Method validation

Validation procedures were carried out according to FDA guidelines [17] as follows

System suitability

System suitability experiment was performed by injecting six consecutive injections using aqueous standard mixture equivalent to MQC concentration of the calibration curve. System suitability was performed at the start of the method validation and on each day as a first experiment. The %CV of retention time (RT) should be ≤ 2.00% and the %CV of area ratio should be ≤ 5.00%.

Carryover Effect

The carryover effect due to the auto sampler was investigated by injecting a sequence of un extracted samples consisting of reconstitution solution (RS), aqueous upper limit of quantification (AQ ULOQ), reconstitution solution (RS), aqueous lower limit of quantification(AQ LLOQ) and extracted samples containing standard blank (STD Blank), ULOQ, STD Blank and LLOQ. The carryover response in subsequent injections of RS or STD Blank after aqueous or extracted ULOQ should be ≤ 20.0% of the equivalent or extracted LLOQ standard.

Specificity

The plasma was obtained from six different blank rabbits that were housed in the same environment as the treated animals. The plasma was analysed by the optimized procedure to check for the interfering compounds. Response of interfering peaks in STD blank at RT of R and S enantiomers should be ≤ 20.0% of that in LLOQ and Response of interfering peaks in STD blank at RT of internal standard should be ≤ 20.0% of that in LLOQ.

Linearity

The linearity of the method was determined by using a 1/x2 weighted least square regression analysis of standard plots associated with a six-point standard curve (20- 1000ng/mL). The linear regression analysis of hydroxyzine enantiomers was performed by plotting the peak area ratio of hydroxyzine enantiomer (R/S) over IS (y) against the hydroxyzine concentration (x) in ng/mL. The % accuracy of all calibration standards should be within 85-115 and for LLOQ standard within 80-120.The regression(r) should be ≥0.99.0 for both the enantiomers.

Intra- and inter-day precision and accuracy

The precision (% CV) and accuracy was evaluated by analysing 6 replicates on two different days at different concentration levels corresponding to HQC, MQC, LQC and LLOQ during the course of validation. The accuracy was calculated as the absolute value of the ratio of the calculated mean values of the quality control samples to their respective nominal values, expressed as percentage. The % CV of intra and inter-day for LQC, MQC, HQC should be ≤15.0% and for LLOQ should be ≤20.0%. The % mean accuracy of intra and inter-day for LQC, MQC, and HQC should be 85-115 and for LLOQ should be 80-120.

Extraction recovery

The % mean recoveries of both enantiomers and the internal standard were determined by measuring the responses of the extracted plasma quality control samples against unextracted quality control samples at HQC, MQC, and LQC levels. The % CV of recovery at each QC level and for internal standard should be ≤ 15.00. The overall mean recovery for all QC levels should be ≤20.00.

Stability of analyte in plasma

Stability studies in plasma were conducted in various conditions using six replicates of LQC and HQC samples against the freshly spiked calibration standards and quality control samples. Freeze thaw stability of the spiked quality control samples was determined after four freeze thaw cycles stored at -28 ± 5 °C. Bench top stability of the spiked quality control samples was determined for a period of 12 hours stored at room temperature. Long Term analyte Stabilities of the spiked quality control samples were determined for a period of 35 days by storing them at -28 ± 5°C. The % mean accuracy for LQC and HQC should be within 85.00-115.00. The % CV should be ≤15.00.

Pharmacokinetic study and data analysis

Male rabbits were obtained from JSS medical college animal house (JSS University, Mysuru) and were kept in a constant temperature (20 ± 20C) and relative humanity (50 ± 10%). Six rabbits weighing 3.0-3.5kg were orally and intramuscularly administered with racemic hydroxyzine at a dose of 5, 2.5mg/kg respectively. Blood samples (1mL) were collected in EDTA-coated tubes at 0 (pre-dose), 0.5, 1, 1.5, 2, 3, 6, 12, and 24 h after drug administration. The blood samples were immediately centrifuged at 10,000 for 15 min, and the plasma samples were then separated and stored at -280C until analysis.

Pharmacokinetic parameters were calculated with the non-compartmental model using PK Solutions 2.0 software. The maximum plasma concentration (Cmax) and the time to the maximum plasma concentration (Tmax) were taken from the concentration-time curves. The area under the plasma concentration-time curve to the last measurable con-centration (AUC0-t) was calculated using the trapezoidal rule. The terminal phase was determined by visually examining the log-transformed concentration-time curves. The elimination rate constant (λz) was determined by linear regression analysis of the terminal phase of the log concentration-time curve. The area under the plasma concentration-time curve to infinity (AUC0-∞) was estimated by combining AUC0-t with AUCt-∞, where AUCt-∞represents the residual area of the drug from time t to infinity and was calculated by dividing the last measurable plasma concentration value by the elimination rate constant (λz). The elimination half-life (t1/2) was calculated as 0.693/ λz. The mean residence time (MRT) was estimated from AUMC/AUC, where AUMC is the area under the first moment concentration—time curve. The differences in pharmacokinetic parameters were determined using Student’s t-test with a significance level of P < 0.05.

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