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

Transcription

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

2 6.1. DEVELOPMENT AND VALIDATION OF AN LC-MS/MS METHOD FOR THE DETERMINATON OF MONTELUKAST IN HUMAN PLASMA INTRODUCTION Drug profile: Montelukast (Fig.1), chemically known as 2-[1-[1(R)-[3-[2(E)-(7-chloroquinolin-2- yl)vinyl] phenyl]-3[2-(1-hydroxy-1-methylethyl)phenyl]propylsulfanylmethyl] cyclo propyl] acetic acid sodium salt 113,114. It is a leukotriene receptor antagonist (LTRA) used for the maintenance treatment of asthma and to relieve symptoms of seasonal allergies. It is usually administered orally. Montelukast is a CysLT 1 antagonist 118, 119, that it blocks the action of leukotriene D4 (and secondary ligands LTC4 and LTE4) on the cysteinyl leukotriene receptor CysLT 1 in the lungs and bronchial tubes by binding to it. This reduces the broncho constriction otherwise caused by the leukotriene and results in less inflammation. It is used for the treatment of bronchial asthma Fig.1 Chemical structure of Montelukast 219

3 Properties: Chemical name : Chemical formula : C 35 H 35 ClNNaO 3 S Molecular weight : CAS Registry Number: pka : 14.6 Solubility : Physical state : white to off-white powder Sodium {1-[({(1R)-1-{3-[(E)-2-(7-chloro-2-quinolinyl) vinyl] phenyl}-3-[2-(2-hydroxy-2-propanyl) phenyl] propyl} sulfanyl) methyl] cyclopropyl} acetate Freely soluble in ethanol, methanol and water and practically insoluble in acetonitrile. Therapeutic Category : Antiasthmatic Trade names : Singulair, Montelo-10 and Monteflo Bioavailability : 63-73% T ½ : hours Plasma protein binding : 99% Metabolism : Hepatic Elimination : Biliary MECHANISM OF ACTION: The cysteinyl leukotrienes (LTC4, LTD4, and LTE4) are products of arachidonic acid metabolism and are released from various cells, including mast cells and eosinophils. These eicosanoids bind to cysteinyl leukotriene (CysLT) receptors. The CysLT type-1 (CysLT1) receptor is found in the human airway (including airway smooth muscle cells and airway macrophages) and on other pro-inflammatory cells (including eosinophils and certain myeloid stem cells). CysLTs have been correlated with the pathophysiology of asthma and allergic rhinitis. In asthma, leukotriene-mediated effects include airway 220

4 edema, smooth muscle contraction, and altered cellular activity associated with the inflammatory process. In allergic rhinitis, CysLTs are released from the nasal mucosa after allergen exposure during both early- and late-phase reactions and are associated with symptoms of allergic rhinitis. Intranasal challenge with CysLTs has been shown to increase nasal airway resistance and symptoms of nasal obstruction. Montelukast is an orally active compound that binds with high affinity and selectivity to the CysLT1 receptor (in preference to other pharmacologically important airway receptors, such as the prostanoid, cholinergic, or - adrenergic receptor). Montelukast inhibits physiologic actions of LTD4 at the CysLT1 receptor without any agonist activity. 221

5 Side effects: Montelukast produces very few side effects like headache, and rashes. Eosinophilia and neuropathy are infrequent. Few cases of Churg-Strauss syndrome (vasculitis with eosinophilia) have been reported. Skin rash, bruising, severe tingling, numbness, pain, muscle weakness; Mood or behavior changes, anxiety, depression, or thoughts about suicide or hurting yourself; Tremors or shaking; Severe sinus pain, swelling, or irritation; or Worsening asthma. Past work on the chromatographic method for montelukast: 1. Balasekhara Reddy Challa et al., 147 (2010) developed a simple, sensitive, and specific LC-ESI MS/MS method for quantification of Montelukast (MO) in human plasma using Montelukast-d 6 (MOD6) as an internal standard (IS) is discussed here. Chromatographic separation was performed on YMC-pack pro C 18, 50 x 4.6 mm, S-3 μm column with an isocratic mobile phase composed of 10mM ammonium formate (ph 4.0):acetonitrile (20:80 v/v), at a flow-rate of 0.8 ml min 1. MO and MOD6 were detected with proton adducts at m/z and in multiple reaction monitoring (MRM) positive mode respectively. MO and MOD6 were extracted using acetonitrile as precipitating agent. The method was validated over a linear concentration range of ng ml 1 with correlation coefficient (r 2 ) The intraday precision and accuracy were within and The inter-day precision and accuracy were within % and % for MO. Both analytes were found to be stable throughout three freeze-thawing cycles, bench top, and autosampler stability studies. This method was utilized successfully for the analysis of plasma samples following oral administration of MO (5 mg) in 31 healthy Indian male human volunteers under fasting conditions. 222

6 2. Yao Huang, Li Ding et.al., 148 (2009)A liquid chromatography electrospray ionization mass spectrometry (LC ESI MS) method was developed for the determination of montelukast in human plasma. After precipitation of plasma proteins the plasma samples were separated on a C 18 column with a mobile phase consisted of 20mmol/ml ammonium acetate buffer solution containing 0.2% formic acid-methanol (12:88, v/v). LC-ECI-MS was performed in selective ion monitoring (SIM) mode using target ions [M+H] + at m/z for montelukast and m/z for telmisartan, the internal standard (IS). The calibration curve was linear over the plasma concentration range of1ng/ml-100ng/ml. The lower limit of quantification (LLOQ) was 1ng/ml for montelukast. The relative standard deviation of intra and inter batches were less than 5.9% and 10.0% respectively. The method was successfully applied to study the pharmacokinetics of montelukast sodium tablets in healthy male volunteers 3. RM Singh et, al., 149 (2010) Describes a simple, precise and accurate HPLC method for estimation of montelukast sodium in bulk and in tablet dosage form. The separation was achieved by using octadecyl silane column (C18) and acetonitrile: 1 mm sodium acetate adjusted to ph 6.3 with acetic acid in proportion of 90:10 v/v as mobile phase, at a flow rate of 1.5 ml/min. Detection was carried out at 285 nm. The retention time of montelukast sodium was found to be 3.4 min. The limit of detection was found 1.31 µg/ml and limit of quantification 3.97 µg/ml. The accuracy and reliability of the proposed method was ascertained by evaluating various validation parameters like linearity (1-100 µg/ml), precision, accuracy and specificity according to ICH guidelines. The proposed method provides an accurate and precise quality control tool for routine analysis of montelukast sodium in bulk and in tablet dosage form. 4. S A Patel et al 150 (2010) describes simple, specific, accurate and precise reverse phase high pressure liquid chromatographic method for the simultaneous determination of Montelukast sodium and Bambuterol hydrochloride in combined dosage form. The sample was analyzed by reverse phase C18 column (Phenomenex C18, 5μ, 250mm x 4.6mm) as stationary phase; methanol: acetonitrile : 1% trichloroacetic acid in the ratio of 80:10:10 v/v/v as a mobile phase at a flow rate of 1.0ml/min. Quantification was achieved with ultraviolet detection at 220 nm. The retention time for Montelukast sodium and Bambuterol hydrochloride was found to be 3.17 and 2.35 min, respectively. The 223

7 linearity for both the drugs was in the range of μg/ml with mean accuracies ± 0.49 and ± 0.63 for Montelukast sodium and Bambuterol hydrochloride, respectively. The method was successively applied to pharmaceutical formulation because no chromatographic interferences from the tablet excipients were found. The method retained its accuracy and precision when the standard addition technique was applied. 5. R.Revathi et. al., 151 (2005) developed a cost-effective and less time consuming method for the estimation of montelukast sodium and doxofylline in combined pharmaceutical formulation as the Reverse phase HPLC methods proposed for single component or multicomponent analysis of montelukast sodium with other drugs are time consuming.the separation was performed on an inertsil C8 (5 μm, mm) column in isocratic mode with the mobile phase consisting a mixture of methanol and sodium phosphate buffer (75:25 v/v, ph 6.5 adjusted with orthophosphoric acid). The mobile phase was pumped at a flow rate of 1 ml min -1 and eluents were monitored at 230 nm. The selected chromatographic conditions were found to separate doxofylline (retention time = 3.4 min) and montelukast sodium (retention time = 5.5 min) with a resolution of The proposed HPLC method was validated with respect to linearity, accuracy, repeatability, specificity, robustness, and ruggedness as per International Conference on Harmonisation guidelines Q2(R1), November 2005 (Validation of Analytical Procedures: Text and Methodology). The percentage recoveries for doxofylline and montelukast sodium ranged from 98.1% to 101.7% and 98.2 to 101.9%, respectively, which indicated that the above method was enough accurate and precise.hence, it was concluded that the developed method is suitable for routine analysis of these combination due to its less analysis time. 6. V. Choudhariet. al 152 developed and validated a simple, accurate, precise, and sensitive spectrophotometric method for simultaneous estimation of Montelukast (MON) and Levocetirizine (LEV) in combined tablet dosage form have been. The ratio derivative spectroscopic method involves measurement of first derivative amplitude of ratio spectra at nm for MON and nm for LEV as two wavelengths for estimation. Beer's law is obeyed in the concentration range of 4-12 and 2-6 μg/ml for MON and LEV, respectively. LOD values for MON and LEV are found to be 0.09 μg/ml and

8 μg/ml, respectively. LOQ values for MON and LEV are found to be 0.277μg/mL and μg/ml, respectively. The results of analysis have been validated statistically and recovery studies carried out in the range % to confirm the accuracy of the proposed method. 7. AlirezaShafaati et. al., 153 (2003) A rapid, simple and sensitive high-performance liquid chromatography (HPLC) method, using a monolithic column and fluorescence detection, has been developed for quantification of Montelukast in human plasma. Ethoxyquine, easily available as a pharmaceutical substance, was selected as internal satandard. The assay enables the measurement of Montelukast for therapeutic drug monitoring with a minimum detectable limit of 5 ng/ ml 1. The method involves simple, one-step extraction procedure and analytical recovery was about 97%. The separation was carried out in reversed phase conditions using a Chromolith RP (RP-18e, 100 mm 4.6 mm) column at ambient temperature. The mobile phase was 56% acetonitrile and 50mM sodium dihydrogen phosphate, and distilled water to 100%, adjusted to ph 7.0at a flow rate of 2 ml/min. The excitation wavelength was set at 350 nm, emission at 450 nm. The calibration curve was linear over the concentration range ng/ml. The coefficients of variation for inter-day and intra-day assay were found to be less than 7%. The method was applied to the determination of montelukast in plasma from 12 subjects dosed with Montelukast 10 mg tablets and pharmacokinetic parameters were determined. 8. SriPalakit P et. al., 154 (2008) developed an analytical method based on highperformance liquid chromatographic (HPLC) for the determination of montelukast in human plasma using mefenamic acid as an internal standard. After precipitation of plasma proteins with acetonitrile, chromatographic separation was carried out using a Zorbax Eclipse XDB C8 (150 mm x 4.6 mm i.d., 5 microm) with mobile phase consisted of methanol-acetonitrile-0.04m disodium hydrogen orthophosphate (22:22:56, v/v, ph 4.9). The wavelengths of fluorescence detection were set at 350 nm for excitation and 450 nm for emission. The linearity was confirmed in the concentration range of ng/ml in human plasma. Intra- and inter-day accuracy determined from quality control samples were and %, and and %, respectively. Intra- and inter-day precision measured as coefficient of variation were < or =4.72 and < or =9.00%, 225

9 respectively. Extraction recoveries of drug from plasma were >48.14%. The protocol herein described was employed in a pharmacokinetic study of tablet formulation of montelukast in healthy Thai male volunteers. 9 P.Radhakrishnanand et.al., 155 (2008)A new and accurate chiral liquid chromatographic method has been developed for determination of the enantiomeric purity of montelukast sodium (R enantiomer) in bulk drugs and dosage forms. Normal phase chromatographic separation was performed on an immobilized amylase-based chiral stationary phase with n-hexane ethanol 1,4-dioxane trifluoroacetic acid diethyl amine 5:25:10:0.3:0.05 (v/v) as mobile phase at a flow rate of 1.0mLmin 1. The elution time was approximately 15min. The resolution (RS) between the enantiomers was >3. The mobile phase additives trifluoroacetic acid and diethyl amine played a key role in achieving chromatographic resolution between the enantiomers and also in enhancing chromatographic efficiency. Limits of detection and quantification for the S enantiomer were 0.07 and 0.2μg, respectively, for a test concentration of montelukast sodium of 1,000μgmL 1 and 10μL injection volume. The linearity of the method for the S enantiomer was excellent (R2>0.999) over the range from the LOQ to 0.3%. Recovery of the S enantiomer from bulk drug samples and dosage forms ranged from 97.0 to 103.0%, indicative of the high accuracy of the method. Robustness studies were also conducted. The sample solution stability of montelukast sodium was determined and the compound was found to be stable for a study period of 48hrs. 226

10 EXPERIMENTAL & ANALYTICAL METHODOLOGY A) MATERIALS i) Instrumentation The author had attempted to develop a liquid chromatographic method for the determination of Montelukast using an isocratic Shimadzu HPLC equipment comprising of two LC10AT VP pumps, VP CTO-10AS VP column oven, a Zorbax Eclipse, XDB phenyl column (4.6 ID X 75 mm, 3.5µ) and an API 3000 (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 Microbalance Vibramax Vacuum pump Refrigerator PH meter Micropipettes, Multipette and Micro tips Vortexer Solid phase extraction chamber Orpheus C mg/1.0mL cartridges Poly propylene tubes Water Purification System Ultra sonicator Nitrogen Evaporator Shimadzu API 3000, MDS Sciex Sanyo (-86 C) VIP Series Sartorius Heidolph Millipore Samsung Orion Brand and Eppendorf Spinix Orochem Orochem Torson s Elix 10 / Milli-Q gradient Bandelinsonorex ZymarkTurbovap LV station, Caliper ii) Drug and Internal standard The reference sample of montelukast was gifted by Hetero drugs Ltd, India and montelukast D6 sodium salt was gifted by Clearsynth labs, India. 227

11 iii) Chemicals and solvents Montelukast reference standard Montelukast D6 reference standard Orpheus C mg/1.0mL cartridges Acetonitrile (HPLC grade) Methanol (HPLC grade) Milli-Q water Ammonium acetate (AR grade) Formic acid (GR grade) Human plasma 0.45µ Membrane filter iv) Preparation of solutions a) Montelukast Stock Solution Weighed accurately, about 5 mg of Montelukast Working standard separately and transferred to a separate 5 ml clean glass volumetric flask, dissolved HPLC grade methanol and made up the volume with the same to produce a solution of ng/ml. Corrected the above concentration of Montelukast solution accounting for its potency and the actual amount weighed for free compound weight. A batch number was provided and the Stock Weighing and Solution Preparation form was completed. The stock solution was stored in refrigerator at 2-8 C and used for maximum of four days. The stock solution was diluted to suitable concentrations using diluents for spiking in to plasma to obtain calibration curve (CC) standards, quality control (QC) samples and DIQC samples. All other dilutions (system suitability dilutions, aqueous mixture, recovery etc.) were prepared in mobile phase. b) Montelukast D6 Stock Solution (Internal Standard) Weighed accurately, about mg of Montelukast D 6 sodium and transferred to a separate 5mL volumetric flask, dissolved in HPLC grade methanol and made up the volume with the same to produce a solution of ng/ml. Corrected the 228

12 above concentration of Montelukast D 6 accounting for its potency and the actual amount weighed. A batch number was provided and the Stock Weighing and Solution Preparation form was completed. The stock solution was stored in refrigerator at 2-8 C and used for maximum of four days. The stock solution was diluted to suitable concentration using diluents for internal standard dilution. c) Biological Matrix: Eight lots of K 2 EDTA human plasma including one hemolytic and one lipemic plasma lots were screened for selectivity test. All eight human plasma lots were found free of any significant interference for Montelukast and Montelukast D 6. Six plasma lots were used to prepare calibration standards and quality control samples and DIQC samples. Selectivity and matrix effect tests were performed as per FDA guidelines, Bioanalytical Method Validation before bulk spiking. After bulk spiking, aliquots of 600 µl for CCs and 600 µl for QCs of spiked plasma samples were pipetted out into a prelabelled polypropylene micro centrifuge tubes and then all the bulk spiked samples were stored to deep freezer at 70 C ± 10 C, except twelve replicates each of LQC and HQC, which were stored in deep freezer at 20 C ± 5 C for generation of stability data at 20 C. 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 Montelukast were prepared. Quality control samples consisted of Montelukast concentrations of 5.036ng/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. 229

13 Standard Concentration Montelukast (ng/ml) Standard I 2-3 times of Cmax 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 (ph: 4.5) 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 followed by ph adjustment to with glacial acetic acid and sonicated in an ultrasonicator for 2 to 5 minutes. f) Mobile Phase 850 ml of HPLC acetonitrile was transferred to a 1000 ml reagent bottle and 150 ml of 5mM Ammonium acetate ph: 4.5 was added it. It was mixed well, sonicated in an ultrasonic bath for 2 to 5 minutes. g) Diluent A mixture of HPLC Acetonitrile and Milli Q water was prepared in the volume ratio of 60:40 as dilution solvent. It was then sonicated in an ultrasonicator for 2 to 5 minutes. 230

14 h) 100mM Ammonium Acetate (ph: 4.5) About g 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 followed by ph adjustment to with glacial acetic acid and sonicated in an ultrasonicator for 2 to 5 minutes. i) 1% Formic acid buffer in water: About 10 ml of Formic acid transferred to a 990 ml reagent bottle and make up to the mark with Milli Q water. Mixed well and sonicated in an ultrasonicator for 5 minutes. A batch number was provided and completed the Solution Preparation form. Stored the solution at room temperature and used within a period of 4 days. j) Rinsing Solution: A mixture of HPLC acetonitrile and Milli Q water was prepared in the volume ratio of 90:10 as rinsing solution. It was then sonicated in an ultrasonicator for 2 to 5 minutes. k) System Suitability Solution A mixture of analyte and internal standards was prepared for system suitability test. The concentration of analyte corresponds to middle concentration of calibration range ( ng/ml for Montelukast) and that of internal standards corresponded to working concentration used for spiking ( ng/ml for Montelukast D 6 ). The system suitability test solution was injected as an aqueous mixture. Aqueous samples were prepared as per recovery basis. 25 µl of analyte ( ng/mL) and 50 µl of working concentration of internal standard ( ng/mL) were mixed with 925 µl of mobile phase to prepare the system suitability sample. 231

15 ANALYTICAL METHOD DEVELOPMENT Optimization of the chromatographic conditions For developing the method for the assay of Montelukast, 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 non- polar zorbax eclipse XDB phenyl 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 acetonitrile and 5 mm Ammonium acetate buffer in a ratio of 85:15 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 0.6 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). 232

16 Fig. 2 Mass Spectra of Montelukast (A) and Montelukast D6 (B) for parent & product masses 233

17 RETENTION TIMES: Montelukast Montelukast D6 : 1.70 ± 0.3 min : 1.70± 0.3 min DETECTION: Drug name Parent mass (amu) Product mass (amu) Montelukast Montelukast D Source/ Gas Parameters (positive mode): API 3000 Parameter Montelukast Montelukast D 6 Ion Spray Voltage (IS) Temperature (TEM 0 C) Curtain Gas (CUR) Collision Gas (CAD) Nebulizer Gas Focusing Potential (FP) Declustering Potential (DP) Collision Energy (CE) Collision Cell Exit Potential (CXP) Entrance Potential (EP)

18 Retention time of Montelukast A model chromatogram showing the separation of Montelukast is presented in Fig. 3 under the above optimized conditions retention time of 1.70 min was obtained for both Montelukast and Montelukast D6. 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 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 ZORBAX Eclipse XDB Phenyl (4.6 X 75 mm, 3.5µ) Acetonitrile: 5mM ammonium acetate buffer (85:15 v/v) 5mM ammonium acetate buffer Isocratic 0.60 ml/min 3.0 min 40 ± 2 0 C 5 0 C 20 μl 700 μl 235

19 Extraction process of plasma samples and their drying Five hundred micro liters of the spiked plasma calibration curve standards and the quality control samples were transferred to a set of pre-labeled polypropylene tubes containing 50 µl of Montelukast D6 dilution (internal standard; ng/ml). The tubes were vortexed for ten seconds. Five hundred micro liters of 1% formic acid solution was added to the tubes and vortexed for another ten seconds. Each of the Orpheus C 18, 100mg/1.0 ml SPE cartridges was conditioned with 1mL of methanol and 1 ml of water followed by equilibrating with 1mL of 1% formic acid on the solid phase extraction chamber. The above samples were loaded on to the cartridges and the cartridges were again washed with 1mL of 100 mm ammonium acetate followed by 2 ml of Milli-Q water. The cartridges were dried for about one minute and eluted with 1mL of mobile phase [Acetonitrile: 5mM ammonium acetate buffer (85:15 v/v)]. The eluents were evaporated in a stream of nitrogen for 20 min at 40 0 C and the residues in the dried tubes were reconstituted with 1.0mL 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 20 μl of the sample was drawn each time from the vials in the auto sampler. 236

20 REPRESENTATIVE CHROMATOGRAMS Fig:3A Representative Chromatogram of an Aqueous Standard and Internal Standard of Mixture of Montelukast Sample Name: "AQM(M-214-SST-02)" Sample ID: "" File: "001.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: N/A Calculated Conc: ng/ml 2.7e5 Acq. Date: 06/01/12 Acq. Time: 15:52:53 2.6e5 Modified: No 2.5e5 Proc. Algorithm: Analyst Classic Bunching Factor: 1 2.4e5 Noise Threshold: 5.00 cps Area Threshold: cps 2.3e5 Num. Smooths: 7 2.2e5 Sep. Width: 0.20 Sep. Height: e5 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: e5 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec 1.9e5 Expected RT: 1.91 min Use Relative RT: No 1.8e Int. Type: Base To Base 1.71 min Retention Time: Area: counts Height: 2.79e+005 cps 1.56 min Start Time: End Time: 2.04 min In te n s ity, c p s 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.0 Sample Name: "AQM(M-214-SST-02)" Sample ID: "" File: "001.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Unknown Concentration: 1.00 ng/ml 2.0e5 Calculated Conc: N/A 06/01/12 Acq. Date: Acq. Time: 15:52:53 1.9e Time, min 1.70 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.89 min Use Relative RT: No Int. Type: Base To Base 1.70 min Retention Time: Area: counts Height: 2.02e+005 cps 1.53 min Start Time: End Time: 2.02 min In te n s ity, c p s 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e Time, min 237

21 Fig: 4 Representative Chromatogram of Blank Sample of Montelukast Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Double Blank Concentration: ng/ml Calculated Conc: N/A 135 Acq. Date: 06/01/ Acq. Time: 15:56: Modified: No In te n s ity, c p s Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Double Blank Concentration: 1.00 ng/ml 130 Calculated Conc: N/A Acq. Date: 06/01/ Acq. Time: 15:56: Modified: No Time, min In te n s ity, c p s Time, min

22 Fig: 5 A Representative Chromatogram of Blank Plasma with Internal Standard Sample of Montelukast Sample Name: "Blank+ISTD" Sample ID: "" File: "003.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Blank Concentration: ng/ml Calculated Conc: N/A Acq. Date: 06/01/ Acq. Time: 16:00: Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 5.00 cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.91 min Use Relative RT: No Int. Type: Base To Base 1.75 min Retention Time: Area: 205 counts Height: 6.24e+001 cps 1.69 min Start Time: End Time: 1.82 min In te n s ity, c p s Sample Name: "Blank+ISTD" Sample ID: "" File: "003.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: Blank Concentration: 1.00 ng/ml Calculated Conc: N/A 3.6e4 Acq. Date: 06/01/12 Acq. Time: 16:00:29 3.4e4 Modified: No Proc. Algorithm: Analyst Classic 3.2e4 Bunching Factor: 1 Noise Threshold: cps Area Threshold: cps 3.0e4 Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: e4 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: e4 RT Window: 30.0 sec Expected RT: 1.89 min Use Relative RT: No 2.4e Time, min 1.73 Int. Type: Base To Base 1.73 min Retention Time: Area: counts Height: 3.69e+004 cps 1.52 min Start Time: End Time: 2.02 min In te n s ity, c p s 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min 239

23 Fig: 6 A Representative Chromatogram of LLOQ Sample of Montelukast Sample Name: "LLOQ QC-1" Sample ID: "" File: "014.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: ng/ml 800 Calculated Conc: ng/ml Acq. Date: 06/01/12 Acq. Time: 16:41: Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 5.00 cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.91 min Use Relative RT: No Int. Type: Base To Base 1.74 min Retention Time: Area: 5733 counts Height: 7.46e+002 cps 1.61 min Start Time: End Time: 1.93 min In te n s ity, c p s Sample Name: "LLOQ QC-1" Sample ID: "" File: "014.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: 1.00 ng/ml Calculated Conc: N/A Acq. Date: 06/01/12 4.0e4 Acq. Time: 16:41:53 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.89 min Use Relative RT: No Int. Type: Base To Base 1.74 min Retention Time: Area: counts Height: 4.16e+004 cps 1.50 min Start Time: End Time: 2.02 min In te n s ity, c p s 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e Time, min e4 1.4e4 1.2e4 1.0e Time, min 240

24 Fig: 7 A Representative Chromatogram of LQC Sample of Montelukast Sample Name: "LQC-1" Sample ID: "" File: "015.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: ng/ml Calculated Conc: ng/ml 2500 Acq. Date: 06/01/12 Acq. Time: 16:45: Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 5.00 cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.91 min Use Relative RT: No Int. Type: Base To Base 1.75 min Retention Time: Area: counts Height: 2.53e+003 cps 1.60 min Start Time: End Time: 1.96 min In te n s ity, c p s Sample Name: "LQC-1" Sample ID: "" File: "015.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: 1.00 ng/ml Calculated Conc: N/A 4.4e4 Acq. Date: 06/01/12 Acq. Time: 16:45:39 4.2e4 Modified: No Proc. Algorithm: Analyst Classic 4.0e4 Bunching Factor: 1 Noise Threshold: cps 3.8e4 Area Threshold: cps Num. Smooths: 7 3.6e4 Sep. Width: 0.20 Sep. Height: e4 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: e4 RT Window: 30.0 sec Expected RT: 1.89 min 3.0e4 Use Relative RT: No Int. Type: Base To Base 1.74 min Retention Time: Area: counts Height: 4.52e+004 cps 1.49 min Start Time: End Time: 2.01 min In te n s ity, c p s 2.8e4 2.6e4 2.4e4 2.2e4 2.0e Time, min e4 1.6e4 1.4e4 1.2e4 1.0e Time, min 241

25 Fig: 8 A Representative Chromatogram of MQC1 Sample of Montelukast Sample Name: "MQC-1-1" Sample ID: "" File: "016.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: ng/ml 1.4e4 Calculated Conc: ng/ml Acq. Date: 06/01/12 1.4e4 Acq. Time: 16:49:26 1.3e4 Modified: No 1.3e4 Proc. Algorithm: Analyst Classic Bunching Factor: 1 1.2e4 Noise Threshold: 5.00 cps Area Threshold: cps 1.2e4 Num. Smooths: 7 Sep. Width: e4 Sep. Height: 1.00 Exp. Peak Ratio: e4 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: e4 RT Window: 30.0 sec Expected RT: 1.91 min Use Relative RT: No Int. Type: Base To Base 1.76 min Retention Time: Area: counts Height: 1.41e+004 cps 1.59 min Start Time: End Time: 2.08 min In te n s ity, c p s Sample Name: "MQC-1-1" Sample ID: "" File: "016.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: 1.00 ng/ml Calculated Conc: N/A 4.2e4 Acq. Date: 06/01/12 Acq. Time: 16:49:26 4.0e4 Modified: No Proc. Algorithm: Analyst Classic 3.8e4 Bunching Factor: 1 Noise Threshold: cps 3.6e4 Area Threshold: cps Num. Smooths: 7 Sep. Width: e4 Sep. Height: 1.00 Exp. Peak Ratio: e4 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: e4 RT Window: 30.0 sec Expected RT: 1.89 min Use Relative RT: No 2.8e Time, min 1.75 Int. Type: Base To Base 1.75 min Retention Time: Area: counts Height: 4.33e+004 cps 1.50 min Start Time: End Time: 2.02 min In te n s ity, c p s 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min 242

26 Fig: 9 A Representative Chromatogram of MQC2 Sample of Montelukast Sample Name: "MQC-2-1" Sample ID: "" File: "017.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: ng/ml Calculated Conc: ng/ml 4.4e4 Acq. Date: 06/01/12 Acq. Time: 16:53:13 4.2e Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 5.00 cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.91 min Use Relative RT: No Int. Type: Base To Base 1.76 min Retention Time: Area: counts Height: 4.49e+004 cps 1.61 min Start Time: End Time: 2.21 min In te n s ity, c p s 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Sample Name: "MQC-2-1" Sample ID: "" File: "017.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: 1.00 ng/ml Calculated Conc: N/A Acq. Date: 06/01/12 4.0e4 Acq. Time: 16:53:13 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.89 min Use Relative RT: No Int. Type: Base To Base 1.75 min Retention Time: Area: counts Height: 4.16e+004 cps 1.51 min Start Time: End Time: 2.02 min In te n s ity, c p s 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e Time, min e4 1.4e4 1.2e4 1.0e Time, min 243

27 Fig: 10 A Representative Chromatogram of HQC Sample of Montelukast Sample Name: "HQC-1" Sample ID: "" File: "018.wiff" Peak Name: "Montelukast" Mass(es): "586.3/422.2 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: ng/ml Calculated Conc: ng/ml 7.0e4 Acq. Date: 06/01/12 Acq. Time: 16:57: Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: 5.00 cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.91 min Use Relative RT: No Int. Type: Base To Base 1.77 min Retention Time: Area: counts Height: 7.23e+004 cps 1.62 min Start Time: End Time: 2.15 min In te n s ity, c p s 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Sample Name: "HQC-1" Sample ID: "" File: "018.wiff" Peak Name: "Montelukast-D6(IS)" Mass(es): "592.4/427.1 amu" Comment: "" Annotation: "" Sample Index: 1 Sample Type: QC Concentration: 1.00 ng/ml 4.4e4 Calculated Conc: N/A Acq. Date: 06/01/12 Acq. Time: 16:57:00 4.2e Time, min 1.76 Modified: No Proc. Algorithm: Analyst Classic Bunching Factor: 1 Noise Threshold: cps Area Threshold: cps Num. Smooths: 7 Sep. Width: 0.20 Sep. Height: 1.00 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: 30.0 sec Expected RT: 1.89 min Use Relative RT: No Int. Type: Base To Base 1.76 min Retention Time: Area: counts Height: 4.31e+004 cps 1.64 min Start Time: End Time: 2.00 min In te n s ity, c p s 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e Time, min 244

28 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 Montelukast) 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. 6. 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. Table: 1 Selectivity Data for Montelukast (LLOQ Area) Montelukast SEL-LLOQ Montelukast Area IS Area Mean SD % CV

29 Table: 2 Selectivity Data for Montelukast (Blank + IS Area) Montelukast (Sample ID) Montelukast Area IS Area Blank+IS (Montelukast D6) Blank+IS (Montelukast D6) Blank+IS (Montelukast D6) Blank+IS (Montelukast D6) Blank+IS (Montelukast D6) Blank+IS (Montelukast D6) Mean Response of Montelukast in presence of Montelukast D6 0.0 Mean Response of Montelukast in SEL- LLOQ % Interference at RT of Montelukast in Presence of Montelukast D6 0.0 Table: 3 Selectivity Data for Montelukast (ULOQ Area) Montelukast Sample ID Montelukast Area IS Area ULOQ (Montelukast) ULOQ (Montelukast) ULOQ (Montelukast) ULOQ (Montelukast) ULOQ (Montelukast) ULOQ (Montelukast) Mean Response of Montelukast D6 in presence of Montelukast Mean Response of Montelukast D6 in SEL-LLOQ % Interference at RT of Montelukast in Presence of Montelukast D Calculation: % Interference of Analyte and IS=Mean Interference at RT of Analytes and ISX 100 Average response in Selectivity LLOQ 246

30 ii) Sensitivity The lowest limit of reliable quantification for Montelukast in human plasma was set at the concentration of the LLOQ, 5.032ng/mL. The precision and accuracy for Montelukast at this concentration was found to be 3.10% and 97.24%. No statistical outlier was found. The results were given in Table: 4. 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: 4 Concentration-response Linearity Data for Sensitivity of Montelukast Montelukast Concentration (ng/ml) STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I Upper Limit Lower Limit Slope Intercept R 2 CC# % Nominal

31 iii) Matrix effect Matrix effect for Montelukast was evaluated by analyzing all the eight batches of plasma at low (LQC) and high (HQC) concentrations. No significant matrix effect was observed in all the eight batches of plasma for Montelukast at low (LQC) and high (HQC) concentrations. The precision for IS normalized matrix factor at LQC and HQC level was found to be 0.69% and 0.61%, respectively. The results were within the acceptable limits and given in tables 5 & 6. Table: 5 Matrix Effect of Montelukast in Absence of Matrix Ions Montelukast LQC HQC AQ-ME Area of Analyte Area of Internal Standard Area Ratio Area of Analyte Area of Internal Standard Area Ratio Mean Mean

32 Table: 6 Matrix Effect of Montelukast in Presence of Matrix Ions Montelukast LQC HQC Matrix Lot. No. Blank (P ) Blank (P ) Blank (P ) Blank (P ) Blank (P ) Blank (P ) Blank (P H, Lipemic) Blank (P K Hemolytic) Area of Analyte Area of Internal Standard Area Ratio IS Normalized MatrixFactor Area of Analyte Area of Internal Standard Area Ratio IS Normalized MatrixFactor Mean SD % CV N 8 8 Calculation:IS normalized Matrix Factor = Peak response area ratio in presence of matrix ions Mean peak response area ratio in absence of matrix ions. Note:No Statistical Outlier was found 249

33 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 Montelukast. The calibration line was linear in the range of 5.032ng/mL to ng/mL of the drug as shown in Fig:11. 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 2 ) was greater than 0.99 and ranged from to for Montelukast. 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 consecutive CCs must fail to meet the above criteria. 75% or atleast 6 non zero CCs including LLOQ & highest concentration must meet above criteria. 250

34 Table : 7 Concentration-response Linearity Data of Montelukast Concentration (ng/ml) Montelukast STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I Slope Intercept r 2 Upper Limit Lower Limit CC# Mean SD CV% % Nominal N

35 Fig: 11 A Representative Calibration Curve for Regression Analysis of Montelukast PA BATCH-1 AND SENSITIVITY.rdb (Montelukast): "Linear" Regression ("1 / (x * x)" wei Analyte Area / IS Area Analyte Conc. / IS Conc. 252

36 v) Precision and Accuracy The precision of the assay was measured by the percent coefficient of variation for QC samples of Montelukast. 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 Montelukast (% CV) Within-batch precision for LLOQ QC ranged from 4.34 to 7.62% Within-batch precision for LQC, MQC1, MQC2 and HQC ranged from 0.93 to 7.47% Intra-day Precision for Montelukast (% CV) Intra-day precision for LLOQ QC was 6.90% Intra-day precision for LQC, MQC1, MQC2 and HQC ranged from 4.67 to 6.24% Between-batch Precision for Montelukast (% CV) Between-batch precision for LLOQ QC was 6.69% Between-batch precision for LQC, MQC1, MQC2 and HQC ranged from 4.69 to 8.78% Within-batch Accuracy for Montelukast (% 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 Montelukast (% Nominal) Intra-day accuracy for LLOQ QC was % Intra-day accuracy for LQC, MQC1, MQC2 and HQC ranged from to % Between-batch Accuracy for Montelukast (% Nominal) Between-batch accuracy for LLOQ QC was % Between-batch accuracy for LQC, MQC1, MQC2 and HQC ranged from to % 253

37 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: 8 Within Batch Precision and Accuracy for Montelukast Concentration (ng/ml) MONTELUKAST LLOQ QC LQC MQC1 MQC2 HQC Upper Limit Lower Limit QC# * * * * Mean S.D C.V.% % Nominal N * * * Mean S.D C.V.% % Nominal N