3.0. EXPERIMENTAL WORK

Transcription

1 3.0. EXPERIMENTAL WORK 3.1. ACTIVE PHARMACEUTICAL INGREDIENTS 1. Aceclofenac was supplied by Amoli Organic Pvt Ltd, Gujarat, 2. Artemether was obtained from Inventis Drug Delivery Systems, Hyderabad. 3. Alverine Citrate and P-Hydroxy Alverine were supplied by Laxai-Avanti Life sciences Pvt. Ltd. 4. Clopidogrel Bisulphate and its Carboxylic acid metabolite were commercially procured from Inventis Drug Delivery Systems, Hyderabad. 5. Carvedilol Phosphate and 4-Hydroxyphenyl Carvedilol were commercially procured from Zach system S.P.A., Italy and Vivan Life Sciences, Mumbai. 6. Propranolol [as Internal Standard for Carvedilol Phosphate and 4- Hydroxyphenyl Carvedilol] was supplied by Toronto Research Chemicals. 7. Clonidine was obtained from Inventis Drug Delivery Systems, Hyderabad. 8. Ketoconazole [as Internal Standard for Clonidine] was obtained from Inventis Drug Delivery Systems, Hyderabad. 9. Lumefantrine was obtained from Inventis Drug Delivery Systems, Hyderabad. 10. Levetiracetam and Rimantadine Hydrochloride [as Internal Standard] were obtained from Inventis Drug Delivery Systems, Hyderabad. 11. Telmisartan was obtained from Inventis Drug Delivery Systems, Hyderabad. 12. Ursodeoxycholic acid was supplied by Inventis Drug Delivery Systems, Hyderabad. 13. Ticlopidine HCL [as Internal Standard for Artemether, Alverine citrate and P- Hydroxy Alverine; Clopidogrel bisulphate and its carboxylic acid metabolite;

2 Lumefantrine and Telmisartan] was commercially procured from Shilpa Medicare Limited. 14. Diclofenac Sodium [as IS for Aceclofenac and Ursodeoxycholic acid] was supplied by Inventis Drug Delivery Systems, Hyderabad CHEMICALS AND SOLVENTS All the solvents were of analytical grade and used without further purification. 1. Acetonitrile and methanol were of HPLC grade and obtained from SD fine chem. and J. T. Bakers. 2. Formic acid [from Rankem], diethyl ether, dichloro methane and diethyl ether, ammonium formate, ammonium acetate, ammonia, isopropyl alcohol and acetone were obtained from Merck [Worli, Mumbai, India]. 3. Water was deionized, filtered and purified on a milli-q reagent grade water system from millipore [St Quentin en Yvelines, France]. 4. Drug free and healthy human plasma was obtained from Actimus Biosciences Pvt Ltd [Visakhapatnam] and Body Care Labs [Hyderabad], where blood was collected from volunteers in tubes containing citrate phosphate dextrose or heparin or K2EDTA. After centrifugation, the plasma was transferred into polypropylene tubes and stored at or below -20ºC INSTRUMENTS USED The High performance liquid chromatography [HPLC] system [Shimadzu Corporation, Kyoto, Japan] is equipped with LC-20ADVP binary pump, a DGU20A3 degasser and a SIL HTC auto sampler equipped with a CTO 10ASVp thermo stated column.

3 Robotic liquid handling system [HTLC] is operated using the software package supplied from the cohesive technologies Aria TM. The HTLC-MS/MS system consists of four pumps for gradient solvent delivery, and a divert valve to direct LC effluent to the mass spectrometer in the analyte elution window. The analytical column effluent is directed through the divert valve to a thermo electron TSQ quantum discovery mass spectrometer. Masses were acquired on a TSQ tandem mass spectrometry [Thermo Finnigan, Sanjose, CA, USA] equipped with electrospray ionization [ESI] and connected to a PC runs with the standard software Xcalibur and LC Quan Mass spectroscopic detection was performed on a triple Quadrapole instrument [Thermo, TSQ Quantum Discovery Max]. Other instruments used for the study were 1. Analytical Balance [Sartorious & CPA225D] 2. Micro Balance [Sartorious & SE-2] 3. Vortex Shaker [Spinix] 4. Ultra Sonicator [Bandolin Sonorex] 5. Micropipettes and Multipettes [Eppendorf] 6. Refrigerators [Godrez and Whirlpool] 7. Membrane Filters [0.22µ/0.45µ] 8. Filtration Assembly 9. Biomedical Deep Freezers [-86ºC & -30ºC][Sanyo] 10. Refrigerated Centrifuge [Eppendorf] 11. Syringe [Hamilton] and Magnetic Stirrer SOFT WARES USED

4 1. LC Quan & 2.5.6[Shimadzu Prominence] 2. Aria OS [Aria OS cohesive Tech.] 3.5. ANALYTICAL COLUMNS USED 1. Agilent Zorbax XDB-C18 [50 x 2.1mm,5μm] 2. Discovery C8 [50 x 2.1mm,5μm] 3. Discovery C18 [50 x 4.6mm,5μm] 4. Agilent C8 [50 x 3.0mm,5μm] 5. Agilent C18[50 x 2.0mm,5μm] 6. Anal micro C18 [4.6 x 50 mm, 50µm, 60Aº] 7. Thermo BDS Hypersil C18, [5μm, 4.6 x 50mm] 8. Cohesive Propel C18 [5μm, 3.0 x 50 mm] 9. Anal Chem.-C18 [50 x 4.6mm, 5μm] 10. HTLC Column-Cohesive Cyclone P [50 x 0.5mm, 50μm], 11. HTLC Column-Cyclone MAX [50 x 2.1mm, 5μm] 12. HTLC Column-Polar Plus [50 x 2.1mm, 5μm] columns OTHER MATERIALS USED 1. Human plasma [anticoagulant-cpda, K 2EDTA, K 3EDTA] 2. HPLC vials 3. Polypropylene tubes and caps 4. Para film 5. Eppendorf tubes 6. Hand gloves 7. Pipette tips [ μl & μl] 8. Multipette tip [Comb tip plus 1 ml and 2.5 ml].

5 3.7. METHOD DEVELOPMENT Prepared stock solutions of analyte and internal standard respectively and their dilutions in suitable solvent such as acetonitrile/methanol for tuning of molecule on mass spectrometer by considering the solubility & stability data from the available literature Tuning of Molecules [To get optimum MS/MS parameters in the ESI/MS/MS mode] 1. Set the syringe pump and injection valve for auto loop injection. 2. Heated the ion transfer tube to 350ºC maximizes the ion transmission to the mass spectrometer to set the capillary [ion transfer tube] temperature so that it is proportional to the flow rate of solution. 3. Set the mass spectrometer for specific compound from tune master. 4. Ran the automatic compound optimization procedure to fine-tune the mass spectrometer parameters that are compound dependent. 5. Set the positive or negative parameters depending upon the structure of the product. 6. Ran the system in scan mode for baseline without interference with HPLC Grade water or methanol. 7. Repeat the run on MS/MS system after getting the baseline with buffered water. No interference was observed. Then, injected the sample with required concentrations and observed the peaks in spectrum. 8. Identified the exact mass in centroid.

6 9. Optimized the tuning of the molecule to establish the stable and intense ion beam by optimizing parameters such sheath gas, auxiliary gas, spray voltage, skimmer offset flow rate and collision gas. 10. Optimized the multiple ion parameters. 11. Confirmed the parent ion mass by recording the spectrum with optimized parameters. 12. Verified the condition by using multiple channel addition Identifying, Optimizing and Confirmation of Parent Ion [Q1 MS]- 1. Entered the exact center mass with scan width [units]. 2. Set the scan time & peak width[0.70µ] with micro scans to 1 3. Ran the system in scan mode [SRM or MRM]. 4. Turned off the Auto SIM. 5. Controlled the source CID, data processing, and Q2 CID Gas check. 6. Optimized the collision gas and collision energy to identify the products. 7. Identified the exact mass in centroid. 8. Optimized the collision energy and skimmer effect. 9. Verified the condition by using multiple channel addition. 10. Optimized the parameters for identifying products by using multiple reaction monitoring. 11. Entered the exact parent ion mass & product ion mass collected from Literature. 12. Recorded the spectrum with optimized parameters. 13. Optimized the multiple ion parameters Identifying, Optimizing and Confirmation of Product/daughter Ion [Q3 MS and MRM]-

7 1. Set the positive or negative parameters depending upon the structure of the product. 2. Ran the system in scan mode. 3. After fragmenting the parent mass with adjusting the collision energy and collision gas [mtorr] observed multiple ions in spectrum. 4. Finally confirmed the product ion mass from multiple ion masses by recording the spectrum with optimized parameters. 5. Verified the condition by using multiple channel addition. 6. Finally recorded the optimized tuning parameters to molecule which are synchronization mode, ion source[esi/apci], scan type[srm/mrm],polarity[positive/negative], SRM transition[q1 mass; Q3 mass [m/z]], spray voltage, sheath gas pressure, auxiliary gas pressure, capillary temperature, collision Energy [CE] and skimmer offset Method Development on HPLC Method development on HPLC was performed for following drugs such as Aceclofenac, Artemether, Alverine and P-Hydroxy Alverine, Clonidine, Lumefantrine, Levetiracetam, Telmnisartan and Ursodeoxy cholic Acid. Sample preparation involved preparation of stocks for drug & internal standard, different mobile phases, calibration curve standards and quality control samples based c max, calibration curve spiking solutions, quality control spiking solutions, spiked plasma calibration curve & quality control standards, and different procedures for extraction [precipitation, liquid liquid extraction, solid phase extraction]. Chromatographic separation of drug and internal standard in matrix by set the chromatographic conditions such as using the different HPLC analytical columns,

8 different mobile phases, pumping mode [binary flow/gradient flow], flow rate, injection volume, pressure limit [0 psi-5000 psi], retention time, run time, auto sampler temperature, column oven temperature, detector, rinsing cycle [rinsing volume, needle stroke, rinsing speed, sampling speed, purge time, rinsing mode] Quantification [recovery and peak shape] was done by altering any chromatographic parameter, standardization, peak height, area and calibrations check Method Development on HTLC Method development on HTLC was performed for Clopidogrel and its metabolite; Carvedilol and $-Hydroxy Carvedilol. Connected the modules for method development on HTLC as follows shown in Fig.No.3.1 and used an UNION in place of analytical column. The development started with use of cyclone turbo column on HTLC. Created three LC methods using 3.5, 6.8 and 8.0 ph Buffers as shown below LC Method with 3.5 ph Buffer- 1. Created first LC Method on HTLC to equilibrate the Column with 3.5 ph buffer and injected air using this method as shown in Fig.No Created first LC Method on HTLC to run the actual sample in 3.5 ph Buffer as shown in Fig.No LC Method with 6.8 ph Buffer- 1. Created second LC Method on HTLC to equilibrate the column with 6.8 ph Buffer and injected air using this method as shown in Fig.No.3.4.

9 2. Created second LC Method on HTLC to run Sample with 6.8 ph Buffer as shown in Fig.No LC Method with 8.0 ph Buffer- 1. Created third LC Method on HTLC to equilibrate the Column with 8.0 ph Buffer and injected air using this method as shown in Fig.No Created third LC Method on HTLC to run Sample with 8.0 ph Buffer as shown in Fig.No.3.7. Purged all the lines with respective Buffers A=3.5 ph, B=6.8 ph and C=8.0 ph and D= Acetonitrile, Checked the detector Status which is inline and placed the samples in Auto Sampler Tray position. Created a batch by entering the proper path, sample name, proper LC method and file names; and injected the samples as shown in below. Injection sequence for LC method Sr.No. Name Method No. of injections 1. Air 3.5 ph Buffer LC method Sample 3.5 ph Buffer LC method Air 6.8 ph Buffer LC method Sample 6.8 ph Buffer LC method Air 8.0 ph Buffer LC method Sample 8.0 ph Buffer LC method 03 Observed the chromatographic results of injected samples with different HTLC columns [i.e. cyclone P, Cyclone MAX, Polar plus] by using three different buffers to get the complete blank peek in loading cycle based. If the peak is seen in the first 60 sec then it implies that the column is not retaining the drug and elutes quickly. If not observes the peak in the first 60 sec then it means that the drug is retained in the

10 column. If observed a big peak in the washing cycle and a small peak in the loading time, it means that the column is not fully retaining the drug. Based on the results the buffer and HTLC column was selected. A program was created to optimize the Transfer Cycle as shown in Fig.No.3.8. The Mobile Phase selected was based on an assumption- 3.5 ph, and the sample was loaded at 2mL/min flow rate for 60 Sec as low as 30 sec. Then it was transferred to the UNION at the flow rate of 0.15mL/min for 60 sec. The Column was washed for 120 sec and followed by loop filling and equilibrium. The trials were made to get complete peak without return to base line during the transfer cycle by increasing the organic percentage to another 10% with selected buffer and HTLC column. Then connected an analytical column and developed a generic method as shown in Fig.No.3.9. by several development trials. Loading Pump A & Eluting Pump A used same P H determined during method development. Also, the ph can vary. Mobile Phase B was Acetonitrile/Methanol which suits analysis. Mobile Phase D= Acetonitrile: Isopropyl Alcohol: Acetone [60:25:15 v/v/v]. Loop filling % was optimized during method development. Tuned finely the LC method by decreasing or increasing the washing with the RT of the peak and the carry over level. A blank was after the drug to find out the carry over.

11 HTLC column Plug Waste A B 5 Eluting Pump 3 4 Union Autosampler Loading Pump Detector Fig.No.3.1. Method Development on HTLC Fig.No ph Buffer Equilibrium LC Method

12 Fig.No ph Buffer LC Method

13 Fig.No ph Buffer Equilibrium LC Method

14 Fig.No ph Buffer LC Method

15 Fig.No ph Buffer Equilibrium LC Method

16 Fig.No ph Buffer LC Method

17 Fig.No.3.8. LC Optimization Method

18 Fig.No.3.9. Generic LC Method Sample Preparation Sample Preparation for Aceclofenac Preparation of Aceclofenac Stock solution Approximately weighed 20 mg of aceclofenac working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given below. Stored the solution in refrigerator [2ºC to 8ºC].

19 Preparation of Diclofenac [IS] stock solution: Approximately weighed 5 mg of diclofenac working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 50 µg/ml from the IS stock solution [IS Stock] by using diluent [50:50 v/v methanol: water] Drug intermediate stock solution: Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the concentration approx 50 µg/ml. Added required volume of diluent [50:50 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples:

20 Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions from the drug intermediate stock using diluent [50:50 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of 0.106, 0.302, 0.605, 1.008, 2.016, 4.032, 6.064, 9.050, , µg/ml by spiking CC spiking solutions and QC standards in the range of , 6.532, 2.939, 0.323, µg/ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.4 ml of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted 0.2mL of CC, QC and subject samples into pre-labelled eppendorf vials. Added 50.0 µl of IS dilution [about 50µg/mL] into eppendorf vials, and vortexed to mix. Added 0.6mL acetonitrile into eppendorf vials, and vortexed to mix. Centrifuged the eppendorf vials at rpm and at 10 C for 10min, transferred approximately 0.5mL of supernatant to prelabelled

21 HPLC vials. Subsequently transferred the HPLC vial to the auto sampler and 5-µL aliquot was injected into the chromatographic system Sample Preparation for Artemether Preparation of Artemether Stock solution Approximately weighed 10 mg of artemether working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Ticlopidine [IS] stock solution: Approximately weighed 5 mg of ticlopidine working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 25 ng/ml from the IS stock solution [IS Stock] by using diluent [0.1% formic acid in methanol] Drug intermediate stock solution:

22 Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the concentration approx 100 µg/ml. Added required volume of diluent [0.1% formic acid in methanol] and Sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples: Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the concentration range 0.5 to 4000ng/mL from the drug intermediate stock using diluent [0.1% formic acid in methanol]. Stored Spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of 10, 20, 81, 162, 362, 482, 643, 804 ng/ml by spiking CC spiking solutions and QC standards in the range of 603, 362, 28.9, 12.5 ng/ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.4mL of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC

23 samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled polypropylene tubes. Added 50.0 µl of IS dilution [about 25 ng/ml] into polypropylene tubes, and vortexed to mix. Added 2.5 ml of 30% v/v dichloromethane in diethyl ether into polypropylene tubes, and vortexed to mix. Centrifuged the polypropylene tubes at 4000 rpm and at 10 C for 10 min. The supernatant layer was transferred in to another pre-labelled glass tube and the solvent was evaporated to dryness at 50 C under nitrogen stream. The residue was dissolved in 200 µl of mobile phase by vortex mixing and the solutions were transferred in to prelabelled conical glass micro vials. Subsequently transferred the vial to the auto sampler and 10-µL solution was injected into the chromatographic system Sample Preparation for Alverine and P-Hydroxy Alverine Preparation of Alverine and P-Hydroxy Alverine Stock solution Approximately weighed 10 mg of alverine [A]/ 10 mg of P-hydroxy alverine [B] working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of acetonitrile and sonicated to aid dissolution. Make up the volume up to the mark with acetonitrile. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using

24 formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Ticlopidine [IS] stock solution: Approximately weighed 10 mg of ticlopidine working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of acetonitrile and sonicated to aid dissolution. Make up the volume to the mark with acetonitrile. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 100 ng/ml from the IS stock solution [IS Stock] by using diluent [80:20 v/v acetonitrile: water] Drug intermediate stock solution: Aliquoted required volume of drug stock solutions [Alverine and P-Hydroxy Alverine] into 10 ml volumetric flask to get the concentration approx 100 µg/ml. Added required volume of diluent [80:20 v/v acetonitrile: water] and Sonicated to aid dissolution. Make up the volume to the mark with Diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with Para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples:

25 Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared Calibration Curve Standards and Quality Control Spiking solutions in the concentration range 1.0 to 250 ng/ml for Alverine and P- Hydroxy Alverine from the drug intermediate stock using diluent [80:20 v/v acetonitrile: water]. Stored Spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards for alverine and P-hydroxy alverine in the range of 20.6, 58.8, 167.9, 373.1, 932.8, , , 4975 pg/ml by spiking CC spiking solutions and QC standards for alverine and P-hydroxy alverine in the range of , , 63.7, 25.5 pg/ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.5mL of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled polypropylene tubes. Added 50.0 µl of IS dilution

26 [about 100 ng/ml] into polypropylene tubes, and vortexed to mix. Added 2.5 ml of 70:30 v/v diethyl ether: dichloromethane into polypropylene tubes, and vortexed to mix. Centrifuged the polypropylene tubes at 4000 rpm and at 10 C for 10 min. The supernatant layer was transferred in to another pre-labelled glass tube and the solvent was evaporated to dryness at 50 C under nitrogen stream. The residue was dissolved in 250 µl of mobile phase by vortex mixing and the solutions were transferred in to prelabelled conical glass micro vials. Subsequently transferred the vial to the auto sampler and 10-µL solution was injected into the chromatographic system Sample Preparation for Clopidogrel and its Metabolite Preparation of Clopidogrel and its Metabolite Stock solution Approximately weighed 5 mg of clopidogrel / 10 mg of carboxylic acid derivative of clopidogrel working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Ticlopidine [IS] stock solution: Approximately weighed 2 mg of ticlopidine working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film.

27 Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 100 ng/ml from the IS stock solution [IS Stock] by using Diluent [80:20 v/v methanol: water] Drug intermediate stock solution: Aliquoted required volume of drug stock solutions [Clopidogrel and its Metabolite] into 10 ml volumetric flask to get the required concentration. Added required volume of diluent [80:20 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples: Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the concentration range 5.0 to 500 ng/ml for clopidogrel and 3.0 to 300µg/mL for its metabolite from the drug intermediate stock using diluent [80:20 v/v methanol: water]. Stored Spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples:

28 Prepared the CC standards in the range of 102, 285, 816, 1632, 3265, 4472, 6577, 8433 pg/ml for clopidogrel and 73, 203, 581, 1163, 2327, 3188, 4689, 6012 ng/ml for its metabolite by spiking CC spiking solutions and QC standards in the range of 5548, 2774, 299, 125 pg/ml for clopidogrel and 3955, 1977, 213, 89 ng/ml for its metabolite by spiking QC spiking solutions in screened human plasma. Aliquoted 0.6mL of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled HPLC vials. Added 50.0 µl of IS dilution [about 100 ng/ml] followed by 0.200mL of 10mM ammonium formate buffer of ph 7.5 into vials, capped it, vortexed to mix and transferred vials to auto sampler. 10-µL solution was injected into the chromatographic system Solutions used for robotic on-line sample extraction system- 10mM ammonium formate buffer was used in pump A, pure acetonitrile was used in pump B, 0.1% formic acid was used in pump C and washing solution in the ratio of 65:20:15[v/v/v] acetonitrile: IPA: acetone was used in pump D.

29 Sample Preparation for Carvedilol & 4-Hydroxyphenyl Carvedilol Preparation of Carvedilol & 4-Hydroxyphenyl Carvedilol Stock solution Approximately weighed 2 mg of carvedilol/ 2 mg of 4-hydroxyphenyl carvedilol working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Propranolol [IS] stock solution: Approximately weighed 2 mg of propranolol working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 100 ng/ml from the IS stock solution [IS Stock] by using Diluent [70:30 v/v methanol: water].

30 Drug intermediate stock solution: Aliquoted required volume of drug stock solutions [carvedilol/ 4- hydroxyphenyl carvedilol] into 10 ml volumetric flask to get the required concentration. Added required volume of diluent [70:30 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples: Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the concentration range 5.0 to 12.5 µg/ml for carvedilol and 4- hydroxyphenyl carvedilol from the drug intermediate stock using diluent [70:30 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of 0.1, 0.7, 1.6, 5.8, 15.0, 55.0, 126.0, ng/ml for carvedilol and 4-hydroxyphenyl carvedilol by spiking CC spiking solutions and QC standards in the range of 200, 100, 1.25, 0.5 ng/ml for carvedilol and 4-hydroxyphenyl carvedilol by spiking QC spiking solutions in screened human plasma.

31 Aliquoted 0.2mL of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled HPLC vials. Added 25.0 µl of IS dilution [about 100 ng/ml] followed by 50µL of 10mM ammonium formate buffer of ph 7.8 into vials, capped it, vortexed to mix and transferred vials to auto sampler. 10-µL solution was injected into the chromatographic system Solutions used for robotic on-line sample extraction system- Pure acetonitrile was used in pump A, 0.15% formic acid was used in pump B, 10 mm ammonium formate buffer was used in pump C and washing solution in the ratio of 10:20:70[v/v/v] acetone: iso propyl alcohol: acetonitrile was used in pump D Sample Preparation for Clonidine Preparation of Clonidine Stock solution Approximately weighed 5 mg of clonidine working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with Para film. Corrected

32 the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Ketoconazole [IS] stock solution: Approximately weighed 5 mg of ketoconazole working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 500 ng/ml from the IS stock solution [IS Stock] by using diluent [80:20 v/v methanol: water] Drug intermediate stock solution: Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the concentration approx 100 ng/ml. Added required volume of diluent [80:20 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples:

33 Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the concentration range as working standards from the drug intermediate stock using diluent [80:20 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of , , , , , , , , , pg/ml by spiking CC spiking solutions and QC standards in the range of , , , , pg/ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.5mL of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted 0.5 ml of CC, QC and subject samples into pre-labelled polypropylene tubes. Added 50.0 µl of IS dilution [about 500 ng/ml] and 100μL of 10mM ammonium acetate ph 6.8 into polypropylene

34 tubes, and vortexed to mix. Added 2.0 ml of 20% v/v dichloromethane in diethyl ether into polypropylene tubes, and vortexed to mix. Centrifuged the polypropylene tubes at 4000 rpm and at 10 C for 10min. The supernatant layer was transferred in to another pre-labelled glass tube and the solvent was evaporated to dryness at 50 C under nitrogen stream. The residue was dissolved in 150 µl of 40:40:20 v/v/v methanol: acetonitrile: 10mM ammonium acetate [ph 6.8] by vortex mixing and the solutions were transferred in to prelabelled conical glass micro vials. Subsequently transferred the vial to the auto sampler and 10-µL solution was injected into the chromatographic system Sample Preparation for Lumefantrine Preparation of Lumefantrine Stock solution Approximately weighed 10 mg of lumefantrine working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Ticlopidine [IS] stock solution: Approximately weighed 5 mg of ticlopidine working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film.

35 Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 2 µg/ml from the IS stock solution [IS Stock] by using diluent [70:30 v/v methanol: water] Drug intermediate stock solution: Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the concentration approx 500 µg/ml. Added required volume of diluent [70:30 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples: Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared Calibration Curve Standards and Quality Control Spiking solutions in the range of to µg/ml from the drug intermediate stock using diluent [70:30 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC].

36 Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of 20, 40, 140, 300, 610, 1020, 1460, 1943, 2430, 3035 ng/ml by spiking CC spiking solutions and QC standards in the range of 2125, 1212, 67, 27 ng/ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.5 ml of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled eppendorf vials. Added 50.0 µl of IS dilution [about 2 µg/ml] into eppendorf vials, and vortexed to mix. Added 1.5 ml acetonitrile into eppendorf vials, and vortexed to mix. Centrifuged the eppendorf vials at rpm and at 10 C for 10min, transferred approximately 0.5mL of supernatant to prelabelled HPLC vials. Subsequently transferred the HPLC vial to the auto sampler and 10-µL aliquot was injected into the chromatographic system.

37 Sample Preparation for Levetiracetam Preparation of Levetiracetam Stock solution Approximately weighed 40 mg of levetiracetam working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Rimantadine [IS] stock solution: Approximately weighed 2 mg of rimantadine working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 500 ng/ml from the IS stock solution [IS Stock] by using 80:20 v/v methanol: water Drug intermediate stock solution: Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the concentration approx 500 µg/ml. Added required volume of diluent [70:30 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to

38 mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples: Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the range as working standards from the drug intermediate stock using diluent [70:30 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of 1, 2, 4, 8, 15, 20, 30, 40 µg/ml by spiking CC spiking solutions and QC standards in the range of 1.2, 2.8, 15.0, 24.7 µg /ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.5 ml of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and

39 subject samples into pre-labelled eppendorf vials. Added 500 µl of IS dilution [about 500 ng/ml] into eppendorf vials, and vortexed to mix. Added 1.0 ml acetonitrile into eppendorf vials, and vortexed to mix. Centrifuged the eppendorf vials at rpm and at 10 C for 10min, transferred approximately 0.8 ml of supernatant to prelabelled HPLC vials. Subsequently transferred the HPLC vial to the auto sampler and 10-µL aliquot was injected into the chromatographic system Sample Preparation for Telmisartan Preparation of Telmisartan Stock solution Approximately weighed 10 mg of telmisartan working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume up to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Ticlopidine [IS] stock solution: Approximately weighed 10 mg of ticlopidine working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of methanol and sonicated to aid dissolution. Make up the volume to the mark with methanol. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution:

40 Prepared the IS dilution of about 100 ng/ml from the IS stock solution [IS Stock] by using 70:30 v/v methanol: water Drug intermediate stock solution: Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the required concentration. Added required volume of diluent [80:20 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples: Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the range as working standards from the drug intermediate stock using diluent [80:20 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of , , , , , , , ng/ml by spiking CC spiking solutions and QC standards in the range of , , , ng/ml by spiking QC spiking solutions in screened human plasma.

41 Aliquoted 0.5 ml of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled eppendorf vials. Added 50 µl of IS dilution [about 100 ng/ml] into eppendorf vials, and vortexed to mix. Added 1.0 ml acetonitrile into eppendorf vials, and vortexed to mix. Centrifuged the eppendorf vials at rpm and at 10 degrees for 10min, transferred approximately 0.8 ml of supernatant to prelabelled HPLC vials. Subsequently transferred the HPLC vial to the auto sampler and 10-µL aliquot was injected into the chromatographic system Sample Preparation for Ursodeoxycholic Acid Preparation of Ursodeoxycholic Acid Stock solution Approximately weighed 10 mg of ursodeoxycholic acid working standard and transferred to 10.0 ml volumetric flask. Added 5.0 ml of acetonitrile and sonicated to aid dissolution. Make up the volume up to the mark with acetonitrile. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Diclofenac [IS] stock solution:

42 Approximately weighed 10 mg of diclofenac working standard and transferred to 10.0 ml volumetric flask. Add 5.0 ml of acetonitrile and sonicated to aid dissolution. Make up the volume to the mark with acetonitrile. Stoppered the flask and shaken gently to mix. Sealed the stopper with para film. Corrected the final concentration by using formula given in section Provide a batch number. Store the solution in refrigerator [2ºC to 8ºC] Preparation of Internal Standard Dilution: Prepared the IS dilution of about 1.5 µg/ml from the IS stock solution [IS Stock] by using 80:20 v/v methanol: water Drug intermediate stock solution: Aliquoted required volume of drug stock solution into 10 ml volumetric flask to get the required concentration. Added required volume of diluent [80:20 v/v methanol: water] and sonicated to aid dissolution. Make up the volume to the mark with diluent. Stopper the flask and shaken gently to mix. Sealed the stopper with para film. Stored the solution in refrigerator [2ºC to 8ºC] Preparation of Calibration Curve Standards and Quality Control Samples:

43 Preparation of Calibration Curve and Quality Control Spiking Solutions: Prepared calibration curve standards and quality control spiking solutions in the range as working standards from the drug intermediate stock using diluent [80:20 v/v methanol: water]. Stored spiking solutions of CC and QC [s] in refrigerator [2ºC to 8ºC] Preparation of Spiked Plasma Calibration Curve and Quality Control samples: Prepared the CC standards in the range of 0.1, 0.202, 0.403, 0806, 1.466, 1.954, 2.443, µg/ml by spiking CC spiking solutions and QC standards in the range of 2.108, 1.265, 0.253, µg/ml by spiking QC spiking solutions in screened human plasma. Aliquoted 0.5 ml of each CC standards and QC sample into pre-labelled polypropylene tubes and capped them tightly. Stored CC standard, QC samples, standard blank and standard zero samples in deep freezer [below 80 C] Sample Preparation Retrieved the frozen CC, QC samples and subject plasma samples from the deep freezer and thawed in water bath maintained at room temperature, vortexed to mix. Removed the caps from the polypropylene tubes. Aliquoted ml of CC, QC and subject samples into pre-labelled eppendorf vials. Added 30 µl of IS dilution [about 1.5µg/mL] into eppendorf vials, and vortexed to mix. Added 1.0 ml acetonitrile into eppendorf vials, and vortexed to mix. Centrifuged the eppendorf vials at rpm and at 10 C for 5min, transferred approximately 0.8 ml of supernatant to prelabelled

44 HPLC vials. Subsequently transferred the HPLC vial to the auto sampler and 10-µL aliquot was injected into the chromatographic system Data Processing Acquired chromatograms using the software supplied by the LC-MS/MS manufacturers. The calibration curve [fitted by first-order y = ax + b, where a=slope, b=intercept, x=concentration and y=peak area ratio of Drug/IS] is plotted as the peak area ratio [Drug/IS] on Y axis vs. the nominal concentration of drug on X axis. The concentrations of the known samples were calculated by using linear regression equation with suitable weighing factor [1/x 2 or 1/x].

45 3.8. METHOD VALIDATION The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose [International Conference on Harmonization Guideline Q2A]. Method validation is the process of demonstrating that analytical procedures are suitable for their intended use [US Food and Drug Administration Draft Guidance for Industry, 2000]. Performed the experiments involved in method validation as discussed below: System Suitability and Auto Injector Carryover 4. System Suitability Processed aqueous standard at MQC level and ran the chromatographic device with injecting six consecutive from aqueous standard. Calculated the mean, standard deviation [SD] and coefficient of variation [CV] for response ratio and retention time of drug and IS. Acceptance Criteria : Coefficient of variation [CV] for Response ratio should be 5% and retention time of drug and IS should be 2%. 5. Auto Injector Carryover- Ran the chromatographic device by injecting by following sample sequence and calculated the % of interference for drug and IS in RS and blank at the retention time of drug and IS compared to the lower concentration level.