l the trials except mobile phase variation remaining parameters were kept constant like flow rate, injection volume, column temperature, autosampler temperature, splitter to flow etc. The chromatograms obtained with the final optimized conditions were shown in Figure 3.

3.2. Optimization of Sample Preparation

After optimization of mass spectrometric conditions and chromatographic conditions next step was tried for optimization of sample processing technique. Keeping the 50 µL plasma volume constant, protein precipitation and liquid-liquid excitation techniques were tried to get better & simple sample processing technique to get better recovery and sensitivity of analytes. Figure 4 depicts the overview of sample preparation methods. It has been found that recovery for PIO with EtOAc and recovery for STG with TBME is less in comparison to protein precipitation technique. Protein precipitation technique and

Figure 4. Sample preparation trails (Trail-A, B and C) for LC-MS/MS injection, because of high recovery and precision protein precipitation technique was finally considered for injection.

both LLE techniques are free of matrix effect to both analytes but because of high recovery and simplicity protein precipitation technique was finalized for sample processing. Ideally, an internal standard should mirror the analytes in as many ways as possible. It should track the analyte during extraction and compensate for any analyte on the column and any inconsistent response due to matrix effects. To compromise the processing level atongoing analysis level due to instrument for quantitation of both analyte by method Tolbutamide was tried as an internal standard (IS). Because of its ionization in positive ionization mode, high & precise recovery from plasma and its ability to give precise results on preliminary precision accuracy trials, Tolbutamide was finalized as IS. The results of method validation support the use of Tolbutamide and were acceptable in this study based on FDA guidelines.

3.3. Sensitivity

The Sensitivity of the method was evaluated by analyzing 6 LOQ QC (Lower Limit of Quantification) at 10.98 ng·mL1 for STG and 8.25 ng·mL1 for PIO The precision and accuracy for STG at LOQ QC level were found to be 7.1% CV and 106.57% nominal respectively. The precision and accuracy for PIO at LOQ QC level were found to be 10.1% CV and 102.81% nominal respectively.

3.4. Linearity, Precision Accuracy and Dilution Integrity

The STG calibration curves were linear from 10.98 - 2091.77 ng·mL1 with correlation coefficient (r2) of 0.9975, while for PIO the linear dynamic range was from 8.25 - 1571.63 ng·mL1 with correlation coefficient (r2) of 0.9975 between five calibration curves, wherein the results are shown in Table 3. The precision values observed for the back-calculated concentrations are presented in Table 4. Intra-day and inter-day precision was less than 9.0% at the three QC levels for both the analytes. The precision values calculated at LLOQ level were 15.07% and 9.59% (intra-day); 10.18% and 6.10% (inter-day) for STG and PIO respectively. Intra-and inter-day accuracy values expressed in terms of %RE were within −5.0% to 11.0% for both the analytes. The mean back-calculated concentrations for 1/4 dilution samples was within 85% - 115% of their nominal concentration, while the coefficient of variation (%CV) for this dilution was less than 3.7% for both the analytes.

3.5. Stability Study

Stability experiments were performed to evaluate their stability in stock solutions and in plasma samples. The conditions which occurred during actual study sample analysis were simulated in method validation stability studies, such as: stock solution stability of STG and PIO; stability in plasma at room temperature; extracted sample stability (Auto-sampler stability at 15˚C); freeze thaw stability and long term stability at −80˚C. Stock solution of STG and PIO were stable at room temperature for 5 h and at 2˚C - 8˚C for 7 days with mean percent change within ±5%. SIT and PIO in control rat plasma were stable for at least 6 h at room temperature; upto 24 h (process stability) in the autosampler maintained at 15˚C and for minimum three freeze and thaw cycles. The long term stability was also established for 14 days at −80˚C. The observed and acceptable percent change for the stability experiments are shown in Tables 5(a) and (b).

3.6. Application of the Method to Pharmacokinetics Study

The method was successfully applied to estimate the plasma concentration versus time profile of selected

Table 3. Back-calculated concentration of Calibration Standards (CS) from respective calibration curves for STG and PIO.

Table 4. Precision and range of the proposed method.

(a)(b)

Table 5. Stability experiments of PIO (a) and STG (b) in plasma.

drugs (STG and PIO) in plasma following simultaneous oral administration at 10 mg·kg1 doses of STG and PIO in Wistar rats (n = 2). Pharmaco-kinetic study was done on two Wistar rats after IAEC (Institutional Animal Ethics Committee) approval. Oral formulation were prepared separately in suspension form (1 mg·mL1) by triturating accurately weighed amount of STG and PIO powdered compound in methyl cellulose solution (0.5%, w/v water) in gravimetric dilution pattern using suspending agent Tween 80 1.25 µL for each 1 mL Oral suspension. Dosesof STG and PIO (10 mg·kg1) were administered simultaneously using an oral gavage at 10 mL·kg1 volume in Wistar rats after overnight fasting (12 hr) and these animals were continued for fasting till 4 hr post dose.

The blood samples (0.17 mL) were collected from retro orbital sinus at predose, 15, 30 min and 1, 2, 4, 6, 10, 12, 24 hrs post dose of simultaneous administration of STG and PIO. After blood collection samples were kept on ice bath till further processing. These samples were separated for plasma by centrifuging at 4˚C for 10 min at 4000 rpm and then stored at −80˚C till further analysis. These samples were analyzed for estimation of the levels of STG and PIO. Results for plasma levels (ng·mL1) of STG and PIO after administration in male Wistar rats respectively. Pharmacokinetic parameters data generated for non-compartment modeling with WinNonlin 5.1 software are tabulated for STG and PIO respectively. Summary PK profile of STG and PIO after single dose administration in male Wistar rats is shown in Table 6 and mean plasma concentration-time profile for STG and PIO is shown in Figure 5.

4. Conclusion

The objective of this work was to develop a simple, high throughput, and sensitive method to simultaneously estimate STG and PIO in plasma following simultaneous oral administration of 10 mg·kg1 doses of STG and PIO in Wistar rats. Moreover, the sensitivity of this method is good for simultaneous estimation of SIT and PIO. Also, the chromatographic run time of 5.0 min makes it possi-

Table 6. Mean Pharmacokinetic profiles of STG and PIO after oral administration at 10 mg·kg−1 doses in Wistar rats.

Figure 5. Mean pharmacokinetic profile of STG and PIO after oral administration of 10 mg·kg−1 doses of STG and PIO in Wistar rats.

ble to analyze 200 samples in a day. From the results of the validation parameters, we can conclude that the method can be very useful for therapeutic drug monitoring both for analysis of routine samples of single dose or multiple dose pharmacokinetics and also for the clinical trial samples with desired precision, accuracy and high throughput.

5. Acknowledgements

The authors are indebted to Dr. Swaroop Kumar Vakkalanka, MD & CEO, Incozen therapeutics Private Limited, for providing the necessary facilities to carry out this work. The authors gratefully acknowledge NIPER-Hyderabad for motivation and assistance during the course of dissertation work.

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NOTES

*Corresponding author.

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