Chapter V. Development and validation of an LC-MS/MS method for the determination of Lansoprazole in human plasma

Transcription

1 Chapter V Development and validation of an LC-MS/MS method for the determination of Lansoprazole in human plasma

2 5.1. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE DETERMINATON OF LANSOPRAZOLE IN HUMAN PLASMA INTRODUCTION Drug profile: Lansoprazole, a benzimidazole derivative, is a proton pump inhibitor that acts on the membrane H+/K+-ATP (adenosine triphosphatase) in gastric parietal cells 133. It is effective in the treatment of various peptic diseases, including gastric and duodenal ulcer, reflux esophagitis, Zollinger Ellison syndrome 134, and other hyperacidic-related conditions. Lansoprazole is metabolized in the liver and the main metabolites are 5- hydroxy lansoprazole and lansoprazole sulphone. Formation of the 5-hydroxy metabolite is mainly by cytochrome P4502C19 (CYP2C19), whereas CYP3A4 is involved in the formation of the sulphone 135, 136. It is clinically important to measure CYP2C19 activity using the hydroxylation and sulfoxidation indexes of lansoprazole, which reflects phenotype, and genotype of CYP2C Lansoprazole is active against Helicobacter pylori. The plasma elimination half-life of lansoprazole does not reflect its duration of suppression of gastric acid secretion. Thus, the plasma elimination half-life is less than two hours, while the acid inhibitory effect lasts more than 24 hours. Fig.1 Chemical structure of Lansoprazole 163

3 Properties: Chemical name : Chemical formula : C 16 H 14 F 3 N 3 O 2 S Molecular weight : CAS Registry Number : pka : 8.73 Solubility : 2-[[[3-methyl-4-(2,2,2-trifluoro-ethoxy)-2- pyridinyl]methyl]sulfinyl]-1h- benzimid-azole Lansoprazole is freely soluble in dimethyl- formamide; soluble in methanol. Physical state : White to brownish-white odorless crystalline powder Therapeutic Category : Anti ulcer agents, Proton-pump inhibitor(ppi) Trade names : Prevacid,Lanzol; Lanzopral; Lanzor; Lasoprol Bioavailability : 80% or more T ½ : hours Plasma protein binding : 97% Metabolism : Hepatic(CYP3A4 & CYP2C19 mediated) Elimination : Renal and Fecal Antisecretory activity: After oral administration, lansoprazole was shown to significantly decrease the basal acid output and significantly increase the mean gastric ph and percent of time the gastric ph was >3 and >4. Lansoprazole also significantly reduced mealstimulated gastric acid output and secretion volume, as well as pentagastrinstimulated acid output. In patients with hypersecretion of acid, lansoprazole significantly reduced basal and pentagastrin- stimulated gastric acid secretion. Lansoprazole inhibited the normal increases in secretion volume, acidity and acid output induced by insulin. 164

4 Drug interactions: The absorption of certain drugs may be affected by stomach acidity, and, as a result, lansoprazole and other PPIs that reduce stomach acid also reduce the absorption and concentration in blood of ketoconazole and increase the absorption and concentration in blood of digoxin. This may lead to reduced effectiveness of ketoconazole or increased digoxin toxicity, respectively. Side effects: Lansoprazole like other PPIs were well-tolerated. The most common side effects are diarrhea, nausea, vomiting, constipation, rash and headaches. Dizziness, nervousness, abnormal heartbeat, muscle pain, weakness, leg cramps and water retention rarely occur. High doses and long-term use (1 year or longer) may increase the risk of osteoporosis-related fractures of the hip, wrist, or spine. Therefore, it is important to use the lowest doses and shortest duration of treatment necessary for the condition being treated. 165

5 Past work on the chromatographic method for lansoprazole: 1. Avgerinos Aetal 138., have reported a simple and rapid (extraction less) highperformance liquid chromatographic method with UV detection at 230 nm for the determination of lansoprazole in biological fluids and pharmaceutical dosage. Niflumic acid was added as internal standard. The separation was performed at ambient temperature on a C18 Spherisorb column with acetonitrile M sodium acetate (40:60, v/v, ph 7) as mobile phase. The retention time was 5.2 min for niflumic acid and 6.7 min for lansoprazole. The detection limit was 20 ng/ml using a 100 µl loop. The method was successfully applied to a pharmacokinetic study of lansoprazole in humans. 2. Isamu Aokietal 139., have reported a simple and sensitive high-performance liquid chromatographic method with ultraviolet detection for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at μg/ml or to urine at 1 20 μg/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human. 3. A. P. Sherjeetal 140., have reported two simple, accurate and precise spectrophotometric methods for simultaneous determination of lansoprazole and domperidone in pharmaceutical dosage form. Method A involves formation of Q- absorbance equation at nm (isoabsorptive point) and at nm while method B is two wavelength method where nm, nm were selected as λ 1 and λ 2 for determination of lansoprazole and nm, nm were selected as λ 1 and λ 2 for determination of domperidone. Both the methods were validated statistically and recovery studies were carried out. The Beer's law limits for each drug individually and in mixture was within the concentration range of 5-50 μg/ml. Linearity of 166

6 lansoprazole and domperidone were in the range of μg/ml and 8-12 μg/ml, respectively. The proposed methods have been applied successfully to the analysis of the cited drugs either in pure form or in pharmaceutical formulations with good accuracy and precision. The method herein described can be employed for quality control and routine analysis of drugs in pharmaceutical formulations. 4. Julie De Smetet al 141., have reported a hydrophilic interaction LC method with MS/MS and validated for the simultaneous quantification of omeprazole and lansoprazole in human plasma. Chromatographic separation was achieved on a Betasil silica column using a high organic mobile phase (eluent A: ACN/formic acid 997.5:2.5 v/v; eluent B: water/formic acid 997.5:2.5 v/v) and gradient elution. The mass spectrometer was operated in the Multiple Reaction Monitoring mode. Prior to chromatography, liquid liquid extraction with ethyl acetate was used and the organic layer was diluted with ACN, allowing direct injection on column. The method showed acceptable linearity, high precision (RSD%<10.5%), accuracy ( %) and selectivity in the two concentration ranges studied: and ng/mL. The LOQ was established at 1.5 and 5ng/mL for the two concentration ranges. Lack of variability in matrix effects was demonstrated and mean extraction recovery for omeprazole and lansoprazole was determined in the low ( %) and high ( %) concentration range, respectively. Additionally, plasma samples were found to be stable after three freeze thaw cycles and for at least 15h after extraction. This assay was successfully applied to a pharmacokinetic omeprazole study in children with cerebral palsy and mental retardation. 5. Takanori Hishinuma et al 142., have reported a rapid, simple and highly sensitive method for the quantitative determination of lansoprazole and rabeprazole concentrations in 20 μl of human serum using high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analytes, along with an internal standard (lansoprazole deuterium derivatives), were separated using a mobile phase of acetonitrile/1 mm ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 0.25 ng/ml. A good linear response was observed for each 167

7 analyte (from 0.25 ngto 2.5 μg/ml). This method was useful for therapeutic drug monitoring and pharmacokinetic studies. 6. PrasannaReddy.Battuet al 143., have reported a simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method and validated for the analysis of Lansoprazole. Chromatographic separation achieved isocratically on a C18 column (Inertsil C18, 5, 150 mm x 4.6 mm) utilizing a mobile phase of acetonitrile/phosphate buffer (70:30, v/v, ph 7.0) at a flow rate of 0.8 ml/min with UV detection at 260 nm. The retention time of lansoprazole was 2.53 min. The method is accurate ( %), precise (intra-day variation % and inter-day variation %) and linear within range µg/ml (R 2 =0.999) concentration and was successfully used in monitoring left over drug. The detection limit of lansoprazole at a signal-to-noise ratio of 3 was 1.80ng/ml in human plasma while quantification limit in human serum was 5.60 ng/ml. The proposed method is applicable to stability studies and routine analysis of lansoprazole in pharmaceutical formulations as well as in human plasma samples. 7. Prasanna Reddy.B, et al 144., have reported a simple, sensitive and precise high performance liquid chromatographic method for the analysis of Pantoprazole sodium and lansoprazole, validated and used for the determination of compounds in commercial pharmaceutical products. The compounds were well separated an isocratically on a C18 column (Inertsil C18, 5µ, 150 mm x 4.6 mm) utilizing a mobile phase consisting of acetonitrile: phosphate buffer (60:40, v/v, ph 7.0) at a flow rate of 1.0 ml/min with UV detection at 230 nm. The retention time of Pantoprazole sodium and lansoprazole was found to be min and The procedure was validated for linearity (Correlation coefficient = 0.999). The study showed that reversed-phase liquid chromatography is sensitive and selective for the determination of Pantoprazole sodium and lansoprazole using single mobile phase. 8. Bhavna Patel et al 145.have reported a RP-HPLC method for simultaneous estimation of lansoprazole and domperidone in combined capsule dosage form. The mobile phase used was a combination of Acetonitrile: Methanol (81:19). The detection of the combined dosage form was carried out at 280 nm and a flow rate 168

8 employed was 1 ml/min. The retention time for lansoprazole and domperidone was found to be 2.8 and 1.57 min respectively. Linearity was obtained in the concentration range of 8-24µg/ml of lansoprazole and 8-24 µg/ml of domperidone with a correlation coefficient of and Detector consists of photodiode array detector; the reversed phase column used was RP-C18 (2.27µm size, 250 mm 4.6 mm i.d.)at ambient temperature. The developed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Thus the proposed method was successfully applied for simultaneous determination of domperidone and lansoprazole in routine analysis. 9. T. K. Ravi et al 146., have reported a simple, fast, precise and accurate high performance thin layer chromatographic method for the simultaneous estimation of lansoprazole and domperidone in tablet formulations. This method allows the determination of ng/spot of lansoprazole and ng/spot of domperidone. The mobile phase composition was n-butanol: glacial acetic acid: water (9.3:0.25:0.5, v/v/v). Densitometric analysis of lansoprazole and domperidone was carried out in the absorbance mode at 288 nm. The limit of detection for lansoprazole and domperidone were found to be 10 and 30 ng/spot, respectively. The limit of quantification for lansoprazole and domperidone were found to be 40 and 65 ng/spot, respectively. The amounts of drug present in the tablet and recovery studies were also carried out. The method was validated for precision, accuracy and reproducibility. 169

9 EXPERIMENTAL & ANALYTICAL METHODOLOGY i) Instrumentation The author had attempted to develop a liquid chromatographic method for the determination of Lansoprazole using an isocratic Shimadzu HPLC equipment comprising of two LC10AT VP pumps, VP CTO-10AS VP column oven, a Hypurity advance C 18 column (4.6 ID X 50 mm, 5µ) and anapi 4000 (MDS Sciex) mass detector was used for chromatographic separation. Data acquisition was done by using Analyst software. The details of the instruments employed in the study are as follows. HPLC System Mass Spectrometer Deep Freezer Refrigerated centrifuge Microbalance Vibramax Vacuum pump Refrigerator PH meter Micropipettes, Multipette and Micro tips Vortexer Poly propylene tubes Water Purification System Ultra sonicator Nitrogen Evaporator Shimadzu API 4000, MDS Sciex Sanyo (-86 C) VIP Series Heraeusmegafuge Sartorius Heidolph Millipore Samsung Orion Brand and Eppendorf Spinix Torson s Elix 10 / Milli-Q gradient Power Sonic510, (Hwashin Technology) ZymarkTurbovap LV station, Caliper ii) Drug and Internal standard The reference samples of lansoprazole and pantoprazole were gifted by Neucon pharma Pvt Ltd., Goa. iii) Chemicals and solvents Lansoprazole reference standard Pantoprazole reference standard Methanol (HPLC grade) Milli-Q water Ammonium acetate (AR grade) 170

10 Sodium hydrogen carbonate (AR grade) Methyl tert-butyl ether (TMBE) (HPLC grade) Human plasma 0.45µ Membrane filter iv) Preparation of solutions a) Lansoprazole Stock Solution About 5 mg of Lansoprazole, working standard was weighed accurately, and transferred to a 5 ml clean glass volumetric flask, dissolved in HPLC grade methanol and made up the volume with the same to produce a solution of 1 mg/ml of Lansoprazole. The stock solutions were diluted to suitable concentrations using diluent for spiking in to plasma to obtain calibration curve (CC) standards, quality control (QC) samples and DIQC samples. All other final dilutions (system suitability dilutions, aqueous mixture, recovery etc.) of Lansoprazole were prepared in mobile phase. b) Pantoprazole Stock Solution (Internal Standard) About 10 mg of Pantoprazole Sodium Reference standard was weighed accurately and transferred to a 10 ml volumetric flask, dissolved in HPLC grade methanol and made up the volume with the same to produce a solution of 1 mg/ml of Pantoprazole. The stock solution was diluted to suitable concentration using diluent for internal standard dilution and mobile phase for system suitability dilution. c) Biological Matrix Six K 2 EDTA human plasma lots were screened for selectivity test. All six human plasma lots were found free of any significant interference for Lansoprazole and internal standard. Selectivity, Matrix Effect and sensitivity tests were performed. After bulk spiking, aliquots (sample contained 0.5M Sodium Hydrogen Carbonate: plasma 1:10) of 600 µl for CCs and 600 µl for QCs of spiked plasma samples were pipetted out in prelabelled polypropylene RIA vials and then all the bulk spiked samples were transferred to deep freezer maintained at -70 C ± 10 C, except twelve sets of LQC and HQC, which were transferred to deep freezer maintained at -20 C ± 5 C for generation of stability data at -20 C. 171

11 d) Calibration Curve Standards and Quality Control Samples Calibration curve standards consisting of a set of nine non-zero concentrations ranging from ng/ml to ng/mL for Lansoprazole were prepared. Quality control samples consisted of Lansoprazole concentrations of ng/ml (LLOQ QC), ng/ml (LQC), ng/ml (MQC1), ng/ml (MQC2) and ng/ml (HQC) were prepared. These samples were stored below -70 C until use. Twelve sets of LQC and HQC were transferred to the -20 C deep freezer to check stability at -20 C. Standard Concentration Lansoprazole (ng/ml) Standard I 2-3 times of C max Standard H 80% of I Standard G 60% of I Standard F 40 % of I Standard E 20% of I Standard D 10% of I Standard C 5% of I Standard B 40% of C conc Standard A 50% of B conc LLOQ QC Conc equal to A LQC times of LLOQ MQC 1 50% of I MQC 2 50% of I HQC 75-90% of I e) 5mM Ammonium Acetate Buffer About mg of ammonium Acetate was transferred to a 1000 ml reagent bottle and dissolved in 1000 ml of Milli Q water. The buffer was mixed well and sonicated in an ultrasonicator for 3 to 5 minutes. The amount weighed depends on total volume of Milli Q water to be used for preparation of 5mM ammonium Acetate buffer. 172

12 Buffer was prepared as and when required and used within a period of 4 days of preparation. f) Mobile Phase 800 ml of HPLC Methanol was transferred to a 1000 ml reagent bottle and 200 ml of 5mM Ammonium Acetate Buffer was added it. It was mixed well, sonicated in an ultrasonic bath for 3 to 5 minutes. The mobile phase was prepared as and when required and used within a period of 7 days of preparation. g) Diluent A mixture of HPLC grade Methanol and Milli Q water was prepared in the volume ratio of 60:40 (v/v) as diluent. It was then sonicated in an ultrasonicator for 3 to 5 minutes. The diluent was prepared as and when required and used within a period of 7 days of preparation. h) 0.5M Sodium Hydrogen Carbonate About 2g of Sodium Hydrogen Carbonate was transferred to a 50 ml reagent bottle and dissolved in 50 ml of Milli Q water. The buffer was mixed well and sonicated in an ultrasonicator for 3 to 5 minutes. The amount weighed depends on total volume of Milli Q water to be used for preparation of 0.5M sodium hydrogen carbonate. Buffer was prepared as and when required and used within a period of 4 days of preparation. i) Rinsing Solution Diluent was used as rinsing solution j) System Suitability Solution A mixture of drug and internal standard was prepared for system suitability test. The concentration of drug corresponds to mean concentration of ng/mL and that of internal standard corresponds to working concentration used for calibration range ( ng/ml). The same solution was injected as an aqueous mixture. Area Ratio was considered for System Suitability. 173

13 ANALYTICAL METHOD DEVELOPMENT Optimization of the chromatographic conditions For developing the method for the assay of lansoprazole, a systematic study of the effect of various factors was undertaken by varying one parameter at a time and keeping all the other conditions constant. The following studies were conducted for this purpose. A hypurity advance C 18 column was chosen as the stationary phase for this study. The mobile phase and the flow rate In order to get sharp peaks and base line separation of the components, the author has carried out a number of experiments by varying the commonly used solvents, their compositions and flow rate. To effect ideal separation of the drug under isocratic conditions, mixtures of commonly used solvents like water, methanol and acetonitrile with or without buffers in different combinations were tested as mobile phases on a C 18 stationary phase. A binary mixture of methanol and 5 mm Ammonium acetate buffer in a ratio of 80:20 v/v was proved to be the most suitable of all the combinations since the chromatographic peaks obtained were well defined and resolved and free from tailing. A mobile phase flow rate of 0.7 ml/min was found to be suitable. The drug molecule was tuned on the mass spectrometer for the detection of the parent ion and the daughter ion (precursor ions) by injecting 0.5 μg/ml concentration. The tuning was carried out in both positive and negative modes of ionization, but better sensitivity with more reproducibility was found to be observed in the positive polarity mode. All the optimized potential parameters in the positive polarity mode have been given in the following table. The mass spectrum of the drug molecule is given (Fig.2). 174

14 Fig. 2 Mass Spectra of lansoprazole for parent & product masses 175

15 RETENTION TIMES: Lansoprazole Pantoprazole : 1.15 ± 0.3 min : 1.20 ± 0.3 min DETECTION: Drug name Parent mass (amu) Product mass (amu) Lansoprazole Pantoprazole Source/ Gas Parameters (positive mode): API 4000 Parameter Value Lansoprazole Pantoprazole NEB(psi) CUR(psi) CAD(psi) IS(v) TEM ( C) DP(v) CE (v) CXP(v) FP(v) EP(v)

16 Retention time of Lansoprazole A model chromatogram showing the separation of lansoprazole is presented in Fig. 3 under the above optimized conditions retention times of 1.15 and 1.20 min were obtained for lansoprazole and pantoprazole respectively. Data acquisition and processing All the integrations were performed by Applied Bio systems Analyst software version The slopes, intercepts, and correlation coefficients were determined by least squares linear regression analysis using the ratios of drug/internal standard peak areas of Calibration curve standards. All the QC samples were also calculated by Applied Biosystems Analyst Software version

17 Optimized Chromatographic Conditions Parameter Column Mobile phase Buffer Isocratic/gradient mode Flow rate Run time Column oven temperature Auto sampler temperature Volume of injection Rinsing volume Value Hypurity advance C 18 (4.6 X 50 mm, 5µ) Methanol: 5mM ammonium acetate buffer (80: 20 v/v) 5mM ammonium acetate buffer Isocratic 0.70 ml/min 2.5 min 40 ± 2 0 C 15 0 C 20 μl 500 μl Extraction process of plasma samples and their drying Four hundred micro liters of spiked plasma calibration curve standards and the quality control samples were transferred to a set of pre-labeled poly propylene tubes containing each 25 µl of pantoprazole dilution (internal standard; μg/ml). The tubes were added with 5mL of TMBE solution and vortexed for ten seconds. To each of the tubes 2.5 ml of extraction solvent was added. The tubes were further vortexed for 20 min at 200 rpm on a vibramax unit and then were centrifuged at 4000 rpm for 10 min in a refrigerated centrifuge at 4 0 C. From the centrifuged tubes approximately 4.0 ml of the supernatant layer was transferred to each of a new set of pre-labeled poly propylene tubes. The contents of the tubes were evaporated in a stream of nitrogen at 40 0 C for 10 min and the residues of the dried tubes were reconstituted with 1000μL of the mobile phase. The contents of the tubes were vortexed and transferred into auto-sampler vials and then analyzed by LC-MS/MS. An aliquot of 10 μl of the sample was drawn each time from the vials in the auto sampler. 178

18 REPRESENTATIVE CHROMATOGRAMS Fig. 3A Representative Chromatogram of an Aqueous Standard and Internal Standard Mixture 179

19 Fig.4A Representative Chromatogram of a Blank Plasma Sample 180

20 Fig. 5A Representative Chromatogram of Blank Plasma with Internal Standard Sample 181

21 Fig. 6A Representative Chromatogram of a LLOQ QC Sample 182

22 Fig.7 A Representative Chromatogram of a HQC Sample 183

23 Fig. 8A Representative Chromatogram of extracted Blank Plasma Sample 184

24 ANALYTICAL METHOD VALIDATION i) Selectivity The selectivity of the present method was evaluated by checking the blank EDTA (Ethylene di-amine tetra acetic acid) plasma (without spiking with Lansoprazole) obtained. Six different lots of blank plasma were screened and all of them were found to have no significant endogenous interferences at the retention times of the analyte and the internal standard. The representative chromatogram of the extracted blank plasma sample was given in Fig.8. The same human EDTA plasma lots free of interfering substances were used to prepare the calibration curve standards and the quality control samples for the validation study. 185

25 ii) Sensitivity The lowest limit of reliable quantification for Lansoprazole was set at the concentration of the LLOQ, ng/ml. The precision and accuracy for Lansoprazole at this concentration was found to be 2.45% and % respectively. The results are within the acceptance limits and given in tables 1 & 2. Acceptance criteria: Accuracy must be ±20% of nominal value & its precision must be 20% 67% of LLOQ samples injected should be within ±20% of respective nominal value. Table -1 Concentration Response Linearity Data for Matrix effect and Sensitivity of Lansoprazole Concentration (ng/ml) STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I Slope Intercept R R 2 ME CC# % Nominal

26 Table 2 Within Batch Precision and Accuracy for Sensitivity of Lansoprazole Conc. (ng/ml) S.No LLOQ AVERAGE SD %CV 2.45 % NOMINAL iii) Matrix effect Matrix effect for Lansoprazole was evaluated by analyzing all the six batches of plasma at low (LQC) and high (HQC) concentrations. No significant matrix effect was observed in all the six batches (batch no.p , P , P ,P ,P ,P ) of plasma for Lansoprazole at low (LQC) and high (HQC) concentrations. The precision and accuracy for Lansoprazole at LQC concentration was found to be 1.95% and % respectively and at HQC concentration was found to be 0.73% and 98.45% respectively. The results are within the acceptable limits and given in tables 1 & 3. Acceptance criteria: The matrix effect is nullified if the mean % nominal is within 15% & CV% 15% at low & high QC. 187

27 Table-3 Matrix Effect of Lansoprazole Plasma (Batch No.) ME QC# Concentration (ng/ml) Concentration (ng/ml) LQC Mean HQC ME QC# Mean P ,2, ,2, P ,2, ,2, P ,2, ,2, P ,2, ,2, P ,2, ,2, P ,2, ,2, Mean S.D C.V.(%) % Nominal N

28 iv) Linearity The linearity of the method was determined by a weighted (1/X 2 where X is concentration) least square regression analysis of the standard plots associated with the eight point standard curve for Lansoprazole. The calibration line was linear in the range of to ng/mL of the drug as shown in Fig.7. A straight-line fit made through the data points by least square regression analysis showed a constant proportionality with minimal data scattering. The correlation coefficient (r) was greater than 0.99 and ranged from to for Lansoprazole. Acceptance criteria: Coefficient of correlation(r 2 ) should be 0.98 Deviation of LLOQ from nominal value can be ±20% Deviation of standards other than LLOQ from nominal value can be ±15% No two concecutive CCs must fail to meet the above criteria. 75% or atleast 6 non zero CCs including LLOQ & highest concentration must meet above criteria. 189

29 Table 4 Concentration-response Linearity Data of Lansoprazole Nominal Concentration (ng/ml) STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I Slope Intercept R R 2 CC# Mean S.D C.V.% % Nominal N

30 Fig. 9A Representative Calibration Curve for Regression Analysis 191

31 v) Precision and Accuracy The precision of the assay was measured by the percent coefficient of variation for QC samples of Lansoprazole. The accuracy of the assay was measured by computing the ratio of the calculated mean values of the QC samples to their respective nominal values, expressed as percentage nominal. Within-batch Precision for Lansoprazole (% CV) Within-batch precision for LLOQ QC ranged from 1.29 to 3.48% Within-batch precision for LQC, MQC1, MQC2 and HQC ranged from 0.68 to 2.28% Intra-day Precision for Lansoprazole (% CV) Intra-day precision for LLOQ QC was 2.50% Intra-day precision for LQC, MQC1, MQC2 and HQC ranged from 2.01 to 2.46% Between-batch Precision for Lansoprazole (% CV) Between-batch precision for LLOQ QC was 2.56% Between-batch precision for LQC, MQC1, MQC2 and HQC ranged from 3.42 to 4.93% Within-batch Accuracy for Lansoprazole (% Nominal) Within-batch accuracy for LLOQ QC ranged from to % Within-batch accuracy for LQC, MQC1, MQC2 and HQC ranged from to % Intra-day Accuracy for Lansoprazole (% Nominal) Intra-day accuracy for LLOQ QC was % Intra-day accuracy for LQC, MQC1, MQC2 and HQC ranged from to % Between-batch Accuracy for Lansoprazole (% Nominal) Between-batch accuracy for LLOQ QC was % Between-batch accuracy for LQC, MQC1, MQC2 and HQC ranged from to % 192

32 Acceptance criteria: Precision: Low, medium & high QC concentrations should be within 15% & 20% for LLOQ conc. Accuracy: Low, medium & high QC concentrations should be within ±15% & ±20% for LLOQ conc of nominal value. Table 5 Within Batch Precision and Accuracy of Lansoprazole Concentration (ng/ml) LLOQ QC LQC MQC1 MQC2 HQC QC# Mean S.D C.V.% % Nominal N Mean S.D C.V.% % Nominal N Mean

33 S.D C.V.% % Nominal N Mean S.D C.V.% % Nominal N Mean S.D C.V.% % Nominal N

34 Table 6 Intra-day Precision and Accuracy of Lansoprazole Concentration (ng/ml) LLOQ QC LQC MQC1 MQC2 HQC QC# Mean S.D C.V.% % Nominal N

35 Table 7 Between Batch / Inter-day Precision and Accuracy of Lansoprazole. Concentration (ng/ml) LLOQ QC LQC MQC1 MQC2 HQC QC# Mean S.D C.V.% % Nominal N

36 vi) Stability a) Room Temperature Stock Solution Stability Room temperature stock solution stability was carried out at 0 and 7 hours for Lansoprazole by injecting six replicates of prepared stock dilutions of Lansoprazole equivalent to the middle concentration (MQC2). Comparison of the mean area response of Lansoprazole at 7 hours was carried out against the zero hour value, the stability was found to be % for Lansoprazole. The stability of Pantoprazole was carried out at 0 and 7 hours by injecting 6 replicates of prepared stock dilutions of Pantoprazole equivalent to working concentration. Comparison of the mean area response of Pantoprazole at 7 hours was carried out against 0 hour value, the stability was found to be % for Pantoprazole. The results are within the acceptable limits and given in tables 8 & 9. Acceptance criteria: %Comparison response must be between 90% & 110% and %CV must be 10% Table 8 Room Temperature Stock Solution Stability of Lansoprazole (0 and 7 Hours) S.No. Area 0 hr 7 hr Mean S.D C.V.% % Stability

37 Table 9 Room Temperature Stock Solution Stability of Pantoprazole (0 and 7 Hours) S.No. Area 0 hr 7hr Mean S.D C.V.% % Stability b) Room Temperature Spiking Solution Stability Room Temperature Spiking Solution Stability was carried out at 0 and 7 hours for Lansoprazole by injecting six replicates of prepared Spiking Solution of Lansoprazole equivalent to the middle concentration (MQC2). Comparison of the mean area response of Lansoprazole at 7 hours was carried out against the zero hour value of Room temperature Spiking Solution, the stability was found to be % for Lansoprazole. The room temperature spiking solution stability of Pantoprazole was carried out at 0 and 7hours by injecting six replicates of prepared Spiking dilutions of Pantoprazole. Comparison of the mean area response of Pantoprazole at 7 hours was carried out against 0 hour value of Room Temperature Spiking Solution, the stability was found to be % for Pantoprazole. The results are within the acceptable limits and given in tables 10 &

38 Table 10 Room Temperature Spiking Solution Stability of Lansoprazole (0 and 7 Hours) S.No. Area 0 hr 7 hr Mean S.D C.V.% % Stability Table 11 Room Temperature Spiking Solution Stability of Pantoprazole (0 and 7 Hours) Area S.No. 0 hr 7hr Mean S.D C.V.% % Stability

39 c) Refrigerated Stock Solution Stability (at 2-8 C) Refrigerated stock solution stability of Lansoprazole and Pantoprazole was carried out by injecting six replicates of stock dilutions. The stock solution was found to be stable for maximum of 6 days. The six days stock Solution stability of Lansoprazole and Pantoprazole was found to be 99.45% and 99.41% respectively. Stability standard stock solution and comparison (fresh) standard stock solution of Lansoprazole were prepared equivalent to the middle concentration (MQC2) level during method validation. The response of comparison (fresh) sample was corrected by multiplying with correction factor. Correction factor was calculated as follows: Correction factor = Corrected concentration of stability standard stock solution Corrected concentration of comparison (fresh) standard stock solution Note: Corrected concentration: Concentration corrected using potency, actual amount weighed and molecular weight of the compound. The results are within the acceptable limits and given in tables 12 & 13. Acceptance criteria: %Comparison response must be between 90% & 110% and %C.V must be 10% 200

40 Table 12 Refrigerated Stock Solution Stability for Lansoprazole 2-8 C (6 days) Stability standard Comparison S.No. stock standard stock Corrected Response Area Area Mean SD %CV N Correcting factor Mean response of Standard stock Mean corrected response % Response Note: Corrected Response =Comparison Response x Correction Factor Correction factor =Corrected conc. of stability standard stock solution Corrected conc. of comparison standard stock solution Correction factor: % Comparison Response =Mean Response of Stability Standard Stock x 100 Mean Corrected Response of Comparison Standard Stock = X % Comparison Response=

41 Table 13 Refrigerated Stock Solution Stability for Pantoprazole 2-8 C (6 days) Stability standard Comparison S.No. Stock standard stock Corrected Response Area Area Mean SD %CV N Correcting factor Mean response of Standard stock Mean corrected response % Response Note: Corrected Response =Comparison Response x Correction Factor Correction factor=corrected concentration of stability standard stock solution Corrected concentration of comparison standard stock solution Correction factor: % Comparison Response =Mean Response of Stability Standard Stock x 100 Mean Corrected Response of Comparison Standard Stock = x % Comparison Response =

42 d) Auto sampler Stability In assessing the auto sampler stability, six sets each of LQC and HQC samples were processed and placed in the auto sampler. These samples were injected after a period of 49 hours and quantified against freshly spiked calibration curve standards of concentration range equivalent to that used for calculation of precision and accuracy, refer, Table 14. The results demonstrate that the processed samples were stable for 49 hours. The percent nominal of LQC and HQC at 49 hours was 91.38% and 91.21% respectively and precision of LQC and HQC at 49 hours was 2.28% and 0.74% respectively. The results are within the acceptance limits and given in tables 14 & 15. Acceptance criteria: Mean % nominal conc at each QC sample level must be between 85% & 115% and the precision must be 15% of C.V% Atleast 67% of QCs injected at each level should be within ±15% of respective nominal value. Table 14 Freshly Spiked Concentration-response Linearity Data for Stabilities of Lansoprazole Concentration (ng/ml) Slope Intercept R R 2 STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I FRESH1 CC % Nominal

43 Table 15 Auto sampler Stability of Lansoprazole in Processed Human Plasma Samples (49 hours) Concentration (ng/ml) LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 e) Bench Top Stability In short-term room temperature stability, six sets each of LQC and HQC, were determined at 5 hours. The quality control samples were quantified against the freshly spiked calibration curve standards of concentration range equivalent to that used for calculation of precision and accuracy, refer, Table 14 Lansoprazole was found to be stable upto 5 hours as per the acceptance criteria. The percent mean nominal of LQC and HQC was 90.37% and 89.30% respectively and the precision of LQC and HQC was 2.86% and 0.43% respectively. The results are within the acceptance limits and given in tables 14 & 16. Acceptance criteria: Mean % nominal conc at each QC sample level must be between 85% & 115% and precision must be 15% of C.V% Atleast 67% of QCs injected at each level should be within ±15% of respective nominal value. 204

44 Table 16 Bench Top Stability of Lansoprazole (5 hours) Concentration (ng/ml) LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 f) Freeze-thaw Stability The stability of QC samples was determined after three freeze-thaw cycles. Six replicates of LQC and HQC were analyzed after three freeze-thaw cycles. The freeze-thaw quality control samples were quantified against the freshly spiked calibration curve standards of concentration range equivalent to that used for the calculation of precision and accuracy, refer, Table 14. The percent nominal for LQC & HQC was 92.11% and 90.07% respectively and the precision for LQC & HQC was 2.97% and 0.56% respectively for three freeze-thaw cycles. The results are within the acceptance limits and given in tables 14 & 17. Acceptance criteria: Mean % nominal conc at each QC sample level must be between 85%&115% and the precision must be 15% of the C.V% Atleast 67% of QCs injected at each level should be within ±15% of the respective nominal value. 205

45 Table 17 Freeze Thaw Stability (FT III Cycle) of Lansoprazole Concentration (ng/ml) LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 g) Plasma samples Stability at C Plasma samples stability at C was determined for 3 days using six sets each of LQC and HQC. The quality control samples were quantified against the freshly spiked calibration curve standards of concentration range equivalent to that used for calculation of precision and accuracy, refer, Table 14. The mean concentration of stability QCs were compared against the mean of the of the I st day QCs when injected for first time after the bulk spiking (first passing P& A Batch). Lansoprazole was found to be stable up to 3 days at 20 0 C as per the acceptance criteria. The percent stability was 88.66% for LQC and 92.72% for HQC and the precision was 1.91% for LQC and 0.64% for HQC. The results are within the acceptable limits and given in tables 14, 18 & 19. Acceptance criteria: Mean % nominal conc at each QC sample level must be between 85% & 115% and the precision must be 15% of C.V% Atleast 67% of QCs injected at each level should be within ±15% of respective nominal value. 206

46 Table: 18Short Term Stability at -20 C Lansoprazole Data for Day 0 Concentration (ng/ml) LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 Table: 19Short Term Stability at -20 C Lansoprazole Data for Day 3 Concentration (ng/ml) LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 % Stability % Stability =Mean concentration of stability LQC or HQC sample x 100 Mean concentration of LQC or HQC samples of first day of stability testing 207

47 h) Wet-Extract Stability In assessing the Wet extract stability, six sets of LQC and HQC samples were processed and placed on the bench at room temperature. These samples were injected after a period of 23 hours from the first set QC s samples loaded time and quantified against freshly spiked calibration curve standards of concentration range equivalent to that used for calculation of precision and accuracy, refer, Table 14. The results demonstrate that the processed samples were stable for 23 hours. The percent nominal at 23 hours was 91.28% and 89.49% respectively and precision was 4.89% and1.60% respectively for LQC & HQC. The results are within the acceptance limits and given in tables 14 & 20. Acceptance criteria: Mean % nominal conc at each QC sample level must be between 85% & 115% and the precision must be 15% of C.V% Atleast 67% of QCs injected at each level should be within ±15% of respective nominal value. Table 20 Wet extract Stability of Lansoprazole (23 hours) Concentration (ng/ml) LQC HQC QC# Mean S.D C.V.(%) % Nominal N

48 i) Reinjection Stability In accessing the reinjection stability, six sets of LQC and HQC samples were processed and analysed with calibration curve standard. The QC samples were retained in the autosampler and reinjected after a period of 28 hours and quantified against the initial calibration curve data, refer, Table 21. The mean concentration of reinjected QCs was compared against the mean of the QCs when injected for first time. The results demonstrate that the reinjected samples were stable for 28 hours. The percent stability at 28 hours was 99.10% for LQC and % for HQC and precision was 0.56% for LQC and 1.45% for HQC for 28 hours. The results are within the acceptable limits and given in tables 21, 22 & 23. Acceptance criteria: Mean % nominal conc at each QC sample level must be between 85% & 115% and the precision must be 15% of C.V% Atleast 67% of QCs injected at each level should be within ±15% of respective nominal value. Table 21 Concentration-response Linearity Data for Reinjection Stability (26 hours) Concentration (ng/ml) STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I CC# % Nominal Slope Interce pt R R

49 Table 22 Reinjection Stability Data for Lansoprazole 0 Hours Concentration LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 Table 23 Reinjection Stability Data for Lansoprazole 28 Hours Calculations: Concentration LQC HQC QC# Mean S.D C.V.(%) % Nominal N 6 6 % Stability % Stability = Mean concentration of QC re-injected over a period of time x 100 Mean concentration of QCs when injected for the 1 st time 210

50 vii) Recovery For evaluating the recovery six sets each of quality control samples (LQC, MQC2 and HQC) were processed and injected (extracted samples). Twenty four blank matrix samples were processed and four sets of six each blank samples were reconstituted with the aqueous QC dilutions at low, middle and high concentration without internal standard, which represents 100 % extraction of analyte(s) (nonextracted samples). Six blanks were reconstituted with the internal standard solution, which represents 100 % extraction of internal standard. (Non-extracted sample). The non-extracted samples were injected. The recovery comparison samples of Lansoprazole were compared against extracted samples of LQC, MQC2 and HQC of PA-batch-6. The recovery comparison samples of internal standard were compared against the response of internal standard in MQC2. The mean overall recovery of Lansoprazole was 56.65% with a precision ranged from 0.29% to 1.31%. The mean recovery of internal standard was % with a precision ranged from 0.95 to 1.11%. The results are within the acceptance limits and given in tables 24 & 25. Acceptance criteria: The C.V% of mean analyte & ISTD recoveries must be 15% for each QC conc level. The difference of % recovery between the lowest % recovery & highest % recovery should not be more than 25% 211