Development and validation of a copper ligand-exchange chromatographic method for the estimation of D-lactic acid in Ringer-lactate solution.

In this study, the chromatographic conditions for separation and determination of L- and D-lactic acid enantiomers by copper ligand exchange chromatography have been examined and optimized statistically using a response surface methodology (RSM). The chromatographic variables: copper sulfate, acetic acid and organic modifier were screened by operating a 2-level full factorial design (FFD). The significant effect of independent chromatographic variables was analyzed using the analysis of variance (ANOVA). Variables proved significant (p < 0.05) were cautiously tuned using RSM with a face-centered central composite design. Moreover, a D-optimality design was employed to minimize the variation in the regression coefficients of the fitted model. The proposed model represented an excellent example of fulfilling the efficiency of factorial designs in optimizing the chromatographic conditions and maximizing the output. The chromatographic separation was achieved on Supelco Astec CLC-D chiral bidentate ligand (5.0 µm, 150.0 × 4.6 mm). The isocratic mobile phase composition was 7 mM anhydrous copper sulfate in 1.0 mM acetic acid containing 4% methanol. A photodiode array detector was used to determine the optimal detection wavelength, which was at 236 nm. A linear calibration curve was obtained in the range of 30.0-3600 µg mL-1 with a high value for the coefficient of determination (R2 ≥ 0.999). The optimized method has been successfully applied to the determination of lactate in the commercial Ringer-lactate solution for injection. The results obtained were in excellent agreement with the label claim with no interference from other additives commonly co-formulated with the drug. Intra- and inter-day precision, detection and quantification limits, as well as percent coefficient of variation, have been estimated according to ICH guidelines for assessment of analytical procedures.

Validated liquid chromatography-tandem mass spectrometry method for simultaneous determination of clopamide, reserpine and dihydroergotoxine: Application to pharmacokinetics in human plasma.

A simple, sensitive and rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the simultaneous quantitation of clopamide, reserpine and dihydroergotoxine (ergoloid mesylates) in human plasma. Under basic conditions, liquid-liquid extraction using ethyl acetate was efficiently used for extraction of the analytes from plasma samples in presence of indapamide as internal standard (IS). The analytes were separated with isocratic elution on Phenomenex(®) Synergi Fusion-RP 80A column (50×4.6mm, 4µm). With positive ion electrospray ionization (ESI), the analytes were quantified and monitored on a triple quadrupole mass spectrometer using Multiple Reaction Monitoring (MRM) scanning mode. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. The method was linear in the concentration range of 0.04-30.00ng/mL for reserpine, 1-96.00ng/mL for clopamide, and 0.05-40.00ng/mL for dihydroergotoxine alkaloids, respectively. For all analytes, the high sensitivity of HPLC-MS/MS method revealed sufficient lower limit of quantification (LLOQ) ranged from 0.04-1ng/mL using 1mL of plasma. The recoveries from spiked control samples were ≥86.16% for all analytes and IS. The intra- and inter-day precision variations were lower than 13.03% while the accuracy values ranged from 91.76% to 111.50%. The developed method was successfully applied to pharmacokinetic study of fixed dose combination of clopamide, reserpine and dihydroergotoxine in healthy male volunteers.

Validated spectrofluorimetric method for the determination of atorvastatin in pharmaceutical preparations.

A rapid, sensitive and simple spectrofluorimetric method was developed for the estimation of atorvastatin. In this method, the native fluorescence characteristics of atorvastatin have been studied in both acidic and basic media. High sensitivity was obtained with 5% acetic acid at 389 nm using 276 nm for excitation. Regression analysis showed a good correlation coefficient (r=0.9995) between fluorescence intensity and concentration over the range of 1.5-4 µg/mL with detection limit of 0.012 µg/mL. The proposed method was successfully applied to the analysis of atorvastatin in pure and pharmaceutical dosage forms with average recovery of 100.29±0.47%. The results were compared favorably with those of the reported method.

Two different spectrofluorimetric methods for simultaneous determination of gemfibrozil and rosiglitazone in human plasma.

Two accurate, reliable, and highly sensitive spectrofluorimetric methods were developed for simultaneous determination of binary mixture gemfibrozil and rosiglitazone in human plasma without prior separation steps. The first method is based on synchronous fluorescence spectrometry using double scans. At Δλ=27nm, gemfibrozil yields detectable signal that is independent of the presence of rosiglitazone. Similarly, at Δλ=120nm the signal of rosiglitazone is not influenced by the presence of gemfibrozil. Signals at two wavelengths, 301 (Δλ=27nm) and 368nm (Δλ=120nm) vary linearly with gemfibrozil and rosiglitazone concentrations over the range 100-700ngmL(-1) (for gemfibrozil) and 20-140ngmL(-1) (for rosiglitazone), respectively. The limits of detection (LOD) were 2.3 and 2.72ngmL(-1) for gemfibrozil and rosiglitazone, respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which 258nm was selected as the excitation wavelength. Two equations are constructed based on the fact that at ( λ(EM)2=302 nm of gemfibrozil) and (λ(EM)2=369 nm of rosiglitazone) the fluorescence of the mixture is the sum of the individual fluorescence of gemfibrozil and rosiglitazone. The limits of detection (LOD) were 28.1 and 23.63ngmL(-1) for gemfibrozil and rosiglitazone, respectively. The proposed methods were successfully applied for the determination of the two compounds in synthetic mixtures and in human plasma with a good recovery.

Colorimetric determination of simvastatin and lovastatin in pure form and in pharmaceutical formulations.

Simple, accurate and precise colorimetric method for the determination of simvastatin and lovastatin in tablets is described. The method is based on the reaction of simvastatin or lovastatin with hydroxylamine in alkaline medium to form the corresponding hydroxamic acid derivatives which, on treatment with ferric ion in acid medium, yield highly colored ferric-chelate complex with maximum absorption at 513nm. Beer's law is obeyed in the concentration ranges 0.04-0.4mgml(-1) for both simvastatin and lovastatin, respectively. Molar absorptivity values, as calculated from Beer's law data, were found to be 1.15x10(3) and 1.09x10(3)lmol(-1)cm(-1) for simvastatin and lovastatin, respectively. Optimal experimental parameters for the reaction have been studied. The validity of the described procedures was assessed. Statistical analysis of the results reflects that the proposed procedures are precise, accurate and easily applicable for the determination of simvastatin and lovastatin in pure form and in pharmaceutical preparation.