Appendix II- Bioanalytical Method Development and Validation

A2. Bioanalytical method development 1. Optimization of chromatographic conditions Method development and optimization of chromatographic parameters is of utmost important for validating a method in biological matrix (Plasma). 1.1 Selection of wavelength A stock solution of 1.0 mg/ml of Silybin was prepared in methanol. Solution was diluted to 10μg/ml and scanned between 200-800nm using PDA detector. The obtained absorption maximum (λ max) for Silybin was 286nm and used as working wavelength for Silybin. 1.2 Selection of chromatographic method Silybin (CAS No. - 65666-07-1) is available as yellow color powder. Its molecular weight is 482.44 Da, molecular formula C 25 H 22 O 10, log value 2.4, melting point 230-233C. It is soluble in methanol and insoluble in water and oil. It is a mixture of flavonoids extracted from seeds of the Silybum marianum. It primarily consists of silybin and its isomers viz., silicristin and silidianin. The RP-HPLC was selected for the initial separation because of its simplicity, suitability, reproducibility, ruggedness, and wide acceptability. Figure AII.1. Structure of Silybin A and Silybin B Page 113

Table AII.1. Physical properties of silybin Name IUPAC name Physical state Melting point Silybin (2R,3S)-3,5,7-Trihydroxy-2-[(2R)-2-(4-hydroxy-3- methoxyphenyl)-3-(hydroxymethyl)-2,3-dihydro-1,4- benzodioxin-6-yl]-2,3-dihydro-4h-chromen-4-one yellowish powder 230-233C Formula C 25 H 22 O 10 Mol. wt. 482.44 Da Solubility Soluble in methanol and insoluble in water and oil LogP/Hydrophobicity 2.4 pka 6.418 2. Chromatographic conditions HPLC Compound Column Chromatographic mode Run time Detection wavelength Flow rate : Shimadzu- SPD-M20A : Silybin (sigma-s0292-10g) Batch no.-107k0762 : RP-C18 (Merck 100 RP-18 endcapped (5µm)) : Reverse phase : 27.0 min : 286nm : 1 ml/min Injection volume : 20 µl Page 114

3. Mobile phase selection The following mobile phases were used and the chromatograms were recorded. Trial (1) Comment: Trial (2) Comment: Trial (3) ACN : Water (50:50%v/v), ph 3.5 adjusted with glacial acetic acid poor resolution and poor recovery ACN : Phosphate buffer (40:60%v/v), ph 3.5 adjusted with glacial acetic acid Poor resolution ACN : Phosphate buffer (20:80%v/v) ph3.5 Comment: poor resolution Trial (4) ACN : Phosphate buffer (70:30%v/v), ph 3.5 Comment: Trial (5) Comment: Trial (6) poor resolution Methanol : Water (40:60%v/v), ph 3.5 adjusted with glacial acetic acid Poor resolution Methanol : Water (50:50%v/v), ph 3.5 adjusted with glacial acetic acid Comment: Better resolution and good recovery From the above trials, trial 6 was found to be better for elution of various component of Silybin. The retention time of Silybin A and Silybin B was 17.7 and 19.8 min respectively in the same condition. Page 115

4. PREPARATION OF SOLUTIONS Rinsing solution [ Methanol : Water (50:50%v/v)] 250 ml of methanol was added into a glass bottle containing 250 ml of water and the resulting mixture was mixed well and sonicated for 10 minutes. Buffer (0.002%v/v Acetic acid) 999.80mL milli-q water and 20 µl glacial acetic acid was mixed and sonicated. Diluent [ Methanol : Water (50:50%v/v) ph3.5] 100 ml of methanol was transferred into a glass bottle containing 100 ml of water, mixed well and sonicated. 5. Selection of internal standard (ISTD) Selection of internal standard (ISTD) was based on structural, physico-chemical and pharmacological similarity of drug and ISTD. Various standard drugs were chromatographed viz., rutin, quercetin and alpha napthol. Among these α- napthol was well resolved. Hence it was selected as internal standard (ISTD) for the estimation of Silybin from plasma. 6. PREPARATION OF STOCK SOLUTION Main Stock solution of 1mg/mL was prepared. From that intermediate solution of 2µg/mL was prepared and was used for the preparation of calibration curve. Calibration Curve A standard plot was made using the following concentrations:- 15, 30, 60, 120, 180, 200 and 320ng/mL of Silybin. Internal Standard Working Solution (250ng/mL) (IS/WS) α-napthol of concentration 250ng/mL was prepared and used as internal standard. 7. OPTIMIZATION OF EXTRACTION TECHNIQUE Various extraction procedures were tried to extract analyte and internal standard from plasma concentrating on better and consistent recovery without interfering with the quantification of analyte and ISTD. Solid phase extraction, liquid liquid extraction and protein precipitation methods were tried. Out of these three methods, protein precipitation was found to be economic, Page 116

simple and gave good recovery. So it was selected for further validation. Various protein precipitants like acetonitrile, methanol, 1:1 ratio of methanol and acetonitrile were used. All these precipitants were also tried in cold condition. 1:1 ratio of methanol and acetonitrile gave the best recovery in cold condition among all tried precipitant. Sample Extraction Procedure (Protein Precipitation Method) To 95μL of plasma, 5μL of analyte and 5μL of internal standard working solution was added. 300mL of 1:1 ratio of methanol and acetonitrile (extraction solution) was added and vortexed for 5min and after that sample was centrifuged at 14000rpm. Supernatant was separated and 40μL was injected into the HPLC system. Page 117

BIOANALYTICAL METHOD VALIDATION Bioanalytical method for silybin was validated as per USFDA guidelines. The parameters evaluated were: system suitability, carryover, specificity, sensitivity, linearity, precision and accuracy, recovery and stability. The acceptance criteria for these parameters are summarized in table AII.2. Table AII.2. Method validation acceptance criteria for following parameters Parameters Acceptance criteria System suitability eak area ratio: CV 5.0. etention times: CV 2.0 Carryover 20 of response of mean extracted LLOQ Area response at retention time of analyte 5 of response of mean extracted IST. Linearity r 2 0.9800 Specificity If any peak is present at RT of analyte its response should be 20 of extracted LLOQ. Sensitivity % deviation from nominal concentration should be within ±20%. CV of calculated concentration should be 20. Precision and Accuracy The calculated concentration at each QC level should be within 85-115% of their nominal concentration except for LLOQC where it must be within 80-120%. The precision determined at each concentration level should not exceed 15% of the coefficient of variation (%CV) except for the LLOQ, where it should not exceed 20% of the %CV. Recovery Recovery across QC level should be ±15% Stock solution stability Mean % change should be ±10% Stability in plasma Mean % change should be ±15% Page 118

Preparation of calibration curve standard and Quality control samples:- Aqueous dilution of Silybin was prepared by taking respective volume of stock solution as mentioned in table AII.3 and diluted with diluents to get the desired concentration. Table AII.3. Calibration curve and quality control sample dilution SILYBIN SPIKING STOCK PREPARATION Weight taken (mg) Silybin 2µg/mL α-napthol 250ng Vol. of Level Intermediate stock Final vol Spiking stock Conc. Plasma Conc taken (ml) (ml) (ng/ml) (ng/ml)* STD-1 0.075 300.000 15.000 STD-2 0.150 600.000 30.000 STD-3 0.300 1200.000 60.000 STD-4 0.600 2400.000 120.000 STD-5 0.900 3600.000 180.000 STD-6 1.000 4000.000 200.000 STD-7 1.600 6400.000 320.000 LLOQC 0.075 300.000 15.000 LQC 0.225 900.000 45.000 MQC 0.800 3200.000 160.000 HQC 1.450 5800.000 290.000 * Percentage of spiking = 5.0%v/v Page 119

8.1. Bulk spiking Preparation of stock solution of silybin A stock solution of Silybin was prepared for bulk spiking of calibration standards and quality control samples for the method validation. The aqueous spiking stock solution of Silybin for seven calibration standard concentrations ranging from 15.000 to 320.00 ng/ml and for LLOQC, LQC, MQC and HQC of concentrations 15.000 ng/ml, 45.00 ng/ml, 160.00 ng/ml and 290.00 ng/ml was prepared in diluent. The working stock solution of α-napthol (ISTD) was prepared in same diluents at concentration of 250 ng/ml. All seven standards and quality control samples were bulk spiked in plasma and stored at -70 ºC. Aliquots of these stock solution were also stored under refrigeration at 2-8ºC for determination of stock solution stability. 8.2. Preparation of plasma calibration curve, quality control samples, system suitability sample The drug free plasma lots stored at -70ºC were withdrawn and allowed to thaw at room temperature and were then vortexed adequately before use. 8.2.1. Preparation of blank sample: To 95 μl of drug free plasma, 5 μl of diluents were added and sample was extracted as per sample extraction procedure. 8.2.2 Preparation of Zero sample: To 95 μl of drug plasma, 5 μl of diluents and 5 μl of Alpha naphthol (250ng/mL) were added and sample was extracted as per sample extraction procedure. 8.2.3 Preparation of calibration standards: The plasma concentration ranges of Silybin are 15, 30, 60, 120, 180, 200 and 320 (ng/ml). These were prepared by adding 5 μl of ST -1 to STD- respectively and 5 μl of Alpha naphthol (250 ng/ml) to 95 μl of plasma. Page 120

8.2.4. Preparation of system suitability: Five microliters of aqueous MQC spiking stock solution of Silybin, 5 µl of working stock solution of alpha napthol (ISTD) and 300 µl of reconstitution solution (RS) was taken into a vial containing 350 µl insert and the auto sampler was programmed to inject 40 µl into the HPLC system. 8. Result: 9.1. System suitability System suitability test was done separately before start of every new sequence. Five injections of above system suitability solution were injected in HPLC. Percentage CV was found to be less than 1.29 for peak area which was within the acceptance criteria. (Table AII.4) Table AII.4. System suitability S. No. Area ratio of Silybin to ISTD Batch-1 Batch-2 Batch-3 Batch-4 1 0.933 0.924 0.932 0.931 2 0.912 0.942 0.935 0.925 3 0.941 0.934 0.941 0.941 4 0.942 0.937 0.94 0.944 5 0.932 0.938 0.935 0.935 Mean 0.932 0.935 0.9366 0.9352 S.D. 0.012 0.007 0.004 0.008 % CV 1.29 0.73 0.40 0.82 9.2. Specificity / selectivity: Specificity of a method is the ability of method to differentiate and quantify the analyte in the presence of other components in the sample. For selectivity, at least six independent lots of Page 121

blank samples of the appropriate biological matrix was analysed. There was no interference of endogenous material in the quantification of Silybin and IS (α- Napthol) (Table AII.5) Table AII.5. Screening and selectivity of silybin in six different lots of Wistar rat plasma Sr. No. Blank Plasma ID Interference at Rt of analyte LLOQC area % of LLOQC Interference at Rt of ISTD ISTD area % ISTD 1 BP-01 0.00 3766 0.00 0.00 192004 0.00 2 BP-02 0.00 3876 0.00 0.00 202900 0.00 3 BP-03 0.00 3789 0.00 0.00 197288 0.00 4 BP-04 0.00 3827 0.00 0.00 196700 0.00 5 BP-05 0.00 3749 0.00 0.00 197217 0.00 6 BP-06 0.00 3687 0.00 0.00 197501 0.00 Mean S.D. % CV 3782 197268 65.237 3458.584 1.72 1.75 Page 122

Figure AII.2. Representative Chromatogram of LLOQ in pooled Wistar rat plasma Figure AII.3. Representative Chromatogram of blank Wistar rat plasma Figure AII.4. Representative Chromatogram of zero sample in Wistar rat plasma Page 123

9.3. Carry over check: Carry over test was performed to find out that there is no carryover of analyte from the previous injection. The experiment was performed at ULOQ level. There was no significant carry over effect observed in blank sample. Results are shown in table- AII.6. Table AII.6. Carry over check for silybin S. Area % area of LLOQ Sample Name No. Silybin α- napthol Silybin α- napthol 1 Blank-RS 0 0 NA NA 2 AQ-ULOQ 55526 185478 NA NA 3 Blank-RS 0 0 0 0 4 AQ-ULOQ 56864 197563 NA NA 5 Blank-RS 0 0 0 0 6 EXT-Blank plasma 0 0 0 0 7 EXT-ULOQ 55896 204765 NA NA 8 EXT-Blank plasma 0 0 0 0 9 EXT-ULOQ 54859 205755 NA NA 10 EXT-Blank plasma 0 0 0 0 11 LLOQ-1 3876 192004 12 LLOQ-2 3789 202900 13 LLOQ-3 3827 197288 Mean 3831 197397 Page 124

9.4. Sensitivity Appendix II- Bioanalytical Method Development and Validation Sensitivity test is performed for establishing the reproducibility and accuracy of bioanalytical method at lowest limit of quantification. Percentage CV determination at LLOQ level was found to be 5.024% for silybin. (Table AII.7) Table AII.7. Sensitivity for silybin LLOQ (ng/ml) Specified conc. (15ng/mL) Sr. No. Calculated concentration % Nominal concentration 1 14.779 98.527 2 3 4 5 6 16.319 108.793 14.543 96.951 16.136 107.571 15.615 104.099 16.294 108.628 N 6 Mean calc. conc. 15.614 S.D. % CV Mean % Nominal concentration 0.784 5.024 104.095 9.5. Linearity / calibration (standard) curve A calibration (standard) curve is the relationship between instrument response and known concentrations of the analyte. (Table AII.8 and Table AII.9) Page 125

A calibration curve should consist of: A blank sample (matrix sample processed without internal standard) A zero sample (matrix sample processed with internal standard), and Six to eight non-zero samples covering the expected range, including LLOQ. Calibration curves were found to be consistently accurate and precise over 15-320 ng/ml. Analyte concentration at each calibration level was back calculated from the calibration curves. The calibration curve meets the acceptance criteria of r 2 0.98. ( Table AII.8) Table AII.8. Summary of calibration curve parameters of silybin Linearity Slope Intercept r 2 1 0.00090 0.00750 0.9993 2 0.00086 0.00851 0.9997 3 0.00091 0.00830 0.9993 4 0.00079 0.00762 0.9992 Page 126

Table AII.9. Back-calculated concentrations of calibration curve standards of silybin in Wistar rat plasma Linearity Conc. (ng/ml) STD-1 STD-2 STD-3 STD-4 STD-5 STD-6 STD-7 Sr. No. 15.000 30.000 60.000 120.000 180.000 200.000 320.000 1 12.842 26.583 60.456 119.752 173.041 191.190 304.272 2 13.453 27.703 66.446 125.816 160.906 185.721 326.792 3 14.186 27.143 68.244 129.455 165.760 190.643 306.524 4 15.287 27.703 66.267 127.636 173.648 187.909 305.961 Mean 13.942 27.283 65.353 125.665 168.339 188.866 310.888 S.D. 1.05 0.54 3.38 4.21 6.12 2.54 10.65 % CV 7.543 1.965 5.179 3.352 3.633 1.345 3.424 % Accuracy 92.95 90.94 108.92 104.72 93.52 94.43 97.15 Page 127

9.6. PRECISION AND ACCURACY (P&A) The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte. It is determined by replicate analysis of samples containing known amounts of the analyte. Precision and accuracy was checked at quality control samples eg. LLOQC, LQC, MQC & HQC. 9.6.1. Intra-batch or within-batch precision & accuracy % CV & % nominal concentration for QC samples i.e. LLOQC, LQC, MQC and HQC samples of Silybin for all four precision and accuracy batches were calculated. % Accuracy for LLOQC was 93.51 and ranging from 98.08 to102.657 for LQC, MQC and HQC. % Precision for LLOQC was 9.30 for LQC, MQC and HQC it ranges from 2.38 to 6.03%. 9.6.2. Inter-batch or between-batch precision & accuracy The inter-day precision and accuracy of the day were estimated from the assay of QC samples of different days. (Table AII.10) Global mean % accuracy for quality control samples i.e. LLOQC, LQC, MQC and HQC samples were 89.31, 98.29, 98.47 and 102.29 respectively, which are within acceptance criteria of 85-115% for LQC, MQC and HQC samples and 80-120% for LLOQC. % global precision for quality control samples i.e. LLOQC, LQC, MQC and HQC samples were 8.74, 3.81, 3.23 and 6.60 respectively, which are within acceptance criteria of 15 for LQC, MQC and HQC samples and of 20 for LLOQC. Page 128

Table AII.10. Inter- day Precision & Accuracy batch for silybin Specified (ng/ml) conc. LLOQC (15ng/mL) LQC (45ng/mL) MQC (160ng/mL) HQC (290ng/mL) 1 16.295 42.619 156.578 279.006 2 14.359 47.053 164.030 299.486 Batch-1 3 12.318 43.499 157.765 303.932 4 13.945 44.055 153.201 312.550 5 13.709 42.276 156.790 273.367 6 13.538 45.321 154.609 317.899 7 13.916 42.619 156.578 264.804 8 10.970 47.053 164.030 299.486 Batch-2 9 12.318 43.499 157.765 303.932 10 13.945 44.055 152.717 312.550 11 13.709 42.276 156.790 273.367 12 12.460 45.321 154.609 317.899 13 15.021 43.745 160.555 259.312 14 11.523 47.618 166.308 302.163 Batch-3 15 12.318 44.006 160.031 308.543 16 14.508 44.448 150.494 313.127 17 14.288 42.389 153.444 273.367 18 13.007 45.491 149.093 321.321 19 13.915 42.410 160.210 273.229 20 13.719 46.888 165.772 313.002 Batch-4 21 12.318 43.481 159.432 297.439 22 13.302 43.919 150.893 313.127 23 12.448 42.184 153.201 273.367 24 13.658 45.327 166.446 313.072 Number 24 24 24 24 Mean SD %CV Mean % nominal conc. 13.396 44.231 157.556 296.639 1.171 1.687 5.096 19.592 8.74 3.81 3.23 6.60 89.31 98.29 98.47 102.29 Page 129

9.7. EXTRACTION RECOVERY: Recovery pertains to the extraction efficiency of an analytical method within the limits of variability. Recovery is calculated by comparing detector response obtained from extracted QC sample to that of true concentration of pure authentic standard. Recovery of silybin at LQC, MQC and HQC level were 102.72%, 98.03% and 98.78% respectively. Mean recovery of α-napthol was found to be 88.44%. Recovery of analyte and ISTD was found to be consistent, precise and reproducible. 9.8. STABILITY STUDIES: Drug stability in a biological fluid depends on the storage conditions, chemical properties of the drug, matrix, and container system. This study evaluated the stability of the analyte during sample collection and handling, after long-term (frozen at the intended storage temperature) and short-term (bench top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process. Normally, as an example, the following stability tests should be evaluated: Freeze and thaw stability of the analyte in the matrix (plasma) from freezer storage conditions to room temperature. Bench top stability of the analyte in matrix (plasma) at room temperature. Long term stability of the analyte in matrix (plasma) stored in the freezer for long time In-injector/ auto sampler stability of the processed sample at injector or auto sampler temperature. Process stability: Process stability was conducted with six replicates of LQC and HQC samples. For this, samples were kept for 2 hours before vortexing at room temperature and 2 hours after reconstitution at room temperature to ensure the stability of analytes and ISTD during sample preparation. Main stock stability was performed at MQC level. Main stock was stable for 6 days at 2-8 C with mean % change of 0.57%. Page 130

The stability tests performed for silybin in plasma were bench top stability, process stability, ininjector stability, freeze and thaw stability and long term stability. Result of stability shows that silybin was stable for all the above stabilities performed. (Table AII.11) Table AII.11. Stability study QC level Stability Type Mean SD %CV %Accuracy % Change LQC Bench top 45.450 2.770 6.094 101.001 2.67 Process for 4hr 45.488 1.476 3.246 101.085 2.76 In-injector 44.976 0.627 1.394 99.946 1.60 3 rd Freeze/Thaw 45.301 1.522 3.359 100.668 2.33 15 days at -70 C 45.724 2.117 4.631 101.608 3.29 HQC Bench top 301.542 16.309 5.409 103.980 1.29 Process for 4hr 305.450 11.224 3.675 105.328 2.60 In-injector 303.436 12.813 4.223 104.633 1.92 3 rd Freeze/Thaw 292.288 7.684 2.629 100.789-1.82 15 days at -70 C 303.606 10.569 3.481 104.692 1.98 9.9. RUGGEDNESS Ruggedness of the method was checked using different lots of column and reagents. One precision and accuracy (P&A) batch was carried out with different column of same type (different lot) and one P&A batch was carried out with reagents of different manufacturer. For column and solvent ruggedness, different lot of columns and solvents of different manufacturers were used and a linearity followed by precision and accuracy was performed for Page 131

quality control samples at LLOQC, LQC, MQC and HQC levels and % Accuracy were 97.18, 101.38, 99.30 and 98.82% respectively for Silybin in column ruggedness. % Accuracy for quality control samples i.e. LLOQC, LQC, MQC and HQC samples were 97.73, 101.53, 98.24 and 98.07% respectively in solvent ruggedness. % CV for quality control samples i.e. LLOQC, LQC, MQC and HQC samples were 6.78, 4.20, 4.40 and 4.60% respectively for Silybin in column ruggedness. % CV for quality control samples i.e. LLOQC, LQC, MQC and HQC samples were 6.16, 3.29, 6.45 and 6.21% respectively for Silybin in solvent ruggedness. All the values were within acceptance criteria of 85-115% for LQC, MQC and HQC samples and of 80-120% for LLOQC. Page 132