU) (p < 0.05) in TBM patients. There was no significant difference in the complement levels in the TBM and control groups. The results show a well developed CMI response but a poor humoral response in TBM and represent an inverse relationship between the CMI and humoral responses.

Serum concentrations of rifampicin and isoniazid in tuberculosis.

Ninety-four patients, 1-13 years of age suffering from different types of tuberculosis were investigated for serum rifampicin (RIF) and isoniazid (INH) concentrations using microbiological and fluorimetric methods, respectively. Of these, 64 (68.1%) had pulmonary primary complex (PPC); 20 (21.3%) progressive primary disease (PPD) and 10 (10.6%) tuberculous meningitis (TBM). Patients with PPC, PPD and TBM were given two-drug (6HR), three drug (2HRZ, 4HR) and four drug (2SHRZ, 4HRE, 3HE) regimens, respectively. RIF and INH were administered in a dose of 12 and 10 mg/kg/day, respectively. After 10-12 days of continuous therapy, their serum concentrations were estimated at 0, 2, 4, 6, 8 hours for RIF and 0, 1, 3, 5, 7 hours for INH. For RIF, the time to achieve maximum concentrations (Tmax) was 2 hours, range of mean of maximum concentration (Cmax) 3.38 to 3.88 micrograms/ml, terminal half life elimination (T1/2) 3.03 to 3.81 hours and area under serum concentration curve (AUC) 0-8 hours 24.7 to 28.3 micrograms/ml hours in different forms of tuberculosis. INH had a Tmax of 1 h, Cmax 4.38 to 8.17 micrograms/ml, T1/2 4.0 to 4.98 hours and AUC 0-7 hours 34.1 to 57.5 micrograms/ml hours. The concentrations achieved at 7-8 hours with these dosages were much above those required for therapeutic efficacy (minimum inhibitory concentration), being 50 to 250 times for RIF and 35-60 times for INH. We recommend pharmacokinetic studies with lower doses of RIF and INH for less toxic, equally effective and cheaper antitubercular chemotherapy.

Hepatic function in relation to acetylator phenotype in children treated with antitubercular drugs.

Sixty-six children suffering from pulmonary primary complex were investigated for evidence of hepatotoxicity (clinical and biochemical), in relation to the type of acetylator. Acetylator phenotype was determined by the sulphadimidine acetylation test in urine. A large proportion (83.0%) of the children were either normally nourished or had only grade-I malnutrition. Estimation of the levels of SGOT and SGPT determined before therapy and at monthly intervals for the first three months, and then three monthly for one year, did not indicate any biochemical evidence of hepatic derangement in relation to the type of acetylator. Hepatotoxicity of antitubercular drugs is greatly reduced when isoniazid and rifampin are used in lower dosages regardless of acetylator phenotype. Mild degree of malnutrition does not predispose the child to more hepatotoxicity.

Comparison between serum isonicotinic acid hydrazide (INH) levels and urinary sulfadimidine (sulfamethazine) acetylation as predictors of INH acetylator status.

The acetylator status of 40 children with pulmonary tuberculosis was investigated by (1) sulfadimidine (SDM; sulfamethazine) acetylation test in urine and (2) estimation of isonicotinic acid hydrazide (INH) levels. The antimode was at 70% based on the frequency distribution of SDM acetylation. Children acetylating less than 70% of administered SDM were taken as slow acetylators while those with more than 70% as rapid acetylators. The serum INH antimode was at 0.85 micrograms/ml. Thus serum values less than 0.85 micrograms/ml categorised a child as rapid and those with more than 0.85 micrograms/ml as slow acetylators. The sensitivity of these two methods was similar with a correlation coefficient r = 0.64. Thus the determination of the type of acetylator by SDM acetylation test is equally reliable and technically simpler and is recommended instead of INH serum concentration.