Jyoti Kumar Paliwal

Choosing the calibration model in assay validation.

Data transformations and weighting schemes are normally used to obtain the best-fit of standard curves in bioanalysis and the calibration model is usually selected during prevalidation. In the present study, a comparison has been made between unweighted and weighted (1/x, 1/x2, and 1/square root of x) regression models with or without an intercept in achieving the best-fit for the standard curve of CDRI compound 81/470, a new anthelmintic agent, in cow milk. Validation samples in milk at the LLOQ, medium, and high concentrations were also analysed by each of the calibration models. An unweighted regression equation with an intercept overestimated the concentrations at the LLOQ. An unweighted equation without intercept and weighted equations with or without an intercept significantly minimized the bias at the LLOQ without distorting the results at higher concentrations. Hence, an unweighted equation for a straight line passing through the origin was found to be the best model for a standard curve of 81/470 in milk. Similar results were obtained for 81/470 and UMF-078 in serum and plasma, respectively. Bioanalysts should routinely test these models to obtain the best fit model for their calibration curves as part of their assay validation not during prevalidation.

High performance liquid chromatographic (HPLC) determination of centchroman in human serum and application to single-dose pharmacokinetics

A simple and sensitive (2 ng/ml) HPLC method with fluorescence detection has been developed to measure serum concentrations of centchroman, a new nonsteroidal antifertility agent. The method was sufficiently sensitive to follow the drug over 21 days in human volunteers. Pharmacokinetic parameters of centchroman were determined after a single oral dose of 60 mg (2 × 30-mg tablets) in two healthy female volunteers. Centchroman is slowly eliminated from serum, showing a biexponential disappearance curve from serum. The terminal half-life of centchroman in the two volunteers was 168 and 175 hr, respectively.

Simultaneous liquid chromatographic determination of centchroman and its 7-demethylated metabolite in serum and milk☆

A precise and sensitive high-performance liquid chromatographic assay was developed and validated for determination of centchroman (I) and its 7-demethylated metabolite (II) in human serum and milk. The serum, at alkaline pH, was extracted with diethyl ether. In the case of milk, after precipitation of the milk protein with acetonitrile, the supernatant was evaporated to dryness and then extracted with diethyl ether at alkaline pH. After solvent evaporation the residue was reconstituted in mobile phase. Separations were accomplished by reversed-phase liquid chromatography using a Spheri-5 cyano column. Recoveries of I and II were always > 95%. Excellent linear relationships (r > 0.999) were obtained between the measured and added concentration ratios of the corresponding serum and milk concentrations over a range of 1 to 1000 ng/ml and 2.5 to 1000 ng/ml for I and II, respectively.

Evaluation of interaction potential of certain concurrently administered drugs with pharmacological and pharmacokinetic profile of centchroman in rats.

Centchroman (Ormeloxifene) is a nonsteroidal, selective estrogen receptor modulator, oral contraceptive and anticancer agent, and is intended for long-term use by women. In view of its vast clinical application and the interaction of steroidal oral contraceptives with certain commonly used therapeutic agents, evaluation of interaction of certain concomitantly administered therapeutic agents (ibuprofen, rifampicin, diazepam, salbutamol, nifedipine, paracetamol, haloperidol, and tetracycline), in terms of both the postcoital contraceptive efficacy and pharmacokinetic profile, with centchroman was undertaken in female Sprague-Dawley rats. Among the representatives from each commonly used therapeutic category, interaction (pharmacokinetic) was observed with ibuprofen (60 mg/kg, twice daily), haloperidol (0.7 mg/kg, twice daily), and tetracycline (140 mg/kg, twice daily) coadministration on Days 1 through 5 postcoitum. Of these three therapeutic agents, only tetracycline interfered with the contraceptive efficacy of centchroman. It reduced the bioavailability of centchroman and its active metabolite by increasing their excretion through bile and feces. Increased metabolite excretion on tetracycline coadministration indicates the enterohepatic recirculation of the metabolite, not the parent drug. However, the effect of tetracycline was negated by the inclusion of lactic acid bacillus spores in the regimen.

Binding of centchroman with human serum as determined by charcoal adsorption method

Protein binding of drugs is an important factor influencing both pharmacokinetic and pharmacodynamic parameters. Thus, knowing the extent of protein binding of drugs is crucial. Centchroman is a non-steroidal once a week oral contraceptive. It has been reported to be useful for the treatment of breast cancer and osteoporosis. Ample data has been generated on pharmacokinetics of centchroman in animals and humans. The extent of protein binding of centchroman has not been established so far. Non-specific adsorption of the drug limits the use of conventional methods like ultrafiltration and equilibrium dialysis. A method of charcoal adsorption as reported by Yuan et al. (method I) was used after modification (method II) to determine its binding to human serum. The extent of protein binding (%) is estimated from decline of percent drug remaining in the supernatant after adding the charcoal. Study was carried out at 1- and 10-microg/ml concentrations in drug free human serum samples and an HPLC assay was used to determine concentration-time data. The percentage of centchroman remaining in serum versus time data was analysed using non-linear fitting programs on WinNonlin software. Method II was found to give higher estimates of protein binding than the former method by preventing the dilution effect. Using this method, the extent of protein binding of centchroman was found to be 101.83+/-1.28 and 94.87+/-3.59% at 1 and 10 microg/ml, respectively. However, it was approximately 90% in the individual serum samples showing intersubject variability in protein binding of centchroman.

Determination of ampicillin in serum by high-performance liquid chromatography with precolumn derivatization☆

A high-performance liquid chromatographic assay method using precolumn derivatization and fluorescence detection has been developed and validated for the determination of ampicillin in serum. The presented method is simple and provides improved selectivity and sensitivity over other existing HPLC methods. It is linear over the concentration range of 100 to 10,000 ng/ml (method 1) and 2 to 1000 ng/ml (method 2) and the extraction recovery is more than 75%. The coefficient of variation is found to be less than 10% over the concentration ranges studied.

Simultaneous determination of a new antimalarial agent, CDRI compound 80/53, and its metabolite primaquine in serum by high-performance liquid chromatography

Compound 80/53 (I) is a new substance being developed as an antimalarial agent. It is unstable in acidic conditions where it is converted into primaquine. A high-performance liquid chromatographic assay for simultaneous determination in serum of I and primaquine has been developed. Conditions were optimized to minimize the conversion of I into primaquine. The method includes extraction of the unchanged compound and primaquine from serum samples with hexane-2-propanol (pH > 8). Separation was accomplished by reversed-phase chromatography on a C18 column with acetonitrile-tetrahydrofuran-phosphate buffer. The recoveries of I and primaquine were always greater than 70%. No interference was observed in extracts obtained from drug-free serum. The detector response was linear with concentrations of I and the metabolite in the ranges 25-400 and 10-180 ng/ml, respectively, and the within-day precision (coefficient of variation) remained less than 13.7% for I and 12.5% for primaquine. The method is suitable for the determination of concentration-time profiles of I and primaquine in human serum.

A rapid and sensitive high performance liquid chromatographic assay of the new antimalarial compound 80/53 in serum with a novel sample clean-up method and its pharmacokinetics in rabbits

The compound 80/53 (AM) is a new antimalarial agent synthesized by this institute as a safer and less toxic analogue of primaquine. It was found to exhibit fluorescence in acetonitrile solution and this finding was exploited to develop a selective and sensitive high performance liquid chromatographic (HPLC) assay of the AM in rabbit serum. The sample clean-up was done in a single step by simultaneous protein precipitation and extraction with acetonitrile in the presence of sodium sulfate. The lower limit of quantitation of the method was 50 ng ml-1 using 100 microliters of serum sample. The method was fully validated from 50 to 1600 ng ml-1 concentration range with a recovery ranging from 70 to 75%. The within- and between-run variability was less than 10% and the drug in serum was stable over four freeze-thaw cycles and up to 24 h in injection solvent at 4 degrees C. The method was applied to determine the pharmacokinetic parameters of AM in 5 rabbits receiving a single bolus intravenous and peroral dose in a crossover study. The concentration-time data after a 5 mg kg-1 i.v. dose in rabbits was best fitted to the two compartment body model with first order absorption and elimination rate constants. The terminal half-life and MRT of AM were 95.3 +/- 43.5 and 104 +/- 10.6 min respectively. After administering a single 20 mg kg-1 oral dose, the serum levels of AM in all the rabbits declined below the quantitation limit by 90 min and it was not possible to fit the data by the compartmental approach. The MRT and AM after oral dose was 31.1+2-8.3 min. Application of the assay has also been extended to analyze the serum samples of rats, monkeys and humans.

Centchroman: A new non-steroidal oral contraceptive in human milk

Centchroman, a non-steroidal oral contraceptive drug, was given to 13 nursing mothers comprising two groups. Each participant in group I (n = 8) received a single 30 mg dose, and in group II (n = 5) each participant received a 30 mg twice a week dose for twelve weeks. Simultaneous blood and milk samples were collected and analyzed for the parent drug by high performance liquid chromatography. In the single dose study (group I), the mean +/- peak centchroman concentrations in milk and serum were 78.7 +/- 28.4 and 63.6 +/- 23.6 ng/ml with milk-to-serum (M/S) ratio of 1.4 +/- 0.9. There was no significant increase in centchroman concentrations in milk after multiple dosing (group II). However, serum concentrations reached up to 112.5 ng/ml at 6 h after the 13th dose. Average M/S ratios were insignificantly different at trough (prior to next dose) and at peak (4-6 h after dose) centchroman levels. Additionally, the breast milk and serum centchroman concentrations showed a significant correlation (r = 0.64, P < 0.01), indicating that the amount of centchroman excreted into breast milk is dependent on serum concentrations. The weekly dose (% of the maternal dose) of centchroman ingested by the breast-fed infant at peak maternal serum and milk levels was in the range of 0.4 to 11.5%, assuming a weekly milk uptake of 1.05 l/kg. There was no significant difference in the dose ingested by the infants between the two dosing groups. These levels of centchroman passing into breast milk and subsequent exposure to the infants are unlikely to be of any physiological consequence.

Tissue distribution and excretion of CDRI-81/470 in rats.

Methyl‐N[5[[4‐(2‐pyridinyl)‐1‐piperazinyl]carbonyl]‐1H‐benzimidazol‐2‐yl] carbamate (CDRI‐81/470) is a broad spectrum anthelmintic agent, effective against both intestinal and systemic parasitism. Tissue distribution and excretion of CDRI‐81/470 were studied in rats after a single oral dose of 100 mg kg−1 CDRI‐81/470.