CHAPTER 2 MATERIALS AND METHODS

34 CHAPTER 2 MATERIALS AND METHODS 2.1 METHOD DEVELOPMENT 2.1.1 Instrumentation The LC system, consisting of a binary solvent manager, sample manager, degasser unit, column manager and a Tandem MS (API-Triple quad- 4000, supplied by Applied Biosystems, MDS Sciex, Foster City, USA) as the detector with Analyst software (version 1.4.2) was used for quantification of drugs. Refrigerated centrifuge (Heraeus Multifuge -3SR, Thermo Scientific, LED, Gmbh, Germany), Vibramax 110 (Heidolph, Germany), SPE unit (Orochem, USA), Turbo LV evaporator (Zymark, USA), ultrasonic bath (Bandelin Sonorex, Germany) and vortex-mixer (Remi, India) were used for plasma sample extraction. Ultra-low temperature deep freezers with temperature range of -50ºC to ± 20ºC (Thermo Electron Corporation, Gmbh, Germany) was used to store the plasma samples. Electronic microbalance (Sartorius, India) was used for weighing RS/WS. Pipettes (Eppendorf) 100 to 1000 µl; 20 to 200 µl, disposable tips and Milli-Q water system (Millipore, USA) were used for preparing stock solution,

35 sample preparation and dilutions. Refrigerator (LG Corporation, India) was used to store the prepared stock solutions. 2.1.2 Nitroglycerin Method Development HPLC grade methanol and other chemicals and solvents of analytical grade were procured from Merck India Limited, India. Milli-Q purified water was used wherever required. 2.1.2.1 Ammonium chloride (0.025 mm) buffer solution Ammonium chloride (66.86 mg) was dissolved in water in a volumetric flask and the volume was made upto 500 ml and then filtered through 0.2 µm nylon membrane to give 2.5 mm ammonium chloride solution. The final conc. of 0.025 mm ammonium chloride buffer solution was prepared by adding 10.00 ml of 2.5 mm ammonium chloride solution to water in a volumetric flask and made the volume upto 1000 ml, mixed well and then sonicated for 5 min. 2.1.2.2 Washing solutions Wash solution was prepared by mixing 5.00 ml of methanol and 95 ml of water in a reagent bottle and sonicated in ultrasonic bath for 5 min. so as to give a ratio of 1:19 of methanol and water (v/v). Strong wash solution was prepared by mixing 900 ml of methanol and 100 ml of water in a reagent bottle and sonicated in ultrasonic bath for 5 min. so as to give a ratio of 9:1 of methanol and water (v/v). Weak wash solution was prepared by mixing 500 ml of methanol and 500 ml of water in a reagent bottle and sonicated in ultrasonic bath for 5 min so as to give a ratio of 1:1 of methanol and water (v/v).

36 2.1.2.3 Reference/working standards stock solutions NTG RS USP, its metabolites 1,2-DNG, 1,3-DNG and 1,2,4-BTTN (IS) from Cerillant Technologies, USA were supplied by LGC Promochem, Hyderabad, India and used for the method development of NTG. solution of NTG was prepared by dissolving 5 mg of diluted NTG WS in methanol and the volume was made upto 5 ml in a volumetric flask to give a final conc. of 1000000 ng/ml of NTG and calculated as follows: Weight of NTG WS M 1000 5 M 100 1 Conc. of NTG x x1000 x 2 where, M 1 is the molecular weight of NTG (salt free) M 2 is the molecular weight of NTG (salt), if applicable. The prepared solution was stored at 2 to 8 C. solution of 1,2-DNG was prepared by dissolving 100 µl of 1,2- DNG WS (1 mg/ml) in methanol and the volume was made up to 10 ml in a volumetric flask to give final conc. of 10000 ng/ml of 1, 2-DNG. The prepared solution was stored at 2 to 8 C. solution of 1,3-DNG was prepared by dissolving 100 µl of 1, 3-DNG WS (1 mg/ml) in methanol and made the volume up to 10 ml in a volumetric flask to give final conc. of 10000 ng/ml of 1, 3-DNG. The prepared solution was stored at 2 to 8 C.

37 solution of IS was prepared by dissolving 50 µl of 1, 2, 4- BTTN in methanol in 50 ml volumetric flask and made up to the volume to give a stock conc. of 100 ng/ml. Thus, prepared stock solution was stored at 2 to 8 C. To prepare the final conc. of 10 ng /ml of IS, 1mL of above stock solution was dissolved in 9 ml of methanol. 2.1.2.4 CC dilutions and spiked dilutions NTG, 1,2-DNG and 1,3-DNG stocks solutions were used for preparation of CC stock dilutions by adding methanol as per the conc. range described in Table 2.1. Prepared CC stock solutions were further diluted in methanol and each dilution was coded alphabetically as illustrated in Table 2.2. Table 2.1 CC stock dilutions of NTG and its metabolites solutions conc. (ng/ml) aliquot Methanol Final volume Final conc. (ng/ml) CC stock identity NTG 97407.00 0.39 1.61 2.00 18994.36 STD-1 STD-1 18994.36 0.83 3153.06 1,2- DNG 10000.00 2.00 0.17 5.00 4000.00 STD-2 1,3- DNG 10000.00 2.00 4000.00

38 Table 2.2 Dilutions of CC stock dilutions of NTG and its metabolites CC stock solutions STD-2 STD SS H STD SS G STD SS F STD SS E STD SS D STD SS C STD SS B conc. (ng/ml) 3153.06 aliquot Methanol Final volume Final conc. (ng/ml) 403.59 4000.00 1.28 8.72 10.00 512.00 4000.00 512.00 403.59 282.51 512.00 7.00 3.00 10.00 358.40 512.00 358.40 282.51 169.50 358.40 6.00 4.00 10.00 215.04 358.40 215.04 169.50 125.43 215.04 7.40 3.60 10.00 159.12 215.04 159.12 125.43 46.41 159.12 3.70 6.30 10.00 58.87 159.12 58.87 46.41 12.53 58.87 2.70 7.30 10.00 15.89 58.87 15.89 12.53 4.01 15.89 3.20 6.80 10.00 5.08 15.89 5.08 4.01 4.01 5.08 5.00 5.00 10.00 5.08 5.08 5.08 Final CC dilutions identity STD SS H STD SS G STD SS F STD SS E STD SS D STD SS C STD SS B STD SS A

39 Final CC dilutions of NTG, 1, 2-DNG and 1,3-DNG were used for preparation of CC standards. A 500 µl of aliquot of corresponding final conc. of CC dilution into 25 ml volumetric flask and volume made up with plasma to achieve the spiked CC standards. These were then coded alphabetically as mentioned in the Table 2.3. Each spiked calibration standard (650 µl) was transferred into polypropylene capped vials and stored at below -50 C. Table 2.3 Spiked CC standards of NTG and its metabolites CC dilutions conc. (ng/ml) Final conc. (pg/ml) Spiked CC NTG 1,2-1,3-1,2-1,3- NTG DNG DNG DNG DNG identity STD SS H 403.59 512.00 512.00 8071.84 10240.00 10240.00 STD H STD SS G 282.51 358.40 358.40 5650.29 7168.00 7168.00 STD G STD SS F 169.50 215.04 215.04 3390.17 4300.80 4300.80 STD F STD SS E 125.43 159.12 159.12 2508.73 3182.59 3182.59 STD E STD SS D 46.41 58.87 58.87 928.23 1177.56 1177.56 STD D STD SS C 12.53 15.89 15.89 250.62 317.94 317.94 STD C STD SS B 4.01 5.08 5.08 80.20 101.74 101.74 STD B STD SS A 2.00 2.54 2.54 40.10 50.87 50.87 STD A 2.1.2.5 QC dilutions and spiked samples Separate NTG, 1,2-DNG and 1,3-DNG stock solutions were prepared and used for preparation of QC dilutions by adding methanol as per the conc. range described in Table 2.4. Prepared QC stock solution was further diluted in methanol and each dilution was coded alphabetically as mentioned in Table 2.5.

40 Table 2.4 QC stock dilutions of NTG and its metabolites solutions conc. (ng/ml) aliquot Methanol Final volume Final conc. (ng/ml) QC stock dilutions identity NTG 97407.00 0.39 1.61 2.00 18994.36 NTG- QC-1 NTG-QC-1 18994.36 0.83 3153.06 1,2-DNG 10000.00 2.00 0.17 5.00 4000.00 QC-2 1,3-DNG 10000.00 2.00 4000.00 Table 2.5 Dilutions of QC stock dilutions of NTG and its metabolites dilutions QC-2 SS HQC SS MQC SS INTQC SS LQC conc. (ng/ml) 3153.06 aliquot Methanol Final volume Final conc. (ng/ml) 283.77 4000.00 0.90 9.10 10.00 360.00 4000.00 360.00 283.77 124.86 360.00 4.40 5.60 10.00 158.40 360.00 158.40 124.86 20.97 158.40 1.68 8.32 10.00 26.61 158.40 26.61 20.97 5.66 26.61 2.70 7.30 10.00 7.18 26.61 7.18 5.66 2.03 7.18 3.60 6.40 10.00 2.58 7.18 2.58 Final QC dilutions identity SS HQC SS MQC SS INTQC SS LQC SS LQC

41 Final QC dilutions of NTG, 1,2-DNG and 1,3- DNG were used for preparation of QC samples. A 500 µl aliquot of corresponding final conc. of QC dilution into 25 ml volumetric flask and volume made up with plasma to achieve the spiked QC samples. These were then coded alphabetically as mentioned in the Table 2.6. Each spiked QC samples (650 µl) were transferred into polypropylene capped vials and stored at below -50 C. For long term stability, QC samples were stored at -15 C and -50 C Table 2.6 Spiked QC samples of NTG and its metabolites QC solutions conc. (ng/ml) Final conc. (pg/ml) Spiked NTG 1,2- DNG 1,3- DNG NTG 1,2- DNG 1,3- DNG QC samples SS HQC 283.77 360.00 360.00 5675.51 7200.00 7200.00 HQC SS MQC 124.86 158.40 158.4 2497.22 3168.00 3168.00 MQC SS INTQC 20.97 26.61 26.61 419.53 532.22 532.22 INTQC SS LQC 5.66 7.18 7.18 113.27 143.70 143.70 LQC SS LOQQC 2.03 2.58 2.58 40.77 51.73 51.73 LOQQC 2.1.2.6 Sample preparation The required number of QC samples and CC standard vials were withdrawn from ultra-low temperature freezer/deep freezer and allowed to thaw in ice-water bath. The thawed samples were vortexed to ensure complete mixing of contents. To all vials except blank vials, 50 µl of IS solution and 500 µl of plasma sample were added and vortexed. Then 500 µl of water was added to all the samples and vortexed.

42 The strata cartridge was conditioned with 1 ml methanol followed by 1mL water. The prepared samples were loaded into cartridges and samples were washed with 1mL of water followed by 1mL of washing solution (5 % methanol in water) twice. The cartridges were dried approximately for 3 min. and eluted with 300 µl of methanol using 48 position solid phase extractor. The samples were vortexed and transferred into autosampler vials. The samples thus processed were loaded into LC-MS/MS for analysis. 2.1.2.7 Chromatographic and MS/MS conditions Column : Eclipse XDB - C8 (100 mm x 4.6 mm, 0.0035 mm) Mobile phase- A : Methanol Mobile phase- B : 0.025 mm ammonium chloride Injection Volume: 10 µl Flow : 0.500 ml/min. Time (min.) Flow Mobile phase A Mobile phase B 0 0.500 35.0 65.0 5.00 0.500 45.0 55.0 5.01 0.500 65.0 35.0 8.00 0.500 65.0 35.0 9.00 0.500 35.0 65.0 14.00 0.500 35.0 65.0 Column oven temperature: 30 C Auto sampler temperature: 4 C

43 Run time : 14.0 min. Mode of ionization : Electrospray in negative mode Q1/Q3 Mass : NTG: 261.90/62.00, 1, 2 DNG: 216.90/62.00 1,3 DNG: 216.90/62.00 1,2,4-BTTN: 275.90/62.00 2.1.3 Ursodiol Method Development HPLC grade methanol and other chemicals and solvents of analytical grade were procured from Merck India Limited, India. Milli-Q purified water was used wherever required. 2.1.3.1 Ammonium formate (5 mm) solution with 0.1 % formic acid Ammonium formate (315 mg) was weighed and transferred to 1000 ml of volumetric flask, dissolved in water and volume made up with water followed by filtration through 0.2 µm nylon membrane to give 5 mm ammonium formate solution. To this 1 ml of formic acid was added, mixed well and sonicated to give 5 mm ammonium formate solution with 0.1 % formic acid. 2.1.3.2 Diluent (methanol : water) Diluent was prepared in the ratio of 50 : 50 (methanol : water) v/v and the prepared diluent was mixed well and sonicated in ultrasonic bath for 5 min.

44 2.1.3.3 Reconstitution solution Reconstitution solution was prepared by mixing methanol (700 ml) and 5 mm ammonium formate solution with 0.1 % formic acid (300 ml) and sonicated in the ultrasonic bath. 2.1.3.4 Washing solutions Strong wash solution was prepared by mixing 900 ml of methanol and 100 ml of water in reagent bottle and sonicated in ultrasonic bath for 5 min. so as to give a ratio of 9:1 of methanol and water (v/v). Weak wash solution was prepared by mixing 500 ml of methanol and 500 ml of water in reagent bottle and sonicated in ultrasonic bath for 5 min. so as to give a ratio of 1:1 of methanol and water (v/v). 2.1.3.5 Reference/working standards stock solutions Ursodiol RS USP, its metabolites TUDCA and GUDCA from TLC Pharmachem USA, Inc. and the IS ursodiol d4 deutriated standard from Vivan Life Sciences, India, were used for the ursodiol method development. Ursodiol stock solution was prepared by dissolving 10 mg of ursodiol RS in methanol in a volumetric flask and the volume was made upto 5 ml, to give a final conc. of 2000 µg/ml of ursodiol and calculated as follows: where, Weight of ursodiol WS M 1000 5 M 100 1 Conc. of ursodiol stock x x1000 x 2 M 1 is the molecular weight of ursodiol

45 M 2 is the molecular weight of ursodiol (salt), if applicable. The prepared solution was stored at 2 to 8 C. TUDCA stock solution was prepared by dissolving 10 mg of TUDCA RS in methanol in a volumetric flask and the volume was made up to 5 ml, to give a final conc. of 2000 µg/ml of TUDCA and calculated using the formula. Weight of TUDCA WS M 1000 5 M 100 1 Conc. of TUDCA stock x x1000 x 2 where, M 1 is the molecular weight of TUDCA M 2 is the molecular weight of TUDCA (salt), if applicable. The prepared solution was stored at 2 to 8 C. GUDCA stock solution was prepared by dissolving 10 mg of GUDCA RS in methanol in a volumetric flask and the volume made upto 5 ml, to give a final conc. of 2000 µg/ml of GUDCA and calculated using the formula. Weight of GUDCA WS M 1000 5 M 100 1 Conc. of GUDCA stock x x1000 x 2 where, M 1 is the molecular weight of GUDCA M 2 is the molecular weight of GUDCA (salt), if applicable. The prepared solution was stored at 2 to 8 C IS stock solution was prepared by dissolving 10 mg of ursodiol-d4 RS in methanol in a volumetric flask and the volume was made upto 5 ml. to give a stock conc. of 2000 µg/ml of ursodiol-d4 using the formula.

46 Weight of ursodiol d4 M 1000 5 M 100 1 Conc. of ursodiol d4 stock x x1000 x 2 where, M 1 is the molecular weight of ursodiol d4 (salt free) M 2 is the molecular weight of ursodiol d4 (salt), if applicable. The prepared stock solution was stored at 2 to 8 C. Final conc. of 20 µg/ml was prepared, by dissolving 0.10 ml of above stock solution in 9.90 ml of diluent. 2.1.3.6 CC dilutions and spiked samples Ursodiol, TUDCA and GUDCA stock solutions were used for preparation of CC stock dilutions by adding diluent as per the conc. range described in Tables 2.7, 2.8 and 2.9. The prepared CC stock solutions were further diluted with diluent and each dilution was coded alphabetically, as illustrated in the below mentioned Tables. Ursodiol, TUDCA and GUDCA stock dilutions were used for the preparation of spiked CC standards. Individual plasma lot samples were stripped with 5 % charcoal for 3 hr to remove the interference from endogenous ursodiol. Then, the plasma lot was pooled prior to spiking and the corresponding final conc. of CC stock dilutions was transferred (5 % spiking) into 10 ml volumetric flask and made upto the volume with pooled plasma to achieve the spiked CC standards as mentioned in the Table 2.10.

47 Table 2.7 CC stock dilutions of ursodiol solutions conc. (ng/ml) aliquot Diluent Final volume Final conc. (ng/ml) identity Ursodiol 1103.56 0.50 0.500 1.00 551.78 AQ I AQ I 551.78 0.75 0.250 1.00 413.83 AQ H AQ H 413.83 0.55 0.450 1.00 227.61 AQ G AQ G 227.61 0.70 0.300 1.00 159.32 AQ F AQ F 159.32 0.50 0.500 1.00 79.66 AQ E AQ E 79.66 0.50 0.500 1.00 39.83 AQ D AQ D 39.83 0.37 0.625 1.00 14.93 AQ C AQ C 14.93 0.12 0.875 1.00 1.86 AQ B AQ B 1.86 0.50 0.500 1.00 0.93 AQ A Table 2.8 CC stock dilutions of TUDCA solutions conc. (ng/ml) aliquot Diluent TUDCA 1012.65 0.06 0.94 Final volume 1.00 Final conc. (ng/ml) identity 60.75 AQ I AQ I 60.75 0.75 0.25 1.00 45.56 AQ H AQ H 45.56 0.55 0.45 1.00 25.06 AQ G AQ G 25.06 0.70 0.30 1.00 17.54 AQ F AQ F 17.54 0.50 0.50 1.00 8.77 AQ E AQ E 8.77 0.50 0.50 1.00 4.38 AQ D AQ D 4.38 0.37 0.62 1.00 1.64 AQ C AQ C 1.64 0.12 0.87 1.00 0.20 AQ B AQ B 0.20 0.50 0.50 1.00 0.10 AQ A

48 Table 2.9 CC stock dilutions of GUDCA solutions conc. (ng/ml) aliquot Diluent Final volume Final conc. (ng/ml) identity GUDCA 1141.48 0.70 0.30 1.00 799.04 AQ I AQ I 799.04 0.75 0.25 1.00 599.28 AQ H AQ H 599.28 0.55 0.45 1.00 329.60 AQ G AQ G 329.60 0.70 0.30 1.00 230.72 AQ F AQ F 230.72 0.50 0.50 1.00 115.36 AQ E AQ E 115.36 0.50 0.50 1.00 57.68 AQ D AQ D 57.68 0.37 0.62 1.00 21.63 AQ C AQ C 21.63 0.12 0.87 1.00 2.70 AQ B AQ B 2.70 0.50 0.50 1.00 1.35 AQ A Table 2.10 Spiked CC standards of ursodiol and its metabolites CC stock solutions AQ I AQ H AQ G CC stock conc. (µg/ml) Spiked CC conc. (ng/ml) 551.78 15040.93 799.04 15300.05 60.75 1587.48 413.83 11280.70 599.28 11475.04 45.56 1190.61 227.60 6204.38 329.60 6311.27 25.06 654.83 Spiked CC identity STD I STD H STD G

49 Table 2.10 (Continued) CC stock Solutions AQ F AQ E AQ D AQ C AQ B AQ A CC stock conc. (µg/ml) Spiked CC conc. (ng/ml) 159.32 4343.07 230.72 4417.89 17.54 458.38 79.66 2171.53 115.36 2208.94 8.77 229.19 39.83 1085.76 57.68 1104.47 4.38 114.59 14.93 407.16 21.63 414.17 1.64 42.97 1.86 50.89 2.70 51.77 0.20 5.37 0.93 25.44 1.35 25.88 0.10 2.68 Spiked CC identity STD F STD E STD D STD C STD B STD A Aliquots (650 µl) of each spiked calibration standard was transferred into polypropylene capped vials and stored below -50 C. 2.1.3.7 QC dilutions and spiked samples Ursodiol, TUDCA and GUDCA stock solutions were prepared and used for preparation of QC dilutions by adding methanol as per the conc. range and each dilution was coded alphabetically, as mentioned in Table 2.11.

50 Table 2.11 QC dilutions of ursodiol and its metabolites dilutions conc. (ng/ml) aliquot Ursodiol 1103.56 0.37 Diluent Final volume Final conc. (ng/ml) 412.73 TUDCA 1141.48 0.52 0.05 1.00 599.28 GUDCA 1012.65 0.04 45.56 AQ HQC AQ MQC AQ INTQC AQ LQC 412.73 227.41 599.28 0.55 0.44 1.00 330.20 45.56 25.10 227.41 56.85 330.20 0.25 0.75 1.00 82.55 25.10 6.27 56.85 2.78 82.55 0.04 0.95 1.00 4.04 6.27 0.30 2.78 0.96 4.04 0.34 0.65 1.00 1.39 0.30 0.11 Final QC dilutions identity AQ HQC AQ MQC AQ INTQC AQ LQC AQ LOQQC Ursodiol, TUDCA and GUDCA stock dilutions were used for preparation of spiked QC samples. Individual plasma lot samples were stripped with charcoal (5 % w/w) for 3 hr to remove the endogenous interferences at the retention time of analyte and IS. Then, the plasma lot was pooled prior to spiking and the equivalent final conc. of QC samples was made (i.e. 5 % spiking) into 10 ml volumetric flask and made upto the volume with pooled plasma to achieve the spiked QC samples, as mentioned in the Table 2.12. Aliquots (650 µl) of each spiked QC samples were transferred into polypropylene capped vials and stored below -50 C. For long term stability, QC samples were stored at -15 C and -50 C.

51 Table 2.12 Spiked QC samples of ursodiol and its metabolites QC dilutions AQ HQC AQ MQC AQ INTQC AQ LQC AQ LOQQC conc. (µg/ml) Final conc. (ng/ml) 412.73 11270.41 599.28 11412.73 45.56 1173.38 227.41 4350.38 330.20 4405.31 25.10 452.92 56.85 1087.59 82.55 1101.32 6.27 113.23 2.78 76.13 4.04 77.09 0.30 7.92 0.96 25.88 1.39 26.21 0.11 2.69 Spiked QC samples HQC MQC INTQC LQC LOQQC 2.1.3.8 Sample preparation The required number of QC samples, CC standards vials were withdrawn from ultra low temperature freezer/deep freezer and allowed to equilibrate with room temperature. The thawed samples were vortexed to ensure complete mixing of contents. To all vials, except the blank vial, 50 µl of IS solution (20 µg/ml ursodiol-d4) and 500 µl of plasma sample were added and vortexed. Then 400 µl of 5 mm ammonium formate solution was added and vortexed.

52 The strata cartridges were conditioned with 1mL of methanol followed by 1 ml of water. The sample was loaded into Strata X Cartridges -30 mg/1ml, washed with 1mL of water three times and eluted with 1mL of methanol. All the samples were dried under nitrogen evaporator at 40 C and 15 psi. The dried residue was reconstituted with 300 µl of reconstitution solution and vortexed. The sample thus, processed were transferred into auto sampler vials and loaded into LC-MS/MS for analysis. 2.1.3.9 Chromatographic and MS/MS conditions Column : ZORBAX SB C8 (50 mm x 4.6 mm, 0.0035 mm) Mobile phase- A : Methanol Mobile phase- B : 5 mm ammonium formate with 0.1 % formic acid Injection Volume : 5 µl Flow : 0.2 ml/min. without splitter Time (min.) Flow Mobile phase A Mobile phase B 0 0.2 75 25 3.51 0.2 90 10 5.51 0.2 75 25 6.50 0.2 75 25 Column oven temperature: 40 C Auto sampler temperature: 10 C Run time : 6.5 min.

53 Mode of ionization : Electro spray in negative mode Q1/Q3 Mass : ursodiol: 391.20/373.2 TUDCA: 498.40/124.10 GUDCA: 448.40/74.00 Ursodiol-d4: 395.30/377.20 2.1.4 Data Processing Chromatograms for both methods were acquired using the computer based Analyst software 1.4.2, supplied by Applied Biosystems. The conc. of unknown sample was calculated from the following equation, using regression analysis of spiked plasma calibration standard with reciprocate of the drug conc. as weight factor (1/X 2 ). y = m x + b Where, x = Conc. of drugs and its metabolites separately m = Slope of CC y = Peak area ratio of drugs and its metabolites separately to its IS b = y-axis intercept of the CC. 2.2 METHOD VALIDATION 2.2.1 Selectivity Selectivity for NTG and its metabolites was performed by spiking 7 normal blank matrices and one haemolysed matrix with LLOQ conc. These spiked blank samples were then processed along with normal and haemolysed

54 blank plasma samples and analyzed to check the interference at the retention time of analyte and IS. Selectivity for ursodiol and its metabolites was performed by spiking 6 normal blank matrices and one haemolysed matrix with LLOQ conc. These spiked blank samples were then processed along with normal and haemolysed blank plasma samples and analyzed to check the interference at the retention time of analyte and IS The response of interfering peaks in blank at the retention time of drugs/metabolites and the IS peak should be 20 % and 5 % of the response of LLOQ samples run along with batch. 2.2.2 Autosampler Carry Over Test Carryover was evaluated during validation from processed blank, lowest CC standard and highest CC standard samples. The different samples were injected in the following steps: extracted blank, extracted LLOQ with IS, extracted ULOQ with IS, reinjection of first blank and calculated using the formula, Analyte carryover = area of analytae in step 4 area of analytein step1 x 100 Area of analyte in step 2 IS carryover = area of IS in step 4 area of analyte in step1 x 100 Area of analyte in step 2 For analyte, the % carryover must be within 20 % of LLOQ area and for IS it must be 5 % of the IS area of ULOQ.

55 2.2.3 Matrix Effect Matrix effect is estimated quantitatively through calculation of matrix factor, which is the ratio of peak response in the presence of matrix ions to the peak response in the absence of matrix ions. Matrix effect can further be evaluated from matrix factor as follows % Matrix effect = 1- mean of the matrix factor for analyte / IS 100 Matrix effect calculation for NTG was carried out by processing 7 normal plasma and one haemolysed plasma lot without adding analyte or IS and reconstituted with AQ LQC that contains both the analyte and IS. The processed and reconstituted plasma samples were then analyzed along with the AQ LQC samples. Matrix effect calculation for ursodiol was carried out by processing 6 normal plasma and one haemolysed plasma lot without adding analyte or IS and reconstituted with AQ LQC that contains both the analyte and IS. The processed and reconstituted plasma samples were than analyzed along with the AQ LQC samples. The acceptance criteria for the matrix factor is that the ratio should be within 0.85-1.15 and % CV should be 15 %, where as the matrix effect should be within ± 15 %. 2.2.4 Sensitivity, Linearity, Precision and Accuracy Sensitivity of the method is defined as the lowest conc. that can be measured with an acceptable limit of accuracy and precision. A CC was generated to evaluate a linear relationship across the range of the expected matrix conc. CC for NTG with its metabolites was generated by preparing AQ dilutions and spiking 8 point standards whereas for ursodiol and its metabolites

56 the curve was generated using 9 standards. For NTG as well as ursodiol at least 2 times greater and at least 10 % of the expected maximum drug conc. was selected as the highest and the lowest standard conc., respectively. The first standard conc. after LLOQ was twice of LLOQ, and the standard before ULOQ was 70-80 % of ULOQ. The prepared standards were analyzed by least square linear regression of the response ratios in the calibration standard using a weight factor of 1/X 2.The calibration line should be linear and the correlation coefficient (r 2 ) was consistently greater than 0.99. The deviation between two standard conc. for all non-zero CC standards should be ± 15 % except for LLOQ where it should be ± 20 %. Precision and accuracy for NTG, its metabolites and ursodiol, its metabolites was assessed by analyzing 3 batches including both intra and inter day runs. Each batch consists of AQ standard, mobile phase, standard blank (blank without IS), standard zero (blank with IS), calibration standards and 6 replicates of QC samples which includes, LOQQC, LQC, INTQC, MQC and HQC. The precision is determined by calculating % CV at each conc. level of QC samples and the accuracy by calculating the % of nominal value at each conc. level of QC samples. Precision (% CV) = 100 x SD / mean conc. Accuracy (% nominal) =100 x mean conc. /nominal conc. The intra-batch precision was determined by calculating the mean, SD and % CV of each conc. in the same batch. The inter-batch precision was determined by calculating the mean, SD and % CV of each QC conc. between batches.

57 The intra-batch accuracy was determined by calculating the mean of each QC in the same batch and then dividing by its nominal conc. multiplying by 100 to get result in %. The inter-batch accuracy was determined by calculating the mean of each QC between batches and dividing by its nominal conc. multiplying by 100 to get result in %. The normal acceptance criteria of within and between batch precision was 15 % for LQC, INTQC, MQC, HQC and 20 % for LOQQC, whereas for accuracy it should be 85-115 % for LQC, INTQC, MQC, HQC and 80-120 % for LOQQC. 2.2.5 Recovery Recovery was determined for NTG/metabolites and ursodiol/ metabolites as well as their IS by directly comparing the detector response (mean area) of analyte or IS added and recovered from the matrix compared to the detector response of unextracted AQ sample. For the calculation of recovery 6 samples at each QC levels (LQC, MQC and HQC) were processed and analyzed along with the 6 replicate of unextracted AQ samples. Analyte % recovery = Average response of analyte in spiked QC samples at individuallevel x 100 Average response of analyte in aqueous samples at individual level IS % recovery = Average response of analyte in spiked IS samples at individuallevel x 100 Average response of analyte in aqueous samples at individual level

58 The acceptance criteria for the % recovery were 50-110 % and the % CV of the mean recovery of all three QCs and difference of % recovery between any two individual QC should be 15 %. 2.2.6 Stability Stability of drugs in biological fluid was determined at different phases, as it is affected by many factors such as, variation in matrix from subject to subject or day to day, presence of endogenous or exogenous biochemical or chemicals, variation in storage conditions and variation in analytical techniques. For short term stability, stock solution of analyte for NTG/metabolites and ursodiol / metabolites as well as their IS were prepared and divided into two portions. One portion was stored in the refrigerator and the other portion was placed on the bench at room temperature for a period of 4 to 6 hr. Then 6 replicates of each portion of stock dilution at LQC and HQC level were prepared and spiked to calculate the mean SD and % CV of the peak response. Mean response of sample stored at room temperature Percent stability x 100 Mean response of sample stored at refrigerator The stability of the stock solution was acceptable, if it is within the range of 90-110 %. For long term stability, analyte stock solution of for NTG/metabolites and ursodiol / metabolites and IS of each were prepared and stored in the refrigerator for 22 days. A fresh stock solution for both the analyte and IS was prepared on the day of analysis. From both the refrigerator and freshly prepared stock solution dilution equivalent to MQC conc. for analyte and IS were prepared as follows:

59 Solution-1: An AQ solution of analyte was prepared from stock in refrigerator or freezer and intended IS conc. was prepared from freshly prepared IS stock. Solution-2: An AQ solution was prepared from IS stock in refrigerator or freezer and analyte solution from freshly prepared analyte stock. Solution-3: An AQ solution of both the analyte and IS was prepared from freshly prepared stock solutions. Mean area ratio Conc.fresh Conc.stored (Analyte/IS) Sol.1 analyte stock IS stock The stability for analyte 100 Mean area ratio Conc.stored Conc.fresh (Analyte/IS) Sol.3 analyte stock IS stock Mean area ratio Conc.fresh Conc.stored (IS/analyte) Sol.1 analyte stock IS stock The stability for IS 100 Mean area ratio Conc.stored Conc.fresh (IS/analyte) Sol.3 analyte stock IS stock The % stability was calculated, by comparing the area ratio of stability stock solution with the freshly prepared stock solutions. The use of stored solution for longer period was acceptable, if % stability range falls within 90-110 %. For validating the FT duration of matrix samples 6 replicates of analyte at LQC and HQC level in the plasma matrix were stored in freezer (at both -50 0 C and -15 0 C) for 24 hr after, which the same samples were withdrawn from the freezer and allowed to thaw at room temperature. The freeze and thaw procedure was repeated for 4 cycles and then the samples were analyzed against a freshly prepared CC along with freshly prepared QC samples at low and high levels. The stability was determined by calculating the accuracy

60 and precision of QC samples that gone through the FT cycles against the freshly prepared calibration standards and QC samples. Bench top stability of analyte in plasma matrix was evaluated with 6 replicates of LQC and HQC samples which were placed at room temperature for 8.73 hr (NTG) and 2 hr (ursodiol) and then analyzed along with freshly prepared CC and freshly prepared QC samples, at low and high levels. The stability of the samples in matrix was evaluated as: the accuracy for the stability samples was bound to fall within ± 15 % and precision at 15 %. The accuracy of the stability samples against compared samples should be within ± 15 % and 67 % of the total QCs per level must be 100 ± 15 % of nominal value. Wet-extract bench-top stability of analyte in plasma matrix was evaluated with 6 replicates of LQC and HQC samples which were processed and placed at room temperature for 27 hr for NTG and 5 hr 30 min. for ursodiol and then analyzed along with freshly prepared CC and freshly prepared QC samples at low and high levels. Dry-extract stability of ursodiol in plasma matrix was evaluated with 6 replicates of LQC and HQC samples which were processed and placed in refrigerator for 24 hr 40 min. in dried state and then reconstituted and analyzed along with freshly prepared CC and freshly prepared QC samples at low and high levels. Autosampler stability of analyte in plasma matrix was evaluated with 6 replicates of LQC and HQC samples which were processed and placed in autosampler for minimum of 126.83 hr for NTG and 55 hr for ursodiol and then analyzed along with freshly prepared CC and freshly prepared QC samples at low and high levels.

61 2.2.7 Re-injection Reproducibility Re-injection reproducibility was validated through 6 sets of calibration standards and QC samples at low and high level conc. which were prepared, analyzed and assessed for the precision and accuracy. The same samples were kept in the autosampler for 24/48 hr and then the whole sequence of samples was re-injected for the second time and precision and accuracy was assessed. The re-injected samples were bound to fall within the precision and accuracy specifications. 2.2.8 Dilution Integrity Dilution integrity was performed for NTG/metabolites and ursodiol /metabolites by preparing the spiked samples which were twice the conc. of HQC and analyzing 6 replicates of QC were diluted by a factor of 2 or 4 times with screened matrix samples against calibration standards prior to extraction. The acceptance criteria were the precision and accuracy of the dilution integrity QCs which is 15 % and should be within ± 15 % of nominal conc. respectively. 2.3 BA AND BE STUDY NTG and ursodiol BE studies were conducted in 18 and 12 healthy human adult male subjects respectively, under fasting condition. The clinical study was carried out, in accordance with the guideline for GCP, schedule Y and the principles enunciated in the Declaration of Helsinki. The studies were conducted at MTR, Chennai, India, according to the protocol approved by the Chennai Ethics Committee, Chennai, India. Healthy Indian male volunteers were selected after a standardized screening procedure and evaluation on basis of inclusion and exclusion criteria. Screening procedures includes: evaluation of medical history, demographic data, substance use history, physical examination,

62 12-lead electrocardiography, chest radiography, and laboratory analysis of hematologic profile, hepatic and renal function, and disease markers for syphilis, human immune virus, and hepatitis B and C. Only medically healthy subjects with clinically normal laboratory profiles were enrolled in the study. Volunteers were instructed not to use any medication before admission and not to consume any alcohol, tobacco, or xanthine containing products, grapefruit and/ or its juice, and poppy containing foods within 48 hrs before admission and during the stay at MTR. Male volunteers of Body Mass Index (BMI) between 18.50 kg/m 2 and 24.99 kg/m 2 and aged 18 45 years, willing to take ova-vegetarian diet with the limits of the above evaluation were allowed to participate in the study, after the submission the written informed consent, voluntarily. The exclusion of volunteers from the study was based on the allergy to any study medication, signs or symptoms of organ dysfunction, positive urinary drug screen (for cannabinoids and opioids), any blood donation / excess blood loss within 3 months of check-in, or any evaluation parameter significantly outside the reference ranges used at MTR. To all the enrolled subjects, information regarding the drugs, adverse effect (if any) dosing method and period, wash out period, blood withdrawal time points and volume was communicated. 2.3.1 Nitroglycerin For NTG study was conducted as an open label, balanced, randomized, single-dose, two-treatment, two-sequence, two-period, two-way crossover BE design with wash out period of at least 7 days between the periods. 2.3.1.1 Clinical phase The BE study was conducted at MTR and the subjects were housed for at least 11 hr prior to first dose of respective period. After sampling for 240 min.

63 post dose, as per schedule, subjects were discharged from MTR facility. All subjects were fasted overnight for at least 10 hr before the drug administration and for 4 hr post-dose. All subjects received standard meals: lunch, snacks and dinner at 4, 9 and 13 hr respectively, post drug administration. During housing, all meal plans were identical for both periods. Drinking water was not allowed until 1 hr post-dose and thereafter allowed without any further restriction. Compliance to proper dosing was assessed by conducting a thorough examination of the oral cavity by trained study personnel after dosing in each period. A total of 21 blood samples comprising of 288.00 ml blood were collected from each subject in K 2 EDTA vacutainer tubes during the course of the study through indwelling cannulae placed in forearm veins. The serial blood sampling was done at from each subject at predose (00.00), 1.00, 02.00, 03.00, 04.00, 05.00, 06.00, 08.00, 10.00, 12.00, 14.00, 15.00, 20.00, 25.00, 30.00, 45.00, 60.00, 75.00, 120.00, 180.00, and 240.00 min. in each period. The postdose samples were collected within 2 min. of the scheduled time. The plasma samples were separated from the collected blood samples using refrigerated (2 C to 8 C) centrifuge at 4000 ± 50 rpm for 10 min. The separated plasma was transferred into pre-labeled capped polypropylene vials and stored at -50 C until analysis. Vital signs of oral temperature, sitting blood pressure and radial pulse were measured during subject admission, prior to each dosing and 30.00, 60.00, 120.00 and 180.00 min. and after administration of study drug in each study period. Vital signs were measured prior to administration of the dose and were taken within 1 hr of the scheduled dosing time. At all other times, vital signs were taken within 40 min. of the scheduled times. Brief clinical examination of the subject was conducted by a qualified medical designate on duty after subject admission, prior to dosing of study drug and before discharge. All the subjects

64 were also monitored throughout the study period for adverse events, if any. At the end of study, physical examination, vital signs, and clinical laboratory tests (hematology and biochemistry) were performed of the participating subjects to ensure their safety. 2.3.1.2 Bioanalytical phase The plasma samples, stored at -50 C were subjected to analysis using validated LC-MS/MS method to determine the plasma conc. of NTG and their metabolites 1, 2-DNG and 1, 3-DNG. Samples of each subject (both the periods) were processed and analyzed along with CC and QC samples in a single batch. Each batch consisted of 8 calibration points (STD A-H) and subject samples interspaced with QC samples (LQC, INTQC, MQC, HQC). A total of 756 samples from 18 subjects for both the periods were subjected to analysis with calibration range of 43.54-8064.21 pg/ml for NTG, 54.85-10160.00 pg/ml for 1, 2-DNG and 1, 3-DNG. 2.3.2 Ursodiol The bioequivalence study was carried out with an open label, randomized, single-dose, three-treatment, three-sequence, three-period, threeway crossover design with a wash out period of at least 7 days between the periods. 2.3.2.1 Clinical phase All the subjects who were qualified through screening procedure were admitted and housed in the MTR at 60 hr prior to the first dose of respective period. After sampling for 48 hr post dose as per schedule, subjects were discharged from MTR facility. All subjects were fasted overnight for at least 10 hr before the drug administration and for 4 hr post-dose. After overnight fasting,

65 the subjects were randomized into two equal groups. Each subject received a single dose of 250 mg tablet of either the test or the reference product orally in sitting posture with 240 ml of water, under the supervision of a medical practitioner. All the subjects received standard meals: lunch, snacks and dinner at 4, 9 and 13 hr, respectively, after drug administration. During housing, all meal plans were identical for both the periods. Drinking water was not allowed, until 1 hr pre-dose and 2 hr post dose in each period. Thereafter, it was allowed at all times. Compliance to proper dosing was assessed by conducting a thorough examination of the oral cavity by trained study personnel after dosing in each period. A total 27 samples, per period comprising 364 ml blood were collected from each subject in K 3 EDTA vacutainer tubes during the course of the study through indwelling cannulae placed in forearm veins in each period. The serial blood samples were collected at: - 48.00, - 42.00, - 36.00, -30.00, - 24.00, -18.00, -12.00, -06.00, 00.00 (pre-dose) and post dose samples at 00.33, 0.67, 01.00, 01.50, 02.00, 02.50, 03.00, 03.50, 04.00, 05.00, 06.00, 08.00, 10.00, 12.00, 16.00, 24.00, 36.00 and 48.00 hr. The post-dose samples were collected within 2 min. of the scheduled time. The actual endpoint time of collection of each blood sample were recorded. After collection, the blood samples were centrifuged under refrigeration, as soon as possible to separate plasma. All plasma samples were then at -50 C on transfer to the analytical facility for assay. Vital signs, such as oral temperature, sitting blood pressure and radial pulse were measured during subject admission, prior to each dosing at - 49.00, - 39.00, -25.00, -15.00, 00.00 pre-dose and 01.00, 02.00, 05.00, 07.00, 12.00, 24.00 and 36.00 hr post dose in each study period. Vital signs were measured prior to administration of the dose and were taken within 1 hr of the scheduled dosing time. At all other times, vital signs were taken within 40 min. of the

66 scheduled time. Brief clinical examination of all subjects was conducted by a qualified medical designate on duty after subject admission, prior to dosing of study drug and before discharge. All the subjects were also monitored throughout the study period for adverse events, if any. To all the enrolled subjects, information regarding the drugs, adverse effect (if any) dosing method and period, wash out period, blood withdrawal time points and volume was communicated and consent signature for their participation in the study was obtained. At the end of study, physical examination, vital signs, and clinical laboratory tests (hematology and biochemistry) were performed to ensure the safety of all study participants. 2.3.2.2 Bioanalytical phase The plasma samples stored at -50 C were subsequently analyzed using validated LC-MS/MS method to determine the plasma levels of ursodiol and its conjugates; TUDCA and GUDCA. Samples of each subject (all periods) were processed and analyzed along with CC and QC samples in a single batch. Each batch consisted of 9 calibration points (STD A-I) and subject samples interspaced with QC samples (LQC, INTQC, MQC, HQC). A total of 1215 samples of 12 subjects from 3 periods were analyzed with calibration range of 25.88 to 15300.05 ng/ml for ursodiol, 2.68 to 1587.48 ng/ml for TUDCA and 25.44 to 15040.93 ng/ml for GUDCA. 2.4 PHARMACOKINETIC EVALUATION Pharmacokinetic parameters, such as C max, AUC 0-48, T max, K el and T 1/2 were determined in a model-independent way, considering sampling time deviation using WinNonlin software version 5.2.1. The highest conc. measured and the time at which it was first registered after each dose in subjects, were regarded as C max and T max, respectively. The T 1/2 was determined by

67 means of a linear regression using the terminal elimination phase of the formulation according to the algorithm of program WinNonlin. Parameters like T 1/2 and K el, were determined only for subjects in which the log-linear terminal phase could clearly be defined. The AUC 0-48 was calculated to determine the total drug exposure using linear trapezoidal rule. All values below the LOQ was considered as zero for the computation of pharmacokinetic parameters and statistical calculations. The Missing sample (marked as Missing ) and the Not Reported sample (marked as NR ), if observed anywhere in the data and they were not taken into account, when calculating the pharmacokinetic parameters. In such case, the previously available value had been automatically connected with the next available value, within the WinNonlin software. 2.5 STATISTICAL EVALUATION Descriptive statistical parameters, such as arithmetic mean, SD, CV %, minimum, maximum and median were calculated for the test and reference formulation for all the primary pharmacokinetic parameters (C max, AUC 0-48 ) as well as for the secondary pharmacokinetic parameters (T max, T 1/2, K el ). The untransformed and the natural log-transformed (i.e. Ln-transformed) values for the pharmacokinetic parameters C max, AUC 0-48 were analyzed for statistical difference between the test and the reference formulations with ANOVA by the use of mixed procedure of the SAS software version: 9.1.3. The untransformed values for the secondary pharmacokinetic parameters T max, Kel, and T 1/2 were analysed for statistical difference between test and reference formulations with ANOVA by use of mixed procedure of the SAS. ANOVA considered the Sequence, Subject (Sequence), Period, and Treatment effects. Level of significance was considered at 5 % for Subject (Sequence), Period, and Treatment effects while Sequence effects were tested at 10 % level of

68 Significance in the ANOVA. The intra-subject CV (based on ANOVA) for the primary parameters were also calculated. The 90 % CI of the relative mean of the test to reference formulation (T/R ratio) for AUC 0-48 and C max were calculated using natural log-transformed data. The CI intervals for these ratios should be contained within the limits 80 125 % for BE.