Niralee Thakar. Department of Chemistry and Biochemistry. University of North Carolina Wilmington. Approve By. Advisory committee.

Transcription

1 DEVELOPMENT AND VALIDATION OF A COMBINED METHOD FOR QUANTITATIVE DETERMINATION OF DISSOLUTION, ASSAY, AND CONTENT UNIFORMITY IN BISOPROLOL FUMARATE/ HYDROCHLOROTHIAZIDE TABLETS USING HPLC AND UPLC Niralee Thakar A Thesis Submitted to the University of North Carolina Wilmington in Partial Fulfillment of the Requirements for the Degree of Masters of Science Department of Chemistry and Biochemistry University of North Carolina Wilmington 2011 Approve By Advisory committee Jeremy Morgan Nadeem Zia John Tyrell Chair Accepted by Dean, Graduate School

2 TABLE OF CONTENTS ABSTRACT... iv ACKNOWLEDGEMENTS...v DEDICATION... vi LIST OF TABLES... vii LIST OF FIGURES...x INTRODUCTION AND BACKGROUND... 1 EXPERIMENTAL...7 Materials and Reagents... 7 Equipment... 7 HPLC Instrumentations and Conditions... 8 UPLC Instrumentations and Conditions... 9 Preparation of the Solution for Dissolution Testing Preparation of the Solution for Assay and CU Testing RESULTS AND DISCUSSIONS Method Development UPLC Method Transfer Assay/CU Validation Linearity/Range Recovery/Accuracy Precision Specificity ii

3 Robustness of Extraction Procedure Filter Study Robustness of Chromatographic Parameter Stability of Sample Solution Forced Degradation Method Comparison Dissolution Method Validation Linearity/Range Recovery/Accuracy Precision Specificity Robustness of Degassing Procedure Robustness of Sampling Procedure (Manual vs. Automated) Filter Study Robustness of Chromatographic Parameters Stability of Sample Solution Method Comparison Teflon Paddles vs. Non Teflon Paddles CONCLUSION REFFERENCES...69 APPENDIX...73 iii

4 ABSTRACT In pharmaceutical and biomedical analysis, the main objective is to reduce analysis time and maintain good efficiency; therefore, there has been substantial focus on high speed chromatography separation. Recently, one of the most promising developments in the area of fast chromatography separation is ultra performance liquid chromatography (UPLC). A precise, accurate, and simple method was developed and validated for bisoprolol Fumarate (BF) and hydrochlorothiazide (HCTZ). The two combined active pharmaceutical ingredients (API) are in a single dosage form. The method was developed and validated for dissolution, assay, and content uniformity (CU) testing using high performance liquid chromatography (HPLC) and UPLC. The method was validated according to ICH guidelines and current Sandoz Standard Operation Procedure (SOP), for accuracy, precision, intermediate precision, linearity, range, specificity, extraction study, filter study, system suitability, stability of solutions, force degradation, and robustness. 1 Also the method was compared with current United States Pharmacopeias (USP) method for Bisoprolol Fumarate and Hydrochlorothiazide Tablets. The results were compared between two different systems, traditional HPLC and new UPLC. Also the advantages and disadvantage of UPLC and HPLC usage will be discussed. iv

5 ACKNOWLEDGEMENTS I would like to thank the following people who were instrumental in the completion of this thesis: Dr. Nadeem B. Zia (Manager QA/QC Compliance, Sandoz) Dr. John A. Tyrell (Department of Chemistry and Biochemistry, UNCW) Dr. Jeremy Morgan (Department of Chemistry and Biochemistry, UNCW) John Bredin (Manager, QC, Sandoz) Richard Uveges (Director, QC, Sandoz) Jason Miller (Chemist IV, QC, Sandoz) Tenika Smith (Chemist II, QC, Sandoz) Kathy Jones (Chemist II, QC, Sandoz) Amber Vogt (Chemist II, QC, Sandoz) Sandoz Inc. Wilson, NC for the financial support and for the opportunity UNCW Faculty and Staff v

6 DEDICATION I would like to dedicate this to my parents and my husband. My parents have always encouraged me to study further. My husband has supported me in all manners. vi

7 LISTS OF TABLES Table Page 1. Retention time using HPLC HPLC gradient Retention time using UPLC UPLC gradient ASSAY CU METHOD VALIDATION 5. Calculated linearity results for BF and HCTZ using HPLC and UPLC Assay/CU accuracy/recovery results for BF and HCTZ using HPLC and UPLC Assay precision and intermediate precision results for BF using HPLC and UPLC Assay precision and intermediate precision results for HCTZ using HPLC and UPLC CU precision and intermediate precision results for BF using HPLC and UPLC CU precision and intermediate precision results for HCTZ using HPLC and UPLC Robustness of Extraction procedure results for BF and HCTZ using HPLC and UPLC Filter study results for BF and HCTZ using HPLC and UPLC Filter study s percent difference results for BF and HCTZ using HPLC and UPLC Robustness Parameter for HPLC Robustness Parameter for UPLC Robustness system suitability results for BF and HCTZ using HPLC Robustness system suitability results for BF and HCTZ using UPLC Solution stability results for standard preparation using HPLC and UPLC Solution stability results for sample preparation using HPLC and UPLC Forced degradation results for BF in blend sample using HPLC Forced degradation results for BF in blend sample using UPLC Forced degradation results for HCTZ in blend sample using HPLC Forced degradation results for HCTZ in blend sample using UPLC vii

8 24. Method comparison results for BF using USP method Method comparison results for HCTZ using USP method Method comparison results for HCTZ using current Sandoz method Method comparison results for BF using current Sandoz method DISSOLUTION METHOD VALIDATION RESULTS 28. Linearity results for BF and HCTZ using HPLC and UPLC Recovery/Accuracy results for BF and HCTZ using HPLC and UPLC Precision and intermediate precision results for BF and HCTZ using HPLC Precision and intermediate precision results for BF and HCTZ using UPLC Robustness of degassing procedure results for BF using HPLC Robustness of degassing procedure results for BF using UPLC Robustness of degassing procedure results for HCTZ using HPLC Robustness of degassing procedure results for HCTZ using UPLC Robustness of sampling procedure results for BF and HPLC using HPLC Robustness of sampling procedure results for BF and HPLC using UPLC Filter study result for BF and HCTZ using HPLC and UPLC Filter study s percent difference result for BF and HCTZ using HPLC and UPLC Robustness parameter for HPLC Robustness parameter for UPLC Robustness system suitability results for BF and HCTZ using HPLC Robustness system suitability results for BF and HCTZ using UPLC Solution stability results for BF standard preparation using HPLC and UPLC Solution stability results for BF sample preparation using HPLC and UPLC Solution stability results for HCTZ standard preparation using HPLC and UPLC Solution stability results for HCTZ sample preparation using HPLC and UPLC Method comparison results for BF using USP method viii

9 49. Method comparison results for HCTZ using USP method Method comparison results for BF using current Sandoz method Method comparison results for HCTZ using current Sandoz method Teflon paddles vs. non Teflon paddles results for BF and HCTZ using HPLC Teflon paddles vs. non Teflon paddles results for BF and HCTZ using UPLC ix

10 LISTS OF FIGURES Figure Page 1. Structure of BF and HCTZ Dissolution sample using HPLC chromatography Dissolution sample using UPLC chromatography Assay sample using HPLC chromatography Assay sample using UPLC chromatography Resolution ID using HPLC chromatography Resolution ID using UPLC chromatography x

11 INTRODUCTION AND BACKGROUND Pharmaceutical dosage formulations containing the beta-blocker bisoprolol fumarate (BF) and the diuretic hydrochlorothiazide (HCTZ) are used for the treatment of high blood pressure. 3 The combined mixture of BF, 1-[4-[[2-(1-methylethoxy) ethoxy]methyl]-phenoxy]-3-[(1-methylethyl)amino] -2-propanolethylene-1,2- dicarboxylic acid and HCTZ, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiazine-7 sulfonamide-1,1-dioxide is a widely used as antihypertensive drug (Figure 1). 4 Bisoprolol Fumarate HCTZ Figure 1: Structure of BF and HCTZ The HCTZ is a thiazide diuretic and is used for treatment of hypertension and oedema. 5 HCTZ is one of the oldest thiazides used as a diuretic and is often prescribed in combination with other drugs such as beta-blockers, ACE inhibitors, or angiotensin II receptor blockers. 6 The bisoprolol fumarate (BF)/ hydrochlorothiazide (HCTZ) tablets, 2.5 mg/6.25 mg, 5 mg/6.25 mg, and 10 mg/6.25 mg have two active pharmaceutical ingredients (APIs).

12 Literature studies show that various analytical methods were reported for the estimation of HCTZ in biological fluids. Several methods were reported for quantification of BF and HCTZ separately. Very few methods were reported for simultaneous estimation of BF and HCTZ using HPLC. There is no method reported for simultaneous estimation of BF and HCTZ using UPLC. There is one method available in 2011 USP for the simultaneous analysis of both APIs using HPLC; but the dissolution and Assay/content uniformity (CU) methods have different HPLC parameters. The retention times and run time have not been mentioned for dissolution tests. The 2011 USP method for dissolution requires a dual wavelength detector (Photo Diode Array, PDA) to quantitate BF and HCTZ, which is restricted to the use of a dual wavelength detector HPLC instrument. The 2011 USP illustrates an HPLC method that uses for the dissolution of BF /HCTZ in tablets L11 packing and aqueous 0.2% triethylamine solution and acetonitrile (4:1 v/v) as the mobile phase in the isocratic mode. The method for assay and CU tests requires the use of gradient mode and the total gradient run time is twelve minutes. The 2011 USP method for assay has a different sample preparation for BF and for HCTZ; therefore each solution needs to be injected separately in order to quantitate each active. The USP illustrates an HPLC method for the assay/cu test of BF/HCTZ tablets that uses L11 packing and aqueous dibutyl ammonium phosphate and acetonitrile as the mobile phase in the gradient mode. 2 Currently Sandoz has a method for quantitating BF and HCTZ in assay, CU, and dissolution test solutions. The disadvantage of the current Sandoz method is that a different set of HPLC parameters are required for each active, BF and HCTZ, in assay/cu, and dissolution tests. This method uses a Phenomenex Prodegy C 18, 3.9 mm x 2

13 300 mm, 10 μm analytical column and a mobile phase consistent of methanol, buffer solution (pentanesulfonic acid and sodium acetate), and acetic acid (470:530:0.5) in an isocratic mode for the quantitation of BF in assay, CU and dissolution test solutions. Under the above conditions, BF elutes at 12.5 minutes with a total run time of 20 minutes for each injection. Sandoz s current HPLC method for HCTZ quantitation in assay, CU, and dissolution tests uses Phenomenex Prodegy ODS(2), 4.6 mm x 100 mm, 5 μm analytical column and buffer solution (potassium phosphate) and acetonitrile (mobile phase A 90:10 and mobile phase B 10:90) as the mobile phase in the gradient mode. Using this column and mobile phases, HCTZ elutes at 4 minutes with a total gradient time of 20 minutes per injection. The assay/cu sample preparations are also different for BF and HCTZ; therefore, multiple instruments and multiple days are needed to analyze one tablet. Dissolution testing of BF and HCTZ also require different HPLC parameters for each BF and HCTZ because pull time for BF is 20 minutes and HCTZ is 30 minutes. During a literature search, one assay combined method 3 was found for BF and HCTZ. The method was developed for a different formulation and for different strengths of HCTZ. The dissolution method and CU test methods are not addressed in this article. This article describes an HPLC method for the assay of BF and HCTZ tablet using sperisorb, 4.6 mm x 250 mm, 5 μm, cyano analytical column and 0.1 M aqueous potassium dihydrogen phosphate buffer, acetonitrile, and tetrahydrofuran as the mobile phase in the isocratic mode. 3 Another combined method was found during literature search for the assay of BF and HCTZ. In this method 0.1 M aqueous potassium dihydrogen phosphate buffer and acetonitrile in the ratio of 70:30 v/v is used as a mobile phase. Inertsil ODS 3V (250 mm x 4.6 mm, 5 μm) analytical column was used as a 3

14 stationary phase. This article does not address dissolution or CU method either. 6 Both of these methods, Sandoz s current method, and the 2011 USP method use an HPLC instrument to quantitate BF and HCTZ. In order to make analysis user friendly, efficient, and cost effective, a new method needs to be developed to analyze both APIs (BF and HCTZ product range) in one HPLC and UPLC chromatographic run for dissolution, assay and CU. Multiple analysts can prepare assay, CU, and dissolution solutions; and all those solutions can be analyzed using one HPLC or UPLC instrument. In order to avoid the disadvantage of having two different methods for dissolution and assay/cu, a study was conducted. The first part of the present study was to develop and validate a simple, precise, specific, accurate, and robust HPLC method for the simultaneous determination of BF and HCTZ in final dosage form. The second part of the study was to transfer the HPLC method to UPLC and validate the UPLC method. The important reasons for developing a UPLC method are to make the laboratory more environmentally green, efficient, and cost effective than when using HPLC. UPLC typically saves about 80 to 95 percent of mobile phase in isocratic and gradient mode compared with HPLC. UPLC provides higher resolution, speed, and potentially higher sensitivity compared to HPLC. 7 For the past 30 plus years, HPLC has proven to be the predominant technology used in pharmaceutical laboratories. Scientists have been searching for a fast LC as a way to speed up analyses. In early 2004, the first commercially available UPLC was invented by the Milford, Massachusetts based company, Waters Corporation. The Waters UPLC is called the ACQUITY UPLC TM System. 8 The fundamental principles of this development are governed by the van Deemter equation (H = A + B/μ + Cμ) where: H is 4

15 the column s plate height, and μ is the mobile phase linear flow rate. A, B, and C are constants. The van Deemter equation describes the relationship between linear velocity (flow rate) and plate height (HETP or column efficiency). 9 Particle size is one of the variables that can be used to investigate chromatographic performance. 10 Chromatographic resolution can be improved by either increasing the column length or reducing the particle size. Reducing the particle size is far more effective than increasing the column length. 11 The efficiency of the earlier HPLCs increased by decreasing particle size in column packing from 10 μm in the 1970s to 3.5 μm in the 1990s. 12 The UPLC system is used with specially designed Acquity UPLC columns containing a particle size of only 1.7 μm. 8 As particle size gets smaller, the column back pressure gets higher because the pressure required to pump mobile phase through the column is inversely proportional to the square of the particle diameter. The back pressure required for use of these small particle columns becomes high and this presents a challenge to the pressure limitations of a conventional HPLC system. 9 UPLC is capable of having about psi back pressure compared to HPLC, which can have about 4000 psi. 7 Efficiency is three times greater with 1.7 μm particles compared to 5 μm particles and two times greater compared to 3.5 μm particles. The resolution is 70 percent higher with 1.7 μm particles than with 5 μm particles. The resolution is 40 percent higher with 1.7 μm particles than with 3.5 μm particles. High speed is obtained because a column length with 1.7 μm particles can be reduced by a factor of 3 compared to 5 μm particles for the same efficiency, and flow rate can be three times higher. Therefore, the separation in the UPLC can be nine times faster with equal resolution. The UPLC also 5

16 has a greater increase in sensitivity due to less band spreading during migration through a column with smaller particles. 12 Today s pharmaceutical industry is looking for new ways to cut cost and shorten time for the development of drugs while at the same time improving the quality of their products. Faster separation can lead to higher output and less time consumption while running multiple samples. The speed allows a greater number of analyses to be performed in a shorter amount of time, thereby increasing sample output and lab productivity. The transfer of the HPLC method to the UPLC method can be accomplished by simply applying a scaling factor to the mobile phase flow rate and the sample injection volume. The scaling factor was derived from the ratio of the column cross sectional area in order to retain the mobile phase linear velocity. 8 The use of the UPLC reduces the amount of waste, mobile phase consumption, cost of analysis, and provides many of the benefits of green chemistry. 13 UPLC also offers significant theoretical advantages in resolution, speed, sensitivity for analytical determination, and is capable of high speed acquisitions for sampling rate. 14 6

17 EXPERIMENTAL Materials and Reagents: BF Reference Standard, HCTZ, Reference Standard, 4-Amino (4-Amino-6- chloro-1,3-benzenedisulfonamide), Reference Standard, CTZ (Chlorothiazide) Reference Standard, and fumaric acid were supplied by the USP. Dimer ([6-chloro-N-[(6-chloro-7- sulfamoyl-2,3-dihydro-4h-1,2,4-benzothiadiazine-4-yl 1,1-dioxide)methyl]3,4-dihydro- 2H-1,2,4-benzothiazine-7-sulfonamide 1,1-dioxide]) was purchased from Gyma and manufacturer was Cambrex. 4-Amino is HCTZ s degraded impurity. CTZ and Dimer are HCTZ s process related impurities. Bisoprolol impurity A (1-(4-hydroxymethyl)- phenoxy-3-isopropylamino propan-2-ol) was purchased from Gyma and manufacturer was Corden. Bisoprolol impurity A is Bisoprolol s degraded impurity. Trifluoroacetic Acid (TFA) was spectrophotometric grade and it was purchased from Aldrich Chemical Company. Acetonitrile was ACS grade and was purchased from Fisher Brand. Concentrated hydrochloric acid (12N) was purchased from Fisher brand. USP purified water was used for making solutions. Equipment: Agilents 1100, Agilents 1260, and Waters 2695 equipped with gradient pump, auto sampler, temperature controlled compartment, dual wavelength detector, and Photo Diode Array detector (PDA) were used for all HPLC experiments. Waters Acquity equipped with gradient pump, auto sampler, temperature controlled compartment, dual wavelength detectors, and PDA was used for all UPLC experiments. Empower 1 and Empower 2 were used as the data acquisition program to collect chromatograms for each 7

18 injection and to measure the retention times and resolutions. Microsoft Excel, Empower1 and Empower2 were used for calculations. HPLC Instrumentations and Conditions: A qualified HPLC systems equipped with an electronic injector were used for analysis. The chromatographic separations were performed using Water Symmetry C 18, 5 µm, 4.6 mm x 150 mm column, eluted with mobile phase at the flow rate of 1.0 ml/min. Column temperature was at 35 C. Measurement was performed with injection volume of 20 µl for dissolution testing and 10 µl for Assay/CU testing. UV detection was at 224 nm wavelength. Mobile Phase A was prepared by pipetting 1.0 ml of Trifluoroacetic acid (TFA) into 1800 ml water and 200 ml acetonitrile. Mobile Phase B was prepared by pipetting 1.0 ml of TFA into 1800 ml acetonitrile and 200 ml water. Table 1 shows the retention time for each active. HPLC Gradient was followed according to table 2 with a run time of 11 minutes. Table 1: Retention Time using HPLC (Note: The related retention times for 4-Amino, CTZ, and dimer are based on the HCTZ peak, and Fumaric acid and Bisoprolol impurity A are based on the Bisoprolol peak) Active/Impurities Retention Time name (min) RRT Fumaric acid Bisoprolol imp A Amino CTZ HCTZ Bisoprolol Dimer

19 Table 2: HPLC Gradient Time (min) Mobile phase A% Mobile phase B% Curve UPLC Instrumentations and Conditions: Qualified UPLC systems equipped with an electronic injector were used for analysis. The chromatographic separations were performed using Water Acquity BEH C 18, 1.7 µm, 2.1 mm x 100 mm column, and eluted with mobile phase at the flow rate of 0.3 ml/min. Column temperature was at 35 C. Measurements were performed with injection volume of 10 µl for dissolution testing and 2 µl for Assay/CU testing. UV detection was set at 224 nm wavelength. Mobile Phase A was prepared by pipetting 1.0 ml of TFA into 1800 ml water and 200 ml acetonitrile. Mobile Phase B was prepared by pipetting 1.0 ml of TFA into 1800 ml acetonitrile and 200 ml water. Mobile phases were the same for HPLC and UPLC instruments. The same mobile phases were used for assay, CU, and dissolution testing for BF and HCTZ. Table 3 shows the retention time for each active. UPLC Gradient was followed according to table 4 with a run time of 7 minutes. 9

20 Table 3: Retention Times using UPLC (Note: The related retention times for 4-Amino, CTZ, and dimer are based on the HCTZ peak, and Fumaric acid and Bisoprolol impurity A are based on the Bisoprolol peak) Active/Impurities name Time Relative Retention Time (RRT) Fumaric acid Bisoprolol imp A Amino CTZ HCTZ Dimer Bisoprolol Table 4: UPLC Gradient Time (min) Mobile phase A% Mobile phase B% Curve Preparation of the Solution for Dissolution Testing: USP apparatus # 2, paddles were used for dissolution testing. Dissolution medium was 0.1N hydrochloric acid. Dissolution volume was 900 ml. Speed for the paddle was 75 rpm. Sampling time for BF was 20 minutes and for HCTZ was 30 minutes. The final solutions of the standards and samples contained about 2.8 µg/ml concentrations for 2.5 mg of BF label claim, 5.6 µg/ml concentrations for 5 mg of BF label claim, 11.2 µg/ml concentrations for 10 mg of BF label claim, and 6.9 µg/ml concentrations for 6.25 mg of HCTZ. Final sample solutions were filtered through a 0.45 µm PVDF Millex filter or 10

21 equivalent, discarding the first 3 ml of the filtrate. HPLC and UPLC chromatograms for dissolution sample are shown in figure 2 and figure bis AU HCTZ Minutes SampleName Bis HCTZ Bis MK P D-1; Vial 11; Injection 1; Result Id 10452; Date Acquired Tuesday, February 08, :58:59 PM EST Figure 2: Dissolution sample chromatogram using HPLC 0.80 HCTZ 0.60 AU bis Minutes SampleName Bis HCTZ bis MK P D-1; Vial 1:B,3; Injection 1; Result Id 10336; Date Acquired Tuesday, February 08, :04:03 PM EST Figure 3: Dissolution sample chromatogram using UPLC 11

22 Preparation of the Solution for Assay and CU Tests: Diluent for Assay and CU was prepared by pipetting 1.0 ml of TFA into 1600 ml water and 400 ml acetonitrile. For assay samples, 20 tablets were ground and the weight equivalent to 12.5 mg of HCTZ added to a 500 ml volumetric flask. For CU samples, 10 tablets were weighed and each tablet was transferred into a separate 250 ml volumetric flask. The flask was filled with diluent to half their volume, sonicated for 15 minutes with frequent swirling, and then shaken on a mechanical shaker for 15 minutes. Samples were diluted to volume with diluent. Final sample solutions for assay and CU were filtered through a 0.45 µm Whatman glass microfiber filter (GMF), discarding the first 5 ml of the filtrate. The final solution of the standards and samples contained about 10 µg/ml concentration for 2.5 mg of BF label claim, 20 µg/ml concentration for 5 mg of BF label claim, 40 µg/ml concentrations for 10 mg of BF label claim, and 25 µg/ml concentration for 6.25 mg of HCTZ. Resolution ID was prepared to contain about 10 µg/ml final concentrations for fumaric acid and bisoprolol impurity A, 6.25 µg/ml final concentrations for CTZ, 4-amino, and dimer, 1000 µg/ml final concentration for BF, and 625 µg/ml final concentration for HCTZ. HPLC and UPLC chromatograms for assay sample are shown in figure 4 and figure 5. HPLC and UPLC chromatograms for resolution ID having all impurities are shown in figure 6 and figure 7. 12

23 0.40 HCTZ 0.30 AU Bisoprolol Minutes SampleName BisHCTZ_IntPrecision_MK061822_Assay1; Vial 1:A,8; Injection 1; Result Id 18105; Date Acquired Tuesday, February 15, :28:15 PM EST Figure 4: Assay sample chromatogram using HPLC 0.60 HCTZ 0.40 AU 0.20 bisoprolol Minutes SampleName BisHCTZ_IntPrecision_MK061822_Assay1; Vial 1:A,8; Injection 1; Result Id 20475; Date Acquired Tuesday, February 15, :57:31 PM EST Figure 5: Assay sample chromatogram using UPLC 13

24 AU Bis A amino CTZ HCTZ bis Minutes Channel 2998; Processed Channel: PDA nm; Result Id: 29684; Processing Method: bis trc md nnt LC02 RC Dimer AU Bis A amino CTZ HCTZ bis Minutes Channel 2998; Processed Channel: PDA nm; Result Id: 29684; Processing Method: bis trc md nnt LC02 RC Dimer Figure 6: Resolution ID chromatogram using HPLC AU AU bis A amino CTZ hctz Minutes Channel PDA Spectrum; Processed Channel: PDA nm; Result Id: 27175; Processing Method: bis trc md nnt up LC04 id bis A amino CTZ hctz Minutes Channel PDA Spectrum; Processed Channel: PDA nm; Result Id: 27175; Processing Method: bis trc md nnt up LC04 id Dimer bis Dimer bis Figure 7: Resolution ID chromatogram using UPLC 14

25 RESULTS AND DISCUSSIONS Method Development: An isocratic method on HPLC and UPLC is generally simpler to follow compared to a gradient method. 15 The isocratic method using 0.1 M potassium phosphate buffer ph 4.5, acetonitrile and tetrahydrofuran (85:10:5 v/v/v) as a mobile phase did not give good separation between 4-amino, CTZ, and HCTZ with Waters Spherisorb cyano column (4.6 mm x 250 mm, 5 μm, CNRC). Using this mobile phase and column, HCTZ eluted at 4.8 minutes and BF eluted at 5.9 minutes. Using the same column and 0.1 M potassium phosphate buffer ph 4.5 and acetonitrile (85:15 v/v) as a mobile phase did not give good resolution between CTZ and HCTZ. Bisoprolol impurity A and 4-amino impurity coelute. Analysis time was increased because tetrahydrofuran was not part of the mobile phase. HCTZ eluted at 6.2 minutes and BF eluted at 18.7 minutes. Thus, adequate resolution was not achieved by the isocratic mode, and a gradient method was developed. There are many advantages of gradient methods. Gradient methods can improve separation and shorten the run time 16. Waters symmetry C 18 column was the perfect fit for the BF and HCTZ method because it gives good reproducibility and has ph range from Acetonitrile was used in the mobile phase because acetonitrile is miscible with water in all proportions. Starting with pure water as the mobile phase and adding acetonitrile to the water progressively makes the mobile phase dispersive in character and gradually elutes more dispersive substances. Acetonitrile does not associate strongly with water and thus, as opposed to methanol, acetonitrile-water mixtures remain binary in character. Methanol also causes a greater back pressure on the system due to the higher viscosity of methanol/buffer 15

26 compare to acetonitrile/buffer. TFA was used in a mobile phase to make a low ph buffer. There are other acids that could be used to make a low ph buffer, but TFA has some advantages as opposed to other acids. The main advantage is TFA helps to improve resolution and sharpens the peaks. TFA is also volatile and easily removed. TFA has low absorption within detection wavelengths; therefore TFA was used in a mobile phase to make ph 2.2 buffer. BF has a pk a value of 9.5, and HCTZ has pk a value of 7.9 and 9.2. For basic solutes, the retention normally decreases with decreasing ph of the mobile phase as long as the ph does not exceed the log of the ionization constant for a base in reverse phase HPLC. Generally, mobile phase and diluent are similar; they both have similar ph and organic level. ph of the mobile phase is low (ph 2.2) in order to keep a short analysis time, because BF and HCTZ are basic solutes. 18,19 Maximum wavelength for the analysis was determined by using a photodiode array (PDA) detector. A PDA detects the absorption in UV to VIS region. A UV-VIS detector has only one sampleside light-receiving section, but a PDA detector has multiple photodiode arrays to obtain information over a wide range of wavelengths at one time. The idea is that spectra are measured at intervals of one second or less during separation by HPLC with continuous eluant delivery. 20 The 224 nm was the maximum wavelength for BF absorbance. The 272 was the maximum wavelength absorbance for HCTZ. HCTZ had second highest maximum wavelength absorbance at 225 nm. HCTZ gave a higher response compared to BF; therefore, samples were analyzed at 224 nm wavelength. The new developed method for assay, CU, and dissolution using HPLC and UPLC met all acceptance criteria in the preliminary validation for filter study, extraction study, linearity, specificity, and recovery were performed. In the assay/cu method, there 16

27 was a higher concentration for HCTZ, and linearity for HCTZ did not meet acceptance criteria. The method was changed to the lower concentration for BF and HCTZ. Assay linearity had a range of 5 μg/ml to 80 μg/ml for BF and a range of 12.5 μg/ml to 50 μg/ml for HCTZ. Both BF and HCTZ linearity had a correlation coefficient > and Y intercept < 3%. The recovery for assay and dissolution was also performed. The dissolution recovery met the acceptance criteria, but the assay recovery did not. Diluent for the assay was changed from 80:20 (water:acetonitrile) diluent to a low ph by adding 1 ml of TFA into 2 L of 80:20 water: acetonitrile diluent. The diluent with low ph helped to elute a sharper peak shape for Bisoprolol impurity A, but did not help with recovery. Originally, the 0.45 μm PVDF Millex filter was used for the assay and CU analysis. Because the assay recovery was giving low results using 0.45 μm PVDF Millex filter, the filter was changed to the 0.45 μm Whatman GMF/ with GMF filter. The 0.45 μm Whatman GMF/ with GMF filter gave good recovery results for the assay. Assay recovery was performed with a range of 5 μg/ml to 80 μg/ml for BF and a range of 12.5 μg/ml to 50 μg/ml for HCTZ (range from 50 % of lowest label claim to 200 % of highest label claim concentration). Each individual assay recovery value was between % for BF and HCTZ. Dissolution recovery was performed with a range of 1.4 μg/ml to 22.2 μg/ml for BF and a range of 3.47 μg/ml to 13.9 μg/ml for HCTZ (range from 50 % of lowest label claim to 200 % of highest label claim concentration). Each individual dissolution recovery value was between % for BF and HCTZ. For assay/cu analysis using UPLC, a 2 µl injection volume was used. With an increased injection volume using UPLC, the HCTZ area got higher and failed to meet 17

28 acceptance criteria for linearity. For 2.5 mg of BF, assay sample concentration was 10 μg/ml. Dissolution sample concentration for 2.5 mg of BF was 2.8 μg/ml, which is about four times lower than the assay sample concentration. Therefore, the injection volume for dissolution was increased to 10 μl to bring the absorbance equivalent to assay concentration, which is about five times more injection volume than the assay sample injection volume. Linearity for dissolution met acceptance criteria for BF and HCTZ with 10 μl injection volume. The 2011 USP and current Sandoz method for BF/HCTZ tablets have the same dissolution instrument parameters. Both methods have 0.1 N hydrochloric acid as the dissolution medium, paddles, speed of 75 RPM and the temperature of the medium is set at the temperature of the body, 37 C. The sample pull time is 20 minutes for BF and 30 minutes for HCTZ. UPLC Method Transfer The transfer of the HPLC method to UPLC method can be accomplished by scaling factor to the mobile phase s flow rate and the sample injection volume. HPLC columns and UPLC columns are not perfectly comparable; therefore, a UPLC method developed using a scaling factor does not give the same chromatography as HPLC chromatography. 21 BF and HCTZ have five impurities; in order to separate all impurities and get better resolution, a 100 mm length column was a better choice. Water Acquity BEH C 18, 1.7 μm, 2.1 mm x 100 mm column is one of the most comparable columns to the Water Symmetry C 18, 5 μm, 4.6 mm x 150 mm. For injection volume scaling factor was follow as equation 1. 18

29 Target Volume on UPLC (1) = Original injection volume on HPLC x (Target Column Volume/ Original column Volume) = 10 μl x (3.14 x mm x 100 mm) / (3.14 x mm x 150 mm) = 1.52 μl Initially flow rate of the mobile phase was calculated based on the scaling factor and mathematical equation 2 was used. 22 Target Flow rate on UPLC (2) = Original flow rate on HPLC X d 2 of target/ d 2 of original = 1.0 ml/min x mm / mm = 0.21 ml/min All other parameters such as column temperature, UV detection, and mobile phase were the same as HPLC. For the sample injection volume, 1.5 μl did not give good response of BF. More than 2.0 μl injection volume was overloading the column with HCTZ and linearity did not meet acceptance criteria. Injection volume of 3.0 μl gave correlation factor and 3.8 percent y-intercept for HCTZ. The % y-intercept value should be no more than 3.0 percent of the target value. In order to make UPLC analysis faster, the flow rate was increased from 0.2 ml/min to 0.3 ml/min. Assay/CU Validation: The assay/cu chromatographic method and sample preparation method were validated by evaluating linearity, range, accuracy, precision, intermediate precision (ruggedness), specificity, extraction study, filter study, robustness of chromatographic 19

30 parameters, system suitability, solution stability, force degradation, and analytical method comparison according to the ICH guidelines Q2 (R1) and current Sandoz SOP. Linearity/Range: The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. Range is normally derived from linearity studies. Range is established by confirming that the analytical procedure provides an acceptable degree of linearity, accuracy and precision when applied to samples containing amounts of analyte within or at the extremes of the specified range of the analytical procedure. According to the ICH guideline, for the establishment of linearity, a minimum of five concentrations is recommended. 1 In this study, ten concentrations were used to established linearity. According to the ICH guidelines, minimum specified ranges for the assay of an active substance or a finished product is normally from 80 to 120 percent of the test concentration, and for content uniformity, covering a minimum of 70 to 130 percent of the test concentration 1. In this linearity study, the range for BF was a 2 to 400 percent, based on 2.5 mg of BF concentration. The range for HCTZ was 1 to 200 percent, based on 6.25 mg of HCTZ concentration. Standard stock solutions of the BF and HCTZ were diluted to prepare linearity of standard solution in the concentration range of μg ml -1 BF and μg ml -1 HCTZ. Each active was analyzed to plot the linearity curve. The slope, intercept, correlation coefficient, and residual sums of square were determined. Linearity acceptance criteria according to the current Sandoz SOP are the correlation coefficient for linearity cannot be less than and the %y-intercept value 20

31 cannot be more than 3.0% of the target value. Assay CU Linearity results are shown in table 5. Linearity plots are shown in Appendix A (1 4). Table 5: Calculated linearity results for BF and HCTZ using HPLC and UPLC. Excel data analysis regression was used for calculation. BF using HPLC BF using UPLC HCTZ using HPLC HCTZ using UPLC Linear Regression equation (y = ax + b) Correlation Coefficient Residual Sum of Square y= x y= x y= 66231x y= x y-intercept Value 0.69% 0.55% 0.26% 0.19% Recovery/Accuracy: The accuracy expresses the closeness of test results obtained by that procedure to the true value. Recovery or accuracy is sometimes termed trueness. 1 Recovery of the drug using this new method was determined by adding specific amount of APIs and placebo to make marketed sample. In the recovery study, the range for BF was 50 to 800 percent (concentration range of 5 80 μg ml -1 BF) based on 2.5 mg of BF concentration. The range for HCTZ was 50 to 200 percent (concentration range of μg ml -1 HCTZ) based on 6.25 mg of HCTZ concentration. The recovery was performed in triplicate for three concentrations. Two 100 percent API (with no placebo) samples were also prepared for potency determination. Based of the APIs potency, percent recovery was calculated using Excel. The recovery acceptance criteria according to the current Sandoz SOP are that each individual recovery value should be between

32 percent and the mean recovery value at each level should be between percent. Assay/CU Accuracy/Recovery results are shown in table 6. Table 6: Assay/CU Accuracy/Recovery results for BF and HCTZ using HPLC and UPLC BF % Level BF using HPLC BF using UPLC HCTZ % Level HCTZ using HPLC HCTZ using UPLC 50% % % % % % Average of 50% Average of 50% % % % % % % Average of 100% Average of 100% % % % % % % Average of 800% Average of 200% Precision: The precision expresses the closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. Precision was performed by preparing six assay preparations and ten CU preparations at 100 percent concentration for each APIs according to the new developed method. Intermediate precision was carried out by analyzing the sample by a different analyst, using different instruments, and on different dates. The assay precision acceptance criteria according to the current Sandoz SOP are that the percent RSD for each of the six assay sample preparation should be no more than 2.0 percent. The assay 22

33 intermediate precision acceptance criteria are that the percent RSD for each of the six assay sample preparations should be no more than 2.0 percent and the difference in absolute mean/average difference between analyst one and analyst two should be no more than 2.0 percent. The CU precision acceptance criteria according to the current Sandoz SOP are that the acceptance value (AV) for each of the ten CU sample preparations should be no more than 15.0 percent. The CU intermediate precision acceptance criteria are that the acceptance value for each of the ten assay sample preparations should be no more than 15.0 percent and the difference in mean between analyst one and analyst two should be no more than 5.0 percent. The assay precision and intermediate precision results for BF are shown in table 7. The assay precision and intermediate precision results for HCTZ are shown in table 8. The CU precision and intermediate precision results for BF are shown in table 9. CU precision and intermediate precision results for HCTZ are shown in table

34 Table 7: Assay precision and intermediate precision results for BF using HPLC and UPLC # of Assay preparations Analyst 1 Analyst 2 Analyst 1 Analyst 2 BF using HPLC BF using HPLC BF using UPLC BF using UPLC % Average % RSD Absolute % Difference in Averages N/A 0.9 N/A 1.8 Table 8: Assay precision and intermediate precision results for HCTZ using HPLC and UPLC # of Assay preparations Analyst 1 Analyst 2 Analyst 1 Analyst 2 HCTZ using HPLC HCTZ using HPLC HCTZ using UPLC HCTZ using UPLC % Average % RSD Absolute % Difference in Averages N/A 0.2 N/A

35 Table 9: CU precision and intermediate precision results for BF using HPLC and UPLC # of CU preparations Analyst 1 Analyst 2 Analyst 1 Analyst 2 BF using HPLC BF using HPLC BF using UPLC BF using UPLC % Average AV Value % RSD Absolute % Difference in Averages N/A 1.2 N/A

36 # of CU preparations Table 10: CU precision and intermediate precision results for HCTZ using HPLC and UPLC Analyst 1 HCTZ using HPLC Analyst 2 HCTZ using HPLC Analyst 1 HCTZ using UPLC Analyst 2 HCTZ using UPLC % Average AV Value % RSD Absolute % Difference in Averages N/A 1.5 N/A 1.7 Specificity: The ICH documents define specificity as the ability to assess unequivocally the analyte in the presence of components that may be expected to be present, such as impurities, degradation products, and matrix components. Specificity is also known as selectivity. In this study, placebo and diluent was analyzed under HPLC and UPLC conditions. The assay specificity chromatogram for the blank and placebo are shown in Appendix B (1 4). The placebo peak (orange dye, which is used only in 5 mg BF/ 6.25 mg HCTZ tablets for coating) eluted at about 4.0 minutes using HPLC, and using UPLC 26

37 the placebo peak eluted at 1.4 minutes. The HCTZ peak eluted at about 4.3 minutes using HPLC. The resolution between the placebo peak and HCTZ peak was 2.5 using HPLC. The peak purity for BF and HCTZ met acceptance criteria using HPLC and UPLC, which proved there was no other component that eluted at the same time as the HCTZ peak and BF peak. The acceptance criteria for peak purity are that purity angle for the peak should be less than the threshold angle. The peak purity is based on the difference in the purity angle and the threshold angle expressed in degree. Both the purity angle and threshold angle were measured by the use of complex algorithms, based on the conversion of the peak spectra to vectors in a multi-dimensional space. For the determination of purity angle, the peak apex is used as the reference spectrum; all the other spectral data contained within that peak are compared to the spectra of the apex. The noise (threshold angle) calculation is based on a segment of the baseline within the chromatographic run 23. Robustness of Extraction Procedure: The robustness of extraction procedure was performed to determine whether the sample preparation procedure has sufficient time for full extraction of the active ingredient from the sample matrix. In this method, sonication and shaking were specified for 15 minutes each. The study was conducted with the sonication and shaking of the assay sample for 10, 15, and 30 minutes and at each time point samples were analyzed using HPLC and UPLC. The acceptance criteria for the extraction procedure are that the percent difference (absolute) should be no more than 2.0 percent from the method suggested time point for the extraction to be considered robust. The robustness of extraction procedure results are shown in Table

38 Table 11: Robustness of extraction procedure results for BF and HCTZ using HPLC and UPLC BF using BF Using HCTZ using HCTZ using Extraction Time Points HPLC UPLC HPLC UPLC 10 min Sonication min Sonication min Sonication Sonication maximum absolute % difference in average min Shaking min Shaking min Shaking Shaking maximum absolute % difference in average Filter Study: For the filter study, one assay sample and one assay standard were prepared as per the new developed method. The standard and sample were filtered through a 0.45 μm Whatman GMF/with GMF filter and a 0.45 μm Millex-HV PDVF filter. The first 3 ml (3 ml total), the next 2 ml (5 ml total), the next 2 ml (7 ml total), and the last 3 ml (10 ml total) were collected from both filters. A portion of the sample was also centrifuged at 2000 RPM for 10 minutes. The results of the filtered samples were compared with the results of the centrifuged sample. The filter study results are shown in table 12. The filter is considered acceptable when the percent difference (absolute) varies no more than 2.0 percent between collections for the standard. The filtered sample is considered acceptable if the percent difference (absolute) is no more than 2.0 percent different from the value obtained from the centrifuged sample. Percent difference results 28

39 are shown in table 13. In the percent difference results table for sample, only 7 ml collected results are shown because in the developed method the first 5 ml of filtrate are discarded. A 0.45 μm Millex-HV PVDF filter gave low results compared to the centrifuged sample, because BF binds with a filter s particles. HCTZ and BF results using 0.45 μm Whatman GMF/with GMF filter are comparable to the centrifuged results; therefore, the new developed method indicates to use 0.45 μm Whatman GMF/with GMF filter for assay/cu. Table 12: Filter study results for BF and HCTZ using HPLC and UPLC Filter Sample BF using BF Using HCTZ using HCTZ using HPLC UPLC HPLC UPLC Centrifuge ml sample Whatman 5 ml sample ml sample ml sample ml sample Millex 5 ml sample ml sample ml sample ml standard Whatman 5 ml standard ml standard ml standard ml standard Millex 5 ml standard ml standard ml standard

40 Table 13: Filter study s percent difference results for BF and HCTZ using HPLC and UPLC Filter Sample Absolute % Difference BF using HPLC Absolute % Difference BF Using UPLC Absolute % Difference HCTZ using HPLC Absolute % Difference HCTZ using UPLC Whatman 7 ml sample Millex 7 ml sample Whatman Standard Millex Standard Robustness of Chromatographic Parameter: The robustness is a measure the capacity to remain unaffected by small deliberate variations in procedural parameters listed in the procedure documentation and provides an indication of its suitability during normal usage. 1 According to the current Sandoz SOP, robustness is considered established, once the system suitability meets the acceptance criteria. If the system suitability criteria are not met under any of the altered conditions the range may be narrowed accordingly until the robustness is established. One assay sample was analyzed using the altered chromatographic conditions as per table 14 and 15. Table 14: Robustness Parameter for HPLC Parameter Column Equivalency Flow Rate Variation Waters Sunfire C mm x 150 mm 5 µm column ± 0.2 ml/min %ACN in Mobile Phase A ± 3 % Temperature ± 5 C Wavelength ± 4 nm 30

41 Table 15: Robustness Parameter for UPLC Parameter Variation Column Equivalency Waters Aquity BEH HSS 2.1 mm x 50 mm 1.8 µm column Flow Rate ± 0.06 ml/min %ACN in Mobile Phase A ± 3 % Temperature ± 5 C Wavelength ± 4 nm For each condition, the system suitability was evaluated and peak purity was measured for each API. The robustness system suitability results for BF and HCTZ using HPLC are shown in table 16, and the robustness system suitability results for BF and HCTZ using UPLC are shown in table