BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF PIRFENIDONE BY RPHPLC METHOD AND ITS APPLICATION TO THE DETERMINATION OF DRUG FOOD INTERACTION STUDY IN WISTER RATS ABSTRACT N. Tamilselvi *, Dona Sara Kurian Department of Pharmaceutical Analysis KMCH college of pharmacy Coimbatore A simple precise, accurate RP-HPLC method has been developed and validated for bioavailability study of pirfenidone in wister rat plasma. The separation and quantization of pirfenidone was achieved on a C18 reversed phase column using the mobile phase in gradient mode constituting of eluant A HPLC grade water (adjusted to ph 3.5) and eluant B 20% acetonitrile and 15% of methanol in the ratio of 60: 40 at a flow rate 1 mlmin -1. Eluted components were detected at 324 nm. The method showed good linearity for Pirfenidone in the range of 50 250ng ml -1, Y=54.97x - 349.5and correlation coefficient R 2 is 0.998 respectively. The limit of quantitation (LOQ) and limit of detection (LOD) were found to be 12 and 20 ng ml -1 respectively.the developed method shows good accuracy and precision.. Accuracy ranges from 98.49% to 99.37% with the precision 6.43% to 7.67% in inter-day method. Intra-day method the accuracy ranges from 98.64% to 99.33% with the precision 5.64% to 6.93 %. For bioanalytical study, parameters like C max, T max, AUC0-t, AUC 0-, K eli and T 1/2 are compared by statistical analysis. The maximum concentration (C max ) obtained for pirfenidone before and after food was found to be 1020.76 ng ml -1 and 836.5ng ml -1 respectively. The half life (T 1/2 ) of pirfenidone before and after food were calculated and found to be 2.732158 h and 4.009485 h respectively. Area under the curve t AUC. 0 of pirfenidone before food was calculated as 3060.95nghr ml-1 and t AUC. 0 3053.07ng ml -1. Area under the curve and AUC was found to be of pirfenidone after food was calculated as 2534.16nghr ml-1 AUC 0 was found to be 2510.64ng ml -1. Elimination rate constant for Pirfenidone was found to be 0.004228 h -1 and 0.002881 h -1 respectively. This method was successfully applied to the bioavailability study of pirfenidone. KEYWORDS: Pirfenidone, Tinidazole, RP-HPLC, Rat plasma, Validation INTRODUCTION Pirfenidone is a novel compound with demonstrated anti-inflammatory, anti-fibrotic and antioxidant activities that makes it a suitable candidate molecule for managing idiopathic pulmonary fibrosis[1]. It is the first and only drug which has been approved for the treatment of IPF.Pirfenidone is a small non-peptide molecule of low molecular weight (185.2 daltons). The chemical name is 5-methyl-1-phenyl-2-(1H)-pyridone and its empirical formula is C 12 H 11 NO 2. Pirfenidone inhibits fibroblast proliferation, differentiation and relatedcollagen synthesis. Pirfenidone inhibited the production and activity of TGF-β, a cytokine that stimulatescollagen synthesis and inhibits its degradation. Pirfenidone reduced the production of other mediators of fibrogenesis, such as fibronectin and connective tissue growth factor (CTGF). 0 ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 132
Earlier publication have described a method for analysing pirfenidone in biological samples. But this method suffer from some disadvantages, like lack of internal standard, estimation of pirfenidone in rat plasma only under fasting condition. No methods have been reported for the estimation of pirfenidone in rat plasma without fasting, by RP-HPLC method. The aim of this study was to develop a more sensitive liquid chromatographic method for the determination of pirfenidone in rat plasma and the effect of food on its pharmacokinetic profile. MATERIALS AND METHOD Pirfenidone standard was a gift from Cipla Ltd Himachal Pradesh, India. Internal standard used was Tinidazole. Methanol (Finar), Acetonitrile(Rankin), Orthophosphoricacid (SD fine chem), Diethyl ether and Ethyl acetate were of HPLC grade. Water used was HPLC grade and double distilled. Shimadzu LC-20 AT HPLC with UV detector which was set at 324 nm. The analytical column was a C-18 reversed phase column(5µm,25cm 4.6mm ID).The mobile phase consisted of water ph 4.5 (adjusted with orthophosphoric acid) as aqueous phase and Methanol: Acetonitrile (15:20) as organic phase in the ratio 60:40.A flow rate of 1mL/min was used for the sample analysis.the temperature of the column was 25 C. Preparation of standard stock solution Stock solution of pirfenidone and internal standard 1000μg/ml were prepared separately by dissolving 10mg of each drug in 10ml standard flasks and the volume was made up to 10 ml with the mobile phase. From the stock solution, 10μg/ml solution was prepared by diluting 1ml to 100ml with mobile phase. 1ml of the above solution was diluted to 10ml with mobile phase to get a concentration of 1μg/ml. Further solutions were made by from the above solution by diluting 1.0ml, 2.0, 3.0ml, 4.0ml and 5.0ml standard solutions to 10ml in a standard flask with mobile phase to get concentrations of 100,200,300,400 and 500ng/ml respectively. Similarly the working standards were prepared for internal standard also. Sample withdrawal According to the body weight of each animal the drug solution was given through oral gavage. Group (B) animals were kept fasted overnight. Blood samples were withdrawn at specified pre-determined time intervals from 0.5 to 6.0 h, usingretro orbital nerve puncture. The blood samples were immediately transferred to collection tubes containing Disodium EDTA and shaken well, centrifuged using ultra cooling micro centrifuge at 5000 rpm/sec to separate plasma. The separated plasma samples were transferred to a labelled air tight sample tubes and kept in deep freezer for further analysis. Preparation of calibration standard To 200µl of blank plasma,100µl of standard solution of pirfenidone and 100µl of internal standard solutions were added to get concentration of 100, 200, 300, 400 and 500ng/ml respectively. To these calibration standards,100µl of precipitating agent (mixture of ethyl acetate and diethyl ether) was added, and centrifuged for 15 minutes at 5000 rpm. After centrifugation the clear supernatant liquid was collected and kept for evaporation. After complete evaporation the residue was reconstituted with mobile phase. A volume of 20µl was injected into the HPLC column and chromatograms were recorded. Standard calibration graph was plotted using peak area of pirfenidone against its concentration. Method validation The method was validated in terms of accuracy, precision, selectivity, linearity, sensitivity,precision, recovery and stability according to the guidelines issued by the food and drug administration (FDA) for the validation of bioanalytical method. Accuracy and Precision The absolute recovery of pirfenidone was estimated by comparing the response factor of the drug obtained from the plasma with that obtained from the direct injection of drug in mobile phase. The response factor obtained from an amount of the drug added to and extracted from the biological matrix, compare to the response factor obtained for the true concentration of the pure authentic standard is known as the recovery of a drug in an assay.recovery studies were carried out for three levels at six times and the percentage recovery, mean standard deviation and coefficients of variation were calculated. Intraday and interday precision studies were conducted. ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 133
The intraday precision was evaluated by analysis of plasma sample containing drug at three different concentration containing the internal standard using nine replicate determinations for three occasion were injected and chromatogram was recorded. The inter -day precision was similarly evaluated over a period of two week and the response factor and RSD were calculated, from that the mean concentration, standard deviation and % CV were calculated. Recovery studies The relative recovery of drug from plasma was calculated by comparing the readings of concentration obtained from the plasma containing drug to that of equal concentration from standard sample. Recovery studies were carried out six times for sample concentration at three levels within the calibration curve. Linearity and range Linearity and range were estimated by using calibration curve. By using calibration standards prepared by spiking drug (pirfenidone) and internal standard (tinidazole) in plasma at different concentrationslike 100ng/ml to 1000ng/ml the calibration graph was plotted taking concentration of spiked plasma on x-axis and peak area on y-axis. The linearity is determined from 50% to 150% of the proposed concentration. Limit of detection and Limit of quantification Using the calibration curve, the LOD and LOQ were determined.limit of quantitation is the concentration of substance in the sample that will give a signal-to-noise ratio of 10:1. Limit of detection is the concentration that will give a signal-to-noise ratio of 3:1. The signal to noise ratio were performed by comparing measured signal of blank plasma sample with those of known low concentration of drug. Stability For a reasonable time, the stability of sample, standard and reagent used in HPLC method is required to generate reproducible and reliable results. Stability of plasma sample spiked with drug were subjected to three freeze-thaw cycles, short term stability at room temperature for 3 hours and long term stability at 20 0 C for four weeks. Stability of standard solution and internal standard were performed at room temperature for 6 hours and under frozen condition for two weeks. The stability of this solution was studied by performing the experiment and looking for change in separation, retention and asymmetry of the peak which were then compared with the pattern of chromatogram of freshly prepared solutions. Selectivity The selectivity was established by two different methods Method I: Chromatograms of six blank plasma samples obtained from six different rats were compared with chromatogram obtained from standard solutions. Interferences due to endogenous plasma component on the retention times of the selected drugs and internal standard were tested. Method ІІ: The peak purity test method using diode array detector is involved in this method. The PDA spectrum, UV spectrum, absorbance ratio curve and first derivative spectrum of the standard and sample peak was recorded using PDA detector and compared for the peak purity of drug. Robustness By changing the chromatographic conditions slightly, the robustness of the method was studied. The standard solutions were injected in these changed chromatographic conditions. ± 1 % difference in the ratio of acetonitrile in the mobile phase. ± 0.5 difference in units of ph of the buffer. ± 1 % difference in flow rate of the mobile phase. In these changed conditions the separation factor, retention time and peak symmetry was calculated. Deviation in results from original run should be less than 2%. System suitability studies In system suitability studies certain parameters such as column efficiency, resolution, capacity factor were calculated, by repeated injection of standard solutions. Capacity factor (k ): It is measurement of sample molecule how good is retained by a column during separation. The ideal k value ranges from 2-10. Resolution (Rs): It is the difference between the retention times of two solutes divided by their average peak width. The ideal value of (Rs) is 1.5. ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 134
Column Efficiency (N): It is measured by the number of theoretical plates per meter. For ideal good separation, column efficiency N value ranging from 5,000 to 100,000 plates/meters. Peak asymmetry factor: For better column performance it was calculated by the formula. When asymmetry factor of value 0.9 to 1.1 then it is achievable for a well packed column. RESULT AND DISCUSSION Chromatographic Conditions Mixture of ethyl acetate and diethyl ether was selected as good separating agent for pirfenidone, since it showed maximum recovery in comparison with acetonitrile and methanol (Table1). The chromatogram was recorded for the standard calibration and plasma sample under developed chromatographic conditions. The retention time of pirfenidone is 6.5 minutes. The chromatogram was well resolved without any interference.peaks not showed any tailing or fronting. The concentration of pirfenidone in rat plasma was determined from the calibration of the spiked plasma by regression analysis. It showed very good linearity in the range of 50-250ng/ml and the R 2 value was found to be 0.998 (fig1) METHOD VALIDATION Accuracy and precision: Accuracy and precision studies were conducted at two levels i.e. intra-day and inter-day. In this present developed method, showed good accuracy and precision. Accuracy ranges from 98.49% to 99.37% with the precision 6.43% to 7.67% in inter-day method. In intra-day method the accuracy ranges from 98.64% to 99.33% with the precision 5.64% to 6.93 %. The data obtained here, was found to be within limits as per ICH guidelines and method was accurate. Intra-day studies: In this plasma concentration of 50-250ng/ml were injected six times and mean peak area was calculated separately for each concentration and from that accuracy and precision percentage RSD values were calculated and shown in table 3. Inter-day studies: In this the plasma concentrations of 50-250ng/ml were injected into HPLC six times in three different days and mean peak areas were calculated and from that accuracy and precision percentage RSD were calculated and shown in table2. The percentage relative standard deviation of precision for pirfenidone was less than 15% for the bioanalytical study. The results obtained were within limits. Acceptance criteria: The percentage RSD value should be less than 15% for bioanalytical study. Linearity and range : According to ICH guidelines, the method proved to be linear between 50-250 ng/ml for pirfenidone with a correlation equation Y=54.97x- 349.5 correlation coefficient R 2 was 0.998 (Fig 1). Limit of Detection and Limit of Quantification: The limit of quantification (LOQ) and limit of detection (LOD) of pirfenidone was found to be 12ng/ml and 20 ng/ml respectively. Recovery from plasma: The extraction efficiency of pirfenidone from rat plasma at the concentrations of 50, 100, and 200 ng/ml was found to be 97.72%, 99.01% and 99.33% with %RSD of 8.2, 7.5, and 7.9. The results of recovery studies are shown in Table 4. Ruggedness: It expresses the precision within laboratories variations like different days, different analyst, and different equipments. Ruggedness of the method was assessed by spiking the standard 6 times in two different days with different analyst and the reports are shown in Table5. System suitability: Column efficiency (theoretical plates), resolution factor and peak asymmetry factor, HETP, tailing factor, LOQ, LOD are the system suitability parameters. These parameters of the optimized methods were found satisfactory. The results of the system suitability studies in plasma were shown in table 6. These parameters were shown to be within specified limits. ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 135
Pharmacokinetic Study After a single oral dose of 30.0mg/kg of pirfenidone sample was in measurable amount in plasma up to 0.5 to 2 hours. The pharmacokinetic parameters of the samples were calculated using semilog graph, graph pad version 5, Microsoft excels software and the results were given in the Table 7 and 8. The maximum concentration (C max ) obtained for pirfenidone before food and after food were found to be 1020.76 and 836.51 ng/ml respectively. The half life (t 1/2 ) of pirfenidone before food and after food were t calculated and found to be 2.732158 h and4.009485 h. Area under the curve AUC. 0 of pirfenidone before food and after food were found to be 3060.95 and 2534.16ng h/ml and AUC 0 was found to be 3053.07 and 2510.64 ng h/ml.. Elimination rate constant (k eli ) was calculated for pirfenidone from the slope of log concentration versus time curve with regression analysis. Elimination rate constant for pirfenidone before food and after food were found to be 0.004228 and 0.002881 h -1. The pharmacokinetic parameters such as Cmax, Tmax, AUC 0-t, AUC 0-, K Eli, andt 1/2 were compared by statistical analysis. The p value obtained from t-test was found to be 0.3408. Hence there is no significant difference between the plasma concentration before and after food. Table 1 Recovery study for extraction method Level Conc. Of drug added (ng/ml) Amt. of drug recovered from plasma (ng/ml) % Recovery ACN Methanol Diethyl ether and ethyl acetate mixture ACN Methanol Diethyl ether and ethyl acetate mixture Level I 50 44.26 39.50 47.61 88.52 79.01 95.22 Level II 100 s86.14 80.38 95.12 86.14 80.38 95.12 Level III 150 135.47 120.79 145.64 90.31 80.52 97.09 ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 136
Calibration curve PEAK AREA 15000 10000 5000 y = 54.978x 349.5 R² = 0.9982 Series1 Linear (Series1) 0 0 100 200 300 CONCENTRATION ( ng/ml) Fig 1Calibration curve forpirfenidone Table 2 Accuracy and Precision Studies of pirfenidone (Inter day) Si.No Conc. of Drug (ng/ml) Mean* Peak Area Accuracy (%) Precision (%) 1. 50 2359 99.19 6.65 2. 100 5108 99.06 6.43 3. 150 7963 98.49 7.42 4. 200 10397 98.72 7.67 5. 250 13459 99.37 6.91 *n = 6 (Mean of 6 values) for inter day studies ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 137
Table 3 Accuracy and Precision Studies of pirfenidone (Intra day) Si.No Conc. of Drug (ng/ml) Mean* Peak Area Accuracy (%) Precision (%) 1. 50 2409 98.65 5.64 2. 100 5093 99.33 6.21 3. 150 8221 98.72 6.54 4. 200 10426 98.64 5.94 5. 250 13459 99.23 6.93 *n = 6 (Mean of 6 values) for intraday studies Table 4 Recovery studies of pirfenidone Levels Conc. of Drug added Amount of drug Relative Recovery % RSD (ng/ml) recovered in plasma (%) sample (ng/ml) Level-I 50 48.86 97.72 8.2 Level-II 100 99.01 99.01 7.5 Level-III 200 198.67 99.33 7.9 ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 138
Table5 Ruggedness studies for pirfenidone Drug Concentration Mean * peak area %RSD Pirfenidone 100 ng/ml Day I 5169 0.51 Analyst 1 Pirfenidone 100ng/ml Day II 5237 0.68 Analyst 2 *n=6 (Mean of 6 values) Table 6 System suitability studies Si.No Parameters Pirfenidone 1. Theoretical Plate 4792.42 2. Tailing Factor 1.216 3. HETP 35.601 4. LOD 12ng/ml 5. LOQ 20ng/ml 6. Resolution 4.658 7. K 3.126 ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 139
Table7 Plasma concentration of pirfenidone with food and without food Plasma Concentration( ng/ml) Time (h) Before Food After Food 0.5 236.42 204.19 1.0 378.68 322.25 2.0 960.76 836.51 4.0 436.15 355.91 6.0 314.47 274.84 Fig 2 Representative chromatogram of pirfenidone and tinidazole in plasma ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 140
Table8 Pharmacokinetic parameters of pirfenidone Pirfenidone Si.No Parameters Before food After food 1 AUC 0-t (ng.h/ml) 3060.95 2534.16 2 AUC 0- (ng.h/ml) 3053.07 2510.64 3 C max (ng/ml) 1020.76 836.51 4 Tmax (h) 2.0 2.0 5 K Eli (h -1 ) 0.004228 0.002881 6 t 1/2 (h) 2.732158 4.009485 Concentration (ng/ml) 1200 1000 800 600 400 200 0 BEFORE FOOD AFTER FOOD 0 2 4 Time (hrs) 6 8 Fig 3 Comparison of plasma concentration of Pirfenidone before and after food ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 141
CONCLUSION The RPHPLC method developed for the determination of pirfenidone in rat plasma and the effect of food on its pharmacokinetic profile, based on protein precipitation, proved to be rapid and sensitive. Moreover the devised method fully meets FDA guidelines and has high sensitivity, reproducibility and specificity. ACKNOWLEDGEMENTS This work was supported by KMCH college of Pharmacy. REFERENCE [1] Wang Y, Zhao X, Zhong J, Chen Y, Liu X, Wang G.,Biomed.2006, 20:1375. [2] Zhan Xiao-ling, MA Feng-yu1,ZONG Guo-jun2, Anhuai Medical and pharmaceutical journal 2009,15,25. [3] Eur J Pharmacol.2008,590, 400-408. [4] IDrugs. 2004, 7,166-172. [5] Westlake W.J, Peace K, Marcel Dekker, Bioavailability and Bioequivalence of Pharmaceutical Formulations Biopharmaceutical Statistics for Drug Development. 1987. [6] Guidance for industry Food effect bioavailability and fed bioequivalence studies. US Department of health and humanservices Food and Drug Administration Centre for Drug Evaluation and Research (CDER), December 2002. [7] Guidance for industry Bioavailability and Fed bioequivalence studies for orally administered Drug products- General considerations US Department of health and humanservices Food and Drug Administration Centre for Drug Evaluation and Research (CDER), March 2003. [8] Guidance for industry: Bioanalytical method validation.us Department of health and humanservices Food and Drug Administration Centre for Drug Evaluation and Research (CDER), Centre for biologics evaluation and Research (CBER),May 2001. [9] ICH,Q2 (R1)Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization of Technical Requirements For Registration of Pharmaceuticals, ICH Harmonized Tripartite guideline November 2005. ISSN : 0976-285X Vol 3 Issue 3 Jun-Jul 2012 142