Estimation and Stress Study of a Potent Anticoagulant Drug Rivaroxaban by a Validated HPLC Method

Transcription

1 2 ESTIMATION AND STRESS STUDY OF A POTENT ANTICOAGULANT DRUG RIVAROXABAN BY A VALIDATED HPLC METHOD II Estimation and Stress Study of a Potent Anticoagulant Drug Rivaroxaban by a Validated HPLC Method 21

2 2.1 INTRODUCTION OF DRUG Rivaroxaban is chemically known as 5-Chloro-N-({(5S)-2-oxo-3-[4-(3-oxo-4- morpholinyl) phenyl]-1,3-oxazolidin-5-yl}methyl)-2thiophene carbox- mide. The molecular formula of Rivaroxaban is C 19 H 18 ClN 3 O 5 S and the molecular weight is g/mol. Rivaroxaban is an oral anticoagulant substance that prevents coagulation (clotting) of blood. It is an odorless, non-hygroscopic, white to yellowish powder and pure (S)- enantiomer. Rivaroxaban is an oral, direct Factor Xa inhibitor that has been shown to exhibit well-defined pharmacokinetic and pharmacodynamic properties, with high oral bioavailability [1]. It is invented and manufactured by Bayer and marketed as Xarelto [2]. Each Xarelto tablet contains 10 mg, 15 mg, or 20 mg of Rivaroxaban. The inactive ingredients of Xarelto are croscarmellose sodium, hypromellose, lactose monohydrate, magnesium stearate, microcrystalline cellulose, and sodium lauryl sulfate [3]. In the United States It is marketed by Janssen Pharmaceutica [4]. Rivaroxaban is the first available orally active direct factor Xa inhibitor from class of oxazolidinone derivative and it has been optimized for inhibiting both free factor Xa and factor Xa bound in the prothrombinase complex [5]. Rivaroxaban is indicated for the prevention of deep vein thrombosis which may lead to pulmonary embolism in patients undergoing knee or hip replacement surgery. The most common adverse reactions with Rivaroxaban are bleeding complications, including major bleeding events, fainting, itching, and muscle spasms have been reported [6]. The introduction of this drug in a surgical department represents a standard change, this drug is not administered preoperatively but postoperatively, and is not given subcutaneously but orally. Rivaroxaban inhibits free and fibrin-bound factor Xa as well as factor Xa in the prothrombinase complex, therefore it prevents clot formation and clot growth. Figure 1: Structural formula of Rivaroxaban 22

3 2.2 LITERATURE REVIEW The literature reviews regarding Rivaroxaban suggested that various analytical methods are reported for estimation of Rivaroxaban in bulk as well as in pharmaceutical formulations and in various biological fluids by using various analytical techniques such as HPLC, HPTLC and Spectrophometric method. The literature reviews regarding these methods are as under. a) Satyanarayana PV and Madhavi AS have been developed and validated RP HPLC method for the analysis of Rivaroxaban in pharmaceutical dosage form. The separation was achieved on symmetry C18 column. The isocratic mobile phase consisted THF: MeOH: ACN (10:40:50, v/v/v). The detection was monitored at 270nm with 1ml/min flow rate. The linear calibration curve was obtained in the concentration range between 10-40ppm with a coefficient of correlation [7]. b) Mustafa C, Tuba R, Engine K and Sacide A have reported validated RP-HPLC method for estimation of Rivaroxaban in pharmaceutical dosage forms. The separation was achieved by Phenomenex Luna C18 (250mm X 4.6mm, 5µm) column with 40 C column oven temperature. The isocratic elution was performed with ACN: water (55:45, v/v). The UV detection was achieved at 249nm wavelength with 1.2ml/min flow rate. The linear calibration curve was obtained in the concentration range between 5-40 ppm with a coefficient of correlation [8] c) Lories IB, Mostafa AA and Griges MA have developed HPLC, TLC densitometry, first derivative and first derivative ratio spectrophotometry method for determination of Rivaroxaban with alkaline degradation in bulk powder and tablets. The HPLC separation was achieved by C18 column with isocratic mobile phase consisted 1.2% w/v Potassium dihydrogen phosphate (ph 3.5) and ACN (70:30, v/v), ph was adjusted by using OPA. The flow rate was adjusted at 1.5ml/min with 20µl injection volume. The detection was monitored at 280 nm. The TLC separation was achieved by using chloroform-isobutyl alcohol (50:50, v/v) solvent system. The first derivative measurement of the drug was monitored at 237.4nm [9] 23

4 d) Pinaz AK and Muralikrishna KS have developed RP-HPLC method of Rivaroxaban with photolytic, acid and base degradation study. An isocratic separation was achieved using a Phenomenex C A (250mm 4.6mm, 5µm) column, with a flow rate of 1ml/min. The elution was monitored at 250nm with PDA detector. The mobile phase consisted of MeOH: ACN (50:50, v/v). The method was validated for specificity, linearity, precision, accuracy and robustness. The method was linear over the concentration range of µg/ml with r 2 = The drug substance was subjected to thermal and photolytic conditions [10][11][12] e) Chandrasekhar K, Satyavani P, Dhanalakshmi A, Devi ChLL, Anupama B and Narendra D have developed and validated RP-HPLC method for analysis of Rivaroxaban in formulations. The separation was achieved by using C-18 (250mm X 4.6mm, 5µm) column at ambient temperature with mobile phase consisted ACN: MeOH: 0.1% OPA (90:8:2, v/v/v) with the ph The flow rate was adjusted at 1.5ml/min and UV detection was performed at 234nm by UV detector. The linear calibration curve was obtained in the concentration range between ppm with a coefficient of correlation [13] f) Chandrabala S, Hima VB, Rupa MD and Sireesha A have developed and validated UV spectroscopic method for the determination of Rivaroxaban. The drug was estimated at 270 nm in DMSO. The linearity range was found between the concentration range 2 20 ppm with coefficient of correlation The regression equation is, Absorbance = X concentration of drug in µg/ml The apparent molar absorptivity and sandell s sensitivity were found to be L mol 1 cm 1 and µg cm 2, respectively [14]. g) Darshna V and Pinak P have reported HPTLC method with densitometry analysis for determination of Rivaroxaban from its tablet dosage form. The drug was spotted on silica gel F254 TLC plates under pure nitrogen stream by Linomat TLC spotter. Separation was carried out by using Methanol, Toluene, and Tri ethanolamine as mobile phase in ratio of (7:2.5:0.5,v/v/v). Developed TLC plates were scanned by CAMAG TLC 24

5 scanner and detection was carried out at 249nm. The Rf value of separated drug was found at The linearity curve of Rivaroxaban was observed in the concentration range between 500 to 3000ng/spot [15]. h) Schmitz EMH, Heuvel DV, Boonen K, Dongen JLJ, Brunsveld L and Kerkhof DV have developed the method for simultaneously determination of Dabigatran, Rivaroxaban and Apixaban using UPLC-MS/MS and comparison with coagulation assays for therapy monitoring. In this method plasma and full blood were spiked with Dabigatran, Rivaroxaban and Apixaban for a 6-point calibration (23-750ng/ml) with correlation coefficient No significant differences could be seen between plasma and full blood calibration lines. Additionally, no bias was found in spiked hemolytic, icteric and lipemic plasma. Stability tests showed adequate stability during three freeze-thaw cycles, 24-hours plasma storage at 20 C and 8-day sample storage at 4 C. Specificity of the UPLC-MS/MS was good and LLOD and LLOQ were <1ng/mL for all three drugs. For Dabigatran, Rivaroxaban and Apixaban, the relative recovery was 73%, 78% and 104%, respectively. This was considered acceptable, due to the addition of the internal standards. Acceptance criteria for accuracy and precision were applied as given by the FDA (100±15% and <15%, respectively) [16]. i) Pinaz AK and Muralikrishna KS have design dissolution profile of Rivaroxaban with validation by using RP-HPLC in dosage form. The conditions established for dissolution were: 900ml of Acetate buffer (ph 4.5) and 0.2 % SLS as dissolution medium, using a paddle type dissolution apparatus at a stirring rate of 75rpm which was able to give drug release of 98.86%. The drug release was evaluated by RP-HPLC method at 250nm and a good linearity was observed in the concentration range of 20-60µg/mL with correlation coefficient of The percentage recovery of Rivaroxaban was found to be The %CV (0.22%; n=6) indicated a good precision of the analytical method. Robustness of the method was performed by using different rotation speeds and temperatures. The validation parameters included linearity, accuracy, precision and robustness [17] 25

6 j) Pinaz AK and Muralikrishna KS have noted spectrophotometric method for determination of Rivaroxaban simultaneously for bulk and tablet formulation. The study was conducted in MeOH between the wavelengths of nm. The developed method was linear in the concentration range of 2-12µg/ml with correlation coefficient and respectively for bulk and tablet dosage form. The drug recovery was 99.31% in bulk drug analysis and in tablet dosage form it was %. The LOD were found at 0.059µg/ml and µg/ml, The LOQ were found 0.179µg/ml and 0.298µg/ml respectively for bulk and tablet dosage form [18][19]. k) Satyanarayana PVV and Madhavi AS have reported spectrophotometric method validation for determination of Rivaroxaban in bulk and tablet formulation. In this proposed research work five spectrophotometric methods such as 2-2 Bipyridine method (M1), 4-Amino phenazone method (M2), Haematoxylin method (M3), Iso nicotanic hydrazide method (M4), Naptha quinone sulphate method (M5) have been developed and applied for routine determination of Rivaroxaban in pharmaceutical formulations and bulk dosage forms. These methods are based on the color formation of the drug on binding with the different reagents. All these method have different linearity ranges between 2-12, 3-21, 30-90, 5-30 and 15-90ppm respectively for M1, M2, M3, M4 and M5 methods. The recovery of drugs were found %, 99.61%, %, 99.94% and % respectively for M1, M2, M3, M4 and M5 methods [20]. l) Rohde G has reported HPLC MS/MS method for determination of Rivaroxaban in human plasma. After precipitation of plasma proteins with methanol containing the internal standard followed by centrifugation, the plasma supernatant was injected directly onto the HPLC MS/MS system. The concentrations could be determined between µg/l. The Inter-assay precision was 7.4% and inter-assay accuracy was between 96.3 and 102.9% throughout the entire working range [21]. m) Vijayabhaskar N and Sekhar reddy BC have reported a novel RP-HPLC method for the quantification of Rivaroxaban in formulations. The Isocratic elution was achieved on a Kromasil C18 (250mmx4.6mm, 5µm) column with a flow rate of 1ml/min and at 26

7 ambient temperature. The mobile phase consisted of MeOH: ACN (80:20, v/v) (ph 4.4). The UV detection wavelength was maintained at 273nm with 20µl injection volumn. The percentage RSD for precision and accuracy of the developed method was found to be less than 2% [22]. 2.3 AIM OF PRESENT WORK The literature survey indicates, there are many methods are available for the estimation of Rivaroxaban as a drug substance as well as in pharmaceutical dosage form, few of the methods are also available which deals with the bioanalytical study. But all of these are not stability indicating. Most of the reported methods either do not include stress degradation studies or are not completely validated and they are time consuming and expensive. Furthermore these methods are not impressionable to achieve the high throughput study which can be possible by optimizing the method in such a way which includes shortest run time with maximum selectivity. The primary object of the present work was to develop and validate a stability indicating HPLC method for estimation of Rivaroxaban under stress condition such as acid and base hydrolysis, oxidative, thermal and photolytic conditions. Hence, it can be maximum utilize for the analysis of formulation development and stability testing as well as at quality control laboratory for routine use. The aim and scope of the proposed work are, to develop rapid and simplest RP-HPLC method for quantification and estimation of the drug substance with highest selectivity. To perform stress degradation study for confirm the stability of the drug substance. The analytical method validation for the proposed method was performed as per ICH guideline [23][24][25]. 2.4 EXPERIMENTAL CONDITION Materials The Reference standard of Rivaroxaban was provided as a gift sample for research purpose by Anlon Research Organization, Rajkot, India and tablet dosage form was purchased from local market. HPLC grade Acetonitrile was purchased from Merck India Limited, Mumbai, India and HPLC grade Orthophosphoric acid was obtained from Spectrochem Mumbai, India. Analytical grade Hydrochloric acid, Sodium hydroxide 27

8 pellets and Hydrogen peroxide were purchased from Ranbaxy fine chemicals, New Delhi, India. High purity deionised water was obtained from Milli-Q (Millipore, Miliford, MA, USA) purification system µm membrane filters were purchased from Pall Life Sciences, Mumbai, India and nylon syringe filters 0.45 µm were purchased from Millex- Hn, Mumbai, India Instrumentation Chromatographic separation was carried out using a HPLC system (Waters 2489, miliford, USA), consisted a quaternary gradient pump (TM 600), Rheodyne manual injector with 20µl loop, column oven and UV detector employed for analysis. The output signal was monitored and processed by Empower 2.0 version software. A microbalance obtained by LCGC Radwag weighing solution Pvt. Ltd. Mumbai, India, An ultra sonic water bath SONICA from Spincotech Pvt. Ltd. Mumbai, India and ph meter LI 610 ELICO Mumbai, India were also used Chromatographic condition The chromatographic analysis was performed by Phenomenax C8 100A (250mm X 4.6mm id, 5µm particle size) column with 30 C column oven temperature. The isocratic mobile phase was consisted 0.1% OPA: ACN (60:40, v/v) and the detection were monitored at wavelength of 280nm. The flow rate was adjusted at 1ml/min with 20µl injection volume. The total analysis time was selected 15minute Mobile phase preparation The mobile phase was consisted 0.1% Orthophosphoric acid and Acetonitrile (60:40, v/v), before use degassed the mobile phase by ultrasonic bath Diluent preparation Water: Methanol (50:50, v/v) used as a diluent Stock solution preparation The stock solution of 500µg/ml Rivaroxaban was prepared by dissolving accurately weighted 50mg of Rivaroxaban working standard in 100ml volumetric flask. Add 50ml of diluent (water: MeOH (50:50, v/v)) to 100ml volumetric flask and dissolve the 28

9 substance by sonication about 10 to 15 minute. The solution was dilute up to mark with diluent. For standard solution preparation pipette out 5ml of above stock solution into 50ml volumetric flask and dilute up to the mark with diluent. This solution contains 50µg/ml Rivaroxaban Test solution preparation The preparation of 500µg/ml stock solution, twenty tablets were accurately crushed, weighted and average weight has been calculated. The portion of powder equivalent to the weight of five tablets has been taken and transferred to a 100ml volumetric flask. Add 50ml of diluent (water: MeOH (50:50, v/v)) to the flask and dissolve the substance by sonication around 10 to 15 minute. The solution was dilute up to mark with diluent. Filter the solution with 0.45µm membrane filter. For test solution preparation pipette out 5ml of above stock solution into 50ml volumetric flask and dilute up to the mark with diluent. This solution contains 50µg/ml Rivaroxaban. 2.5 RESULT AND DISCUSSION Development and optimization of the HPLC method This chapter describes assay method development and validation of Rivaroxaban tablet with forced degradation study. For accurate, precise and robust method development, the analytical conditions were selected after testing the different parameters that influence LC analysis, such as analytical column, detection wavelength, aqueous and organic mobile phase, mobile phase proportion, analyte concentration, injection volume, flow rate and other factors were exhaustively studied. Phenomenax C8 100A (250mm X 4.6mm id, 5µm particle size) column was used because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. For mobile phase selection, the preliminary trials using different compositions of mobile phases consisting of water and ACN (50:50, v/v) gave poor peak shape. The representative chromatogram for the same is shown as under. 29

10 Figure 2: Chromatogram of standard preparation [water: ACN (50: 50v/v)] Above chromatogram clearly indicate that the peak is not symmetrical and value of theoretical plates is lower side. In focus to develop good symmetrical peak, water was replaced by 0.1% OPA. Thus, better peak shape was obtained. The representative chromatogram for the same is shown as under Figure 3: Chromatogram of standard preparation [0.1% OPA: ACN (50:50, v/v)] Further, the mobile phase proportion was optimized to retain analyte properly that provide good resolution between Rivaroxaban and its degradation impurities. Proportion of ACN is finalized to 40% of the mobile phase. The detection wavelength was decided after screened the standard solution over nm using the advantage of photo diode array detector. On the basis of peak absorption maxima and peak purity index, the 280nm was decided as the detection wavelength which was provided the maximum chromatographic compatibility to the method. As a diluent the mixture of water-methanol (50:50, v/v) was made. Injection volume was fixed to 20µl and the flow rate of the 30

11 mobile phase was set to 2ml/min. On this finalized chromatographic condition, obtained chromatogram was having of good peak symmetry and higher theoretical plates. The representative chromatogram for the same is shown as under Figure 4: Chromatogram of standard preparation [0.1% OPA: ACN (60:40, v/v)] Figure 5: Chromatogram of test preparation [0.1% OPA: ACN (60:40, v/v)] The drug substance was easily extracted from pharmaceutical dosage using diluent as water-meoh (50:50, v/v). Tablet was easily isolated using water and MeOH. The drug substance is freely to very soluble in MeOH. Extraction trials are finalized to keep sonication time for around minute. Solutions of standard preparation and test preparation were found stable in diluent. All validation parameters were performed by taking same diluent concentration Stress degradation study Degradation study was performed by transferring powdered tablets equivalent to 50mg Rivaroxaban into 250ml round bottom flask. Then prepared samples were employed for 31

12 acidic, alkaline, oxidative, thermal and photolytic conditions. After the degradation treatments were completed, the stress content solutions were allowed to equilibrate to room temperature and diluted with mobile phase to attain 50µg/ml concentrations of Rivaroxaban. The placebo was also subjected to same stress conditions to identify any response due to the forced degradation conditions. Specific conditions were described as follow. a) Acidic condition Acidic degradation study was performed by heating the drug content with 20ml 0.5N HCl at 50ºC for 30 minute. After heating equilibrate the solution at room temperature and the mixture was neutralized with 0.5N NaOH solutions. Further the mixture was diluted with diluents to achieve 50µg/ml drug concentration. The resulting chromatogram obtained by acidic degradation described the drug was degraded around 13%. The major impurity peak was found at 3.6 minute and the degradation product resulting from the stress studies did not interfere with the detection of Rivaroxaban Figure 6: Chromatogram obtained by acidic stress degradation study b) Alkali condition Alkaline degradation study was performed by heating the drug content with 10ml 0.01N NaOH for 20 minutes at 60ºC. After heating equilibrate the solution at room temperature and the mixture was neutralized with 0.01N HCl solutions. Further the mixture was diluted with diluents to achieve 50µg/ml drug concentration. The resulting chromatogram obtained by alkali degradation described the major degradation was found in alkali condition and the drug was degraded up to 50%. The major impurity peak was found at 32

13 2.5 minute and the degradation products resulting from the stress studies did not interfere with the detection of Rivaroxaban Figure 7: Chromatogram obtained by alkali stress degradation study c) Oxidative condition Oxidation degradation study was performed by heating the drug content with 30% (v/v) H 2 O 2 at 80 C for 90 minute. Further the mixture was diluted with diluents to achieve 50µg/ml drug concentration. The resulting chromatogram obtained by oxidative degradation described the drug was degraded around 6% and separate degraded product was not found by thermal degradation Figure 8: Chromatogram of oxidative stress degradation study d) Thermal condition Thermal degradation was performed by exposing solid drug at 80ºC for 36 hour in hot air oven. The resulting chromatogram obtained by thermal degradation described the drug 33

14 was degraded around 3% and separate degraded product was not found by thermal degradation Figure 9: Chromatogram obtained by thermal stress degradation study e) Photolytic condition Photolytic degradation study was performed by exposing powder drug directly to sunlight for 72 hour (day hour). It was found that around 1.5% of the drug was degrading in photolytic condition and separate degraded product was not found by photolytic degradation Figure 10: Chromatogram of photolytic stress degradation study From the above chromatogram it can be concluded that there is no interference of any degradation product to the peak of interest and impurity has been generated by each stress condition. 34

15 2.5.3 Method validation parameters The developed LC method for determination of Rivaroxaban in bulk drug as well as in pharmaceutical dosage form is further validated as per ICH guidelines. The method was validated by several validation parameters such as Accuracy, Precision, Linearity, Limit of Detection, Limit of Quantitation, Robustness and Specificity. Whole validation was performed as per ICH guideline Q2A, Q2B and Q2(R1) [23][24][25] to ensuring that the present method was suitable for its intended purpose Specificity study The specificity of the method was evaluated by checking the interference of placebo components with drug. The additives of tablet are practically insoluble in diluent whereas the active pharmaceutical ingredients are freely soluble. So, the active pharmaceutical ingredient is easily isolated from placebo. Further the specificity of the method toward the drug was established by means of the interference of diluent at the retention time of the drug peak, and the specificity of the method toward the drug was also established by means of the interference of the degradation products against drug during the forced degradation study. From the specificity experiment and subsequently achieved chromatograms it has proven that any interference was not observed from blank or placebo to the peak of interest, Hence it authenticate there was no interference of any peak of degradation product with drug peak. So, the proposed method is highly specific with respect to the diluent and excipients in the commercial sample. Data for standard preparation Data for test preparation Replicate Area Replicate Area Average Standard weight (mg) Test weight (mg) Mean Mean test weight (mg) Stdv Label claim 10 % RSD 0.32 % Assay Table 1: Specificity study standard and test preparation data 35

16 Figure 11: Chromatogram of diluent obtained by specificity experiment Figure 12: Chromatogram of placebo obtained by specificity experiment Figure 13: Chromatogram of test preparation obtained by specificity experiment Prototype calculation formula for relative standard deviation is as under: % #$%= $&'()'*) )+,-'&-.( /+'(,'

17 % #$%= % #$%=0.32 Prototype calculation formula for assay determination is as under: % ;<<'== ;> ;$ 2?$?> 2 B %;<<'== % ;<<'== Where, AT = Average Area of Test Preparation. AS = Average Area of Standard Preparation. WS = Weight of Working Standard (mg). WT = Weight of Test Sample (mg). AW = Average Weight of Formulation (mg). LC = Label Claim Weight of Formulation (mg). P = Potency of Working Standard (%) Linearity study The linearity study was determined by analyzing seven solutions in the concentration range between 20-80µg/ml (20, 30, 40, 50, 60, 70 and 80µg/ml of Rivaroxaban). These concentration levels were respectively corresponding to 40, 60, 80, 100, 120, 140 and 160% of test solution concentration. The plot of peak areas against concentration data were evaluated by linear regression analysis. The response of the drug was found to be linear in the investigation and the correlation coefficient of the linearity study was with linear regression equation y=21577x , where y is the peak area in absorbance units; x is the concentration in µg/ml. which proves the method is highly linear over the working range between 20-80µg/ml. 37

18 Data for standard preparation Replicate Standard peak area Mean Std. dev % RSD 0.12 Table 2: Linearity study standard preparation Linearity Level Replicate Area Mean area Level (40%) Level (60%) Level (80%) Level (100%) Level (120%) Level (140%) Level (160%) Table 3: Linearity study level preparation data 38

19 Concentration level (%) Volume of linearity stock solution taken (ml) Diluted to (ml) Final concentration (µg/ml) Correlation co-efficient Slope Intercept Mean area Table 4: Linearity study concentration Vs peak area response data X axis = Concentration (µg/ml), Y axis = Peak Area in absorbance units Chart 1: Linearity curve for Rivaroxaban 39

20 Figure 14: Chromatogram of linearity level-1 (40%) Figure 15: Chromatogram of linearity level-2 (60%) Figure 16: Chromatogram of linearity level -3 (80%) 40

21 Figure 17: Chromatogram of linearity level-4 (100%) Figure 18: Chromatogram of linearity level-5 (120%) Figure 19: Chromatogram of linearity level-6 (140%) 41

22 Figure 20: Chromatogram of linearity level-7 (160%) Prototype calculation formula for relative standard deviation is as under: % #$%= $&'()'*) )+,-'&-.( /+'(,' % #$%= % #$%= Limit of Detection & Limit of Quantitation study LOD is the lowest amount of the drug content which can be detected by the proposed method while LOQ is the lowest amount which can be quantified by the method. The guideline suggest minimum signal to noise ratio (S/N) more than 3.3 for LOD and more than 10 for LOQ. The LOD and LOQ concentrations were found at 0.08µg/ml and 0.175µg/ml respectively. It has been established by evaluating the minimum level at which the analyte could be readily detected and quantified accurately. On the basis of linearity data theoretically it can be also calculated by the given formula. LOD = 3.3(σ /S) LOQ = 10(σ /S) Where, σ = Standard deviation of regression line S = Slope of the calibration curve 42

23 All the results of LOD and LOQ data were within the acceptance criteria, hence it can be conclude that the LOD and LOQ of the method was 0.08µg/ml and 0.175µg/ml respectively which correspond to 0.16% and 0.35% of working concentration. Data for standard preparation Data for LOQ preparation Replicate Peak area Replicate Peak area Mean Mean 4015 Stdev Stdev % RSD 0.33 % RSD 1.11 Table 5: LOQ and standard preparation data Linearity level Concentration level (%) Final concentration (µg/ml) Mean area 1 LOQ Correlation co-efficient Slope Intercept Table 6: LOQ study by evaluating linearity up to LOQ concentration LOQ of the analytical method can evaluated by establish linearity up to LOQ value. Hence linearity study is extended to LOQ value, 43

24 X axis = Concentration (µg/ml) Y axis = Peak Area Chart 2: Regression analysis chart of linearity study included with LOQ level Figure 21: Chromatogram obtained by limit of detection study 44