QbD-Driven Development and Validation of Liquid Chromatography Tandem Mass Spectrometric Method for the Quantitation of Sildenafil in Human Plasma.

The present work was employing the Quality by Design approach for the development and validation of a LC-MS-MS method to support the clinical advancement in determination of sildenafil in human plasma using lorazepam as an internal standard. Sample preparation involved solid phase extraction and calibration range observed between 3 and 1,700 ng/mL. The method was systematically optimized by employing Box-Behnken design and used mobile phase flow rate, pH and composition of mobile phase as the critical factors, and assessing the design for retention time and peak area as the responses. A substantial decrease in the variability associated with the method variables was shown in optimization studies and confirmed enhanced method robustness. The present studies revealed that developed method achieves all the regulatory requirements for linearity, accuracy, precision, selectivity, sensitivity and stability for the determination of sildenafil in human plasma. There was not any significant change in the stability of the drug shown by stability studies, performed in human plasma through freeze-thaw cycles, bench-top stability, short-term stability, long-term stability and auto sampler stability. In short, this method shows satisfactory results for the analysis of sildenafil in human plasma and possesses high degree of utility in pharmacokinetic and bioequivalence studies.

QbD-Driven Development and Validation of a Bioanalytical LC-MS Method for Quantification of Fluoxetine in Human Plasma.

The current studies describe the Quality by Design (QbD)-based development and validation of a LC-MS-MS method for quantification of fluoxetine in human plasma using fluoxetine-D5 as an internal standard (IS). Solid-phase extraction was employed for sample preparation, and linearity was observed for drug concentrations ranging between 2 and 30 ng/mL. Systematic optimization of the method was carried out by employing Box-Behnken design with mobile phase flow rate (X1), pH (X2) and mobile phase composition (X3) as the method variables, followed by evaluating retention time (Rt) (Y1) and peak area (Y2) as the responses. The optimization studies revealed reduction in the variability associated with the method variables for improving the method robustness. Validation studies of the developed method revealed good linearity, accuracy, precision, selectivity and sensitivity of fluoxetine in human plasma. Stability studies performed in human plasma through freeze-thaw, bench-top, short-term and long-term cycles, and autosampler stability revealed lack of any change in the percent recovery of the drug. In a nutshell, the developed method demonstrated satisfactory results for analysis of fluoxetine in human plasma with plausible utility in pharmacokinetic and bioequivalence studies.

Development and validation of an improved LC-MS/MS method for the quantification of desloratadine and its metabolite in human plasma using deutrated desloratadine as internal standard.

PURPOSE: For the determination of desloratadine (DES) and 3-OH desloratadine (3-OHD) in human plasma using deutrated desloratadine (DESD5) as internal standard (IS), a novel stability indicating liquid chromatography-tandem mass spectrometric method was developed and validated to support the clinical advancement. MATERIALS AND METHODS: The solid-phase extraction method used for sample preparation and calibration range was 100-11,000 pg/ml, for which a quadratic regression (1/x(2)) was best fitted. The blank plasma was screened and observed free from any endogenous interference. RESULTS: The accuracy (% nominal) at low limit of quantification LLOQ level for DES and 3-OHD was 100.4% and 99.9% whereas precision (%CV) was 4.6 and 5.1%. They (DES and 3-OHD) were stable in human plasma after five freeze-thaw cycles, at room temperature for 23.8 hour, bench top stability for 6.4 hour. CONCLUSION: This method fulfills all the regulatory requirements for selectivity, sensitivity, precision, accuracy, stability, goodness of fit, and ruggedness of the method for the determination of DES and 3-OHD in human plasma.

Development and validation of LC-MS/MS method for the quantitation of lenalidomide in human plasma using Box-Behnken experimental design.

For the determination of lenalidomide using carbamazepine as an internal standard, an ultra-fast stability indicating LC-MS/MS method was developed, validated and optimized to support clinical advancement. The samples were prepared by solid-phase extraction. The calibration range was 2-1000 ng mL(-1), for which a quadratic regression (1/x(2)) was best fitted. The method was validated and a 3(2) factorial was employed using Box-Behnken experimental design for the validation of robustness. These designs have three factors such as mobile phase composition (X(1)), flow rate (X(2)) and pH (X(3)) while peak area (Y(1)) and retention time (Y(2)) were taken as response. This showed that little changes in mobile phase and flow rate affect the response while pH has no affect. Lenalidomide and carbamazepine were stable in human plasma after five freeze thaw cycles, at room temperature for 23.7 h, bench top stability for 6.4 h. This method competes with all the regulatory requirements for selectivity, sensitivity, precision, accuracy, and stability for the determination of lenalidomide in human plasma, as well as being highly sensitive and effective for the pharmacokinetic and bioequivalence study of lenalidomide.

Pharmacokinetics of a fixed-dose combination of atorvastatin and metformin extended release versus concurrent administration of individual formulations: a randomized, open-label, two-treatment, two-period, two-sequence, single-dose, crossover, bioequivalence study.

BACKGROUND: Type 2 diabetes mellitus is associated with a 2- to 4-fold increased risk of coronary heart disease (CHD). Combined therapy with an antihyperglycaemic agent and an HMG-CoA reductase inhibitor (statin) is indicated for the treatment of diabetic patients at risk of CHD. Patients with type 2 diabetes are generally considered to be at equivalent cardiovascular disease risk to patients with established CHD, and should have low-density lipoprotein (LDL) cholesterol levels reduced to <100 mg/dL or by 30-40%. Atorvastatin is the drug of choice for lowering LDL cholesterol levels. Metformin is the first therapeutic option in type 2 diabetes patients who are overweight or obese because it may also prevent vascular complications and mortality. Hence, a fixed-dose combination (FDC) of atorvastatin 10 mg and metformin 500 mg extended release (ER) was developed for patients with type 2 diabetes with or without hyperlipidaemia. OBJECTIVES: This study set out to establish bioequivalence between treatment 1 (test) - atorvastatin/metformin ER 10 mg/500 mg FDC, and treatment 2 (reference) - atorvastatin 10 mg (Lipitor®) and metformin 500 mg (Glucophage® XR) administered concurrently as individual tablets. METHODS: The study was a randomized, open-label, two-treatment, two-period, two-sequence, single-dose, crossover, bioequivalence study in 40 male subjects of Asian origin aged 18-45 years. The order of receiving the test and reference treatments for each subject during both the periods of the study was determined according to an SAS®-generated randomization schedule. The two treatments were separated by a washout period of 11 days. Blood samples were collected pre-dose and up to 72 hours post-dose in each period for determination of plasma atorvastatin/metformin concentrations and calculation of the respective pharmacokinetic parameters. ANOVA was performed on the lognormal-transformed pharmacokinetic parameters. A 90% confidence interval (CI) for the ratios of the test and reference product averages (least squares means) was calculated for atorvastatin and metformin to establish bioequivalence. RESULTS: The 90% CIs for atorvastatin and metformin were within the bioequivalence acceptance criteria of 80-125%. The 90% CIs obtained for atorvastatin for maximum plasma concentration (C(max))(,) area under the plasma concentration-time curve (AUC) from time zero to time of last measurable concentration (AUC(last)) and AUC from time zero to infinity (AUC(∞)) were (88.11, 106.93), (91.18, 107.94) and (89.25, 106.60), respectively. The 90% CIs observed for metformin for C(max,) AUC(last,) AUC(∞) and AUC from time zero to 24 hours (AUC(24)) were (113.3, 124.0), (102.65, 117.97), (101.87, 116.82) and (102.44, 117.53), respectively. The two treatments were well tolerated by the study subjects. CONCLUSION: Atorvastatin/metformin ER 10 mg/500 mg FDC has similar bioavailability to the co-administration of separate atorvastatin 10 mg and metformin 500 mg tablets. The FDC tablets show similar safety and tolerability profiles to their individual components. Therefore, atorvastatin/metformin ER 10 mg/500 mg FDC tablets can be used safely in clinical settings to decrease the pill burden and increase patient compliance with therapy.