![International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470
@ IJTSRD | Unique Paper ID - IJTSRD23582 | Volume – 3 | Issue – 4 | May-Jun 2019 Page: 327
Observation: Crizotinib eluted with good peak shape and
retention time and tailing was passed
Intermediate precision: Six working sample solutions of
100 ppm are injected on the next day of the preparation of
samples and the % Amount found was calculated and %RSD
was found to be 0.93 and chromatogram was shown in fig.3.
Table3. Data of Intermediate precision
Sl. No Peak Area
1 1439917
2 1470663
3 1472005
4 1477149
5 1473073
6 1462129
Average 1465823
Standard deviation 13618.0
% RSD 0.93
Method of Linearity: To demonstrate the linearity of assay
method, inject 5 standard solutions with concentrations of
about 37.5 ppm to 225 ppm of Crizotinib. Plot a graph to
concentration versus peak area. Slope obtained was 21035
Y-Intercept was 892.5 and Correlation Co-efficient was
found to be 0.999 and Linearity plot.
Table4. Linearity Concentration and Response
Table5. Summary of results
Parameters Crizotinib
Calibration range (mcg/ml) 25-150 ppm
Optimized wave length 267 nm
Retention time 2.563 min
Regression equation (Y) y = 16091x + 1403.4
Correlation coefficient (r
2
) 0.999
Precision (% RSD*) 1.26
Percentage Recovery 100.24
Limit of Detection (µg/ml) 0.080
Limit of Quantization (µg/ml) 0.243
CONCLUSION
Chromatographic conditions used are stationary phase BDS
250 x 4.6 mm, 5µ. Mobile phase buffer: Acetonitrile in the
ratio of 60:40 and flow rate was maintained at 1ml/min,
detection wave length was267 nm,column temperaturewas
set to 30o C and diluents was methanol: Water (50:50),
Conditions were finalized as optimized method. System
suitability parameters were studied by injecting the
standard five times and results were well under the
acceptance criteria. Linearity study wascarriedoutbetween
25 % to150 % levels, r2 value was found to be as 0.999.
Precision was found to be 1.26 for repeatability and 0.93 for
intermediate precision. LOD and LOQ are 0.080 µg/ml and
0.243 µg/ml respectively. By using above method assay of
marketed formulation was carried out 100.24 % was
present.
ACKNOWLEDGEMENT
I am very thankful to Mr. L. Matsyagiri, Associate Professor,
Swami Vivekananda Institute of Pharmaceutical Sciences,
Vangapally, Yadagirigutta, Yadadri Bhongir-506286,
Telangana, India, for his support for the study.
REFERENCES
[1] Sethi PD., Quantitative analysis of Drugs &
Pharmaceuticals. CBS publishersanddistributors, New
Delhi, 3rd edition, 2001 Pg. no 1-120.
[2] Crizotinib.
https://pubchem.ncbi.nlm.nih.gov/compound
(accessed Oct 21, 2015).
[3] Ramachandra and N.V.S. Naidu, Validation of RP -HPLC
Method for Estimation of Dasatinib In Bulk and Its
Pharmaceutical Dosage Forms, Internationaljournalof
pharmacy and biological sciences, 2014; 61-68: 2230-
7605.
[4] Kishore konam & Dharmeshwar jadhav, Development
and validation of a RP- HPLC methodfordetermination
of dronedarone in bulk and pharmaceutical
formulation, International Journal of Pharmacy and
Biological Sciences, 2014; 4(1): 179-185.
[5] InternationalConferenceon Harmonization(ICH),Q2A.
“Validation of Analytical Procedures: Methodology”.
Geneva, 1994.
[6] Skoog et al., Principles of Instrumental Analysis.
Barkhanath publishers, 8th edition, Pg. no. 973-995.
[7] Prapulla Putta, Analytical methods Development and
Validation of Crizotinib by RP-HPLC Technique,
International Journal of Biochemistry and
Pharmacology, 2019; 1(1); 1-4.
[8] Selvakumar. S, Ravichandran. S, Matsyagiri. L,
Development and Validation of analytical method for
Simultaneous estimation of Ornidazole and Cefixime
trihydrate tablet dosage forms by UV spectroscopy.
Asian J. Pharm. Ana. 2016; 6(4): 246-252.
[9] Chatwal GR, and Anand SK. Instrumental Methods of
Chemical Analysis. 5th Revised and Enlarged ed.,
Himalaya Publication House, Mumbai, 2002; 2: 630.
[10] Matsyagiri. L, Jagadeesh. P, Mounika. B, Srinivas. V,
Theja.V, Madhiha Jabeen and Dr. K. Hemamalini,Effect
of solvents on Spectrophotometric Estimation of
Tinidazole in bulk and dosage forms, World Journal of
Pharmaceutical Research, 2018; 7(9): 1742-1754.
[11] JadhavPB, Vivek Shejwal, Development and Validation
of an RP-HPLC Method for Crizotinib, International
Journal of Pharmacy and Pharmaceutical Research.
Human, 2017; 9(2):100-106.
Sl. No
Linearity
Level (%)
Concentration
(ppm)
Area
1 0 0 0
2 25 25 400600
3 50 50 808783
4 75 75 1203917
5 100 100 1624787
6 125 125 2009776
7 150 150 2409818](https://image.slidesharecdn.com/69analyticalmethodsdevelopmentandvalidationofnaproxenandsumatriptanbyrphplc-190705052436/85/Analytical-Methods-Development-and-Validation-of-Naproxen-and-Sumatriptan-by-RP-HPLC-3-320.jpg)
Analytical Methods Development and Validation of Naproxen and Sumatriptan by RP HPLC
- 1. International Journal of Trend in Scientific Research and Development (IJTSRD) Volume: 3 | Issue: 4 | May-Jun 2019 Available Online: www.ijtsrd.com e-ISSN: 2456 - 6470 @ IJTSRD | Unique Paper ID – IJTSRD23582 | Volume – 3 | Issue – 4 | May-Jun 2019 Page: 325 Analytical Methods Development and Validation of Naproxen and Sumatriptan by RP HPLC Prapulla Putta Associate Professor, Department of Pharmaceutical Chemistry, Mother Teresa College of Pharmacy, N.F.C Nagar, Ghatkesar, Medchel, Telangana, India How to cite this paper: Prapulla Putta "Analytical Methods Development and Validationof Naproxenand Sumatriptan by RP HPLC" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456- 6470, Volume-3 | Issue-4, June 2019, pp.325-327, URL: https://www.ijtsrd.c om/papers/ijtsrd23 582.pdf Copyright © 2019 by author(s) and International Journal of Trend in Scientific Research and Development Journal. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0) (http://creativecommons.org/licenses/ by/4.0) ABSTRACT Aim and objective: A simple, précised, accurate method was developed for the estimation of Crizotinib by RP-HPLC technique. Chromatographic conditions used are stationary phase BDS 250x4.6 mm, 5µ. Mobile phase buffer: Methodology: Acetonitrile in the ratio of 60;40 and flow rate was maintained at1ml/min, detection wave length was 267 nm, column temperature was set to 30o C and diluents was methanol: water System suitability parameters were studied by injecting the standard five times and results were well (50:50), Results & Discussion: Conditions were finalized as optimized method under acceptance criteria. Linearity study was carried out between 25 % to 150 % levels, r2 value was found to be as 0.999. Precision was found to be 1.26 for repeatability and 0.93 for intermediate precision.LOD andLOQare0.080 ug/ml. By using above method assay of marketedformulation wascarriedout100.24 % was present. Keywords: Crizotinib, HPLC, ICH Guidelines, Method development INTRODUCTION Chromatography is a method of separating a mixture of components into individual components through equilibrium distribution between two phases1. The technique of chromatography is based on the differences in the rate atwhich components of a mixture move through a porous medium (stationary phase) under the influence of some solvent or gas (mobile phase)2. Crizotinib: Crizotinib, is an anti-cancer drugactingasanALK (anaplastic lymphoma kinase) and ROS1 (c-rosoncogene1) inhibitor3. The aim and objective of the work is to develop and validate a simple and economical RP-HPLC method as per ICH guidelines for the estimation of Crizotinib in drug and pharmaceutical dosage forms, and to study the linearity of the method. Fig.1- Structure of Crizotinib MATERIALS AND METHODS Crizotinib was obtained as a gift sample from Wintac Ltd., (Bangalore). Acetonitrile served as solvent mixturewasalso obtained from CDH, New Delhi. Allother chemicals/reagents were of analytical grade and were used without further purification. Preparation of Buffer: (0.1% OPA) 1ml of Ortho phosphoric acid solution in a 1000 ml of Volumetric flask add about 100 ml of milli-Q water and final volume make up to 1000 ml with milli-Q water4. Optimized Chromatographic Conditions Column : BDS (250*4.6 μm) Mobile phase : OPA buffer: Acetonitrile (60:40) Flow rate : 1.0 ml/min Detector : PDA 267 nm Temperature : 300 C Injection Volume : 10µL Method of validation: The proposed method was validated for various parameters such as linearityandrange, accuracy, precision, robustness, ruggedness, sensitivityandspecificity according to ICH Q2 (R1) guideline and USP guidelines5. Method of Linearity and range: The linearity of an analytical procedure is its ability (within a given range) to obtain test result which are directly proportional to the concentration of an analyte in the sample. The range of an analytical procedure is the interval between the upper and lower concentration of an analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The linearity of the analyticalmethodwasdemonstratedoverthe IJTSRD23582
- 2. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Paper ID - IJTSRD23582 | Volume – 3 | Issue – 4 | May-Jun 2019 Page: 326 concentration range investigated by triplicate analysis (n = 3) at a concentration range of 2-20 μg/ml. The absorbance obtained at respective concentration was recorded, and the graph is plottedasconcentration(μg/ml) versus absorbance. The linear regression equation and the coefficient correlation were obtained from the UV probe software6. Method of Accuracy: The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. This is sometimes termed trueness. The accuracy of proposed method was determined on the basis of recovery study. Recovery study was carried out by spiking standard working solution to sample solution (formulation) at three different levels 80%, 100% and 120%. The final concentration of Crizotinib was determined at each levelsof the amount; three determinations were performed. The percentage recovery was calculated as mean ± standard deviation7. Method of Precision: The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the homogeneous sample under the prescribed conditions. The precision of the method was demonstrated by intra-day and inter-day variation studies. In the intra-day precision study, three different solutions of same concentration were prepared andanalysed inthesame day (morning, noon and evening), whereas in the inter-day precision study, the solutions of same concentration were prepared and analysed, for three consecutive days, and the absorbance were recorded. All study was performed in triplicates. The result was indicated by calculating percentage RSD8. Method of Robustness: The robustness of an analytical procedure is a measure of its capacity remainsunaffected by small, but deliberate variations in method parameters and provides an indication of its reliabilityduringnormalusage9. Method of Ruggedness: The ruggedness is a degree of reproducibility of test result under verification of condition like a different analyst, different instruments and different days10. Assay Procedure Column is equilibrated for 30 min with mobile phase. 20 µl of diluent as blank was injected into the system and recorded the chromatogram for a run timeof 30min.20µlof standard preparation-1 was injected into the system and recorded the chromatogram for a run timeof 30min.20µlof standard preparation-2 was injected into the system and recorded the chromatogram for a run time of 30 min. Test is valid only when the match factor is in between 0.98 to 1.02. 20 µl of standard preparation-2 into the system was separately injected for four times and recorded each chromatogram for a run time of 30 min. Test is valid only when the five standard preparation-2 injections pass the system suitability11. RESULTS AND DISCUSSIONS Table1. System suitability - System suitability data Sl. No Peak Name RT Area USP Plate Count USP Tailing 1 Crizotinib 2.556 1458811 3949 1.37 2 Crizotinib 2.563 1412479 3927 1.34 3 Crizotinib 2.565 1450647 3811 1.33 4 Crizotinib 2.574 1465621 3719 1.37 5 Crizotinib 2.577 1463605 3784 1.35 6 Crizotinib 2.617 1444216 3885 1.33 Mean 1449230 Standard deviation 19722.4 % RSD 1.4 Fig.2- blank Chromatogram Fig.3- System suitability Chromatogram Method of Precision: Table2. Data of Repeatability Sl. No Peak area 1 1450617 2 1423806 3 1448914 4 1456113 5 1469303 6 1476272 Avg. 1454171 Std. devi 18349.4 % RSD 1.26 Fig.4- Optimized chromatogram
- 3. International Journal of Trend in Scientific Research and Development (IJTSRD) @ www.ijtsrd.com eISSN: 2456-6470 @ IJTSRD | Unique Paper ID - IJTSRD23582 | Volume – 3 | Issue – 4 | May-Jun 2019 Page: 327 Observation: Crizotinib eluted with good peak shape and retention time and tailing was passed Intermediate precision: Six working sample solutions of 100 ppm are injected on the next day of the preparation of samples and the % Amount found was calculated and %RSD was found to be 0.93 and chromatogram was shown in fig.3. Table3. Data of Intermediate precision Sl. No Peak Area 1 1439917 2 1470663 3 1472005 4 1477149 5 1473073 6 1462129 Average 1465823 Standard deviation 13618.0 % RSD 0.93 Method of Linearity: To demonstrate the linearity of assay method, inject 5 standard solutions with concentrations of about 37.5 ppm to 225 ppm of Crizotinib. Plot a graph to concentration versus peak area. Slope obtained was 21035 Y-Intercept was 892.5 and Correlation Co-efficient was found to be 0.999 and Linearity plot. Table4. Linearity Concentration and Response Table5. Summary of results Parameters Crizotinib Calibration range (mcg/ml) 25-150 ppm Optimized wave length 267 nm Retention time 2.563 min Regression equation (Y) y = 16091x + 1403.4 Correlation coefficient (r 2 ) 0.999 Precision (% RSD*) 1.26 Percentage Recovery 100.24 Limit of Detection (µg/ml) 0.080 Limit of Quantization (µg/ml) 0.243 CONCLUSION Chromatographic conditions used are stationary phase BDS 250 x 4.6 mm, 5µ. Mobile phase buffer: Acetonitrile in the ratio of 60:40 and flow rate was maintained at 1ml/min, detection wave length was267 nm,column temperaturewas set to 30o C and diluents was methanol: Water (50:50), Conditions were finalized as optimized method. System suitability parameters were studied by injecting the standard five times and results were well under the acceptance criteria. Linearity study wascarriedoutbetween 25 % to150 % levels, r2 value was found to be as 0.999. Precision was found to be 1.26 for repeatability and 0.93 for intermediate precision. LOD and LOQ are 0.080 µg/ml and 0.243 µg/ml respectively. By using above method assay of marketed formulation was carried out 100.24 % was present. ACKNOWLEDGEMENT I am very thankful to Mr. L. Matsyagiri, Associate Professor, Swami Vivekananda Institute of Pharmaceutical Sciences, Vangapally, Yadagirigutta, Yadadri Bhongir-506286, Telangana, India, for his support for the study. REFERENCES [1] Sethi PD., Quantitative analysis of Drugs & Pharmaceuticals. CBS publishersanddistributors, New Delhi, 3rd edition, 2001 Pg. no 1-120. [2] Crizotinib. https://pubchem.ncbi.nlm.nih.gov/compound (accessed Oct 21, 2015). [3] Ramachandra and N.V.S. Naidu, Validation of RP -HPLC Method for Estimation of Dasatinib In Bulk and Its Pharmaceutical Dosage Forms, Internationaljournalof pharmacy and biological sciences, 2014; 61-68: 2230- 7605. [4] Kishore konam & Dharmeshwar jadhav, Development and validation of a RP- HPLC methodfordetermination of dronedarone in bulk and pharmaceutical formulation, International Journal of Pharmacy and Biological Sciences, 2014; 4(1): 179-185. [5] InternationalConferenceon Harmonization(ICH),Q2A. “Validation of Analytical Procedures: Methodology”. Geneva, 1994. [6] Skoog et al., Principles of Instrumental Analysis. Barkhanath publishers, 8th edition, Pg. no. 973-995. [7] Prapulla Putta, Analytical methods Development and Validation of Crizotinib by RP-HPLC Technique, International Journal of Biochemistry and Pharmacology, 2019; 1(1); 1-4. [8] Selvakumar. S, Ravichandran. S, Matsyagiri. L, Development and Validation of analytical method for Simultaneous estimation of Ornidazole and Cefixime trihydrate tablet dosage forms by UV spectroscopy. Asian J. Pharm. Ana. 2016; 6(4): 246-252. [9] Chatwal GR, and Anand SK. Instrumental Methods of Chemical Analysis. 5th Revised and Enlarged ed., Himalaya Publication House, Mumbai, 2002; 2: 630. [10] Matsyagiri. L, Jagadeesh. P, Mounika. B, Srinivas. V, Theja.V, Madhiha Jabeen and Dr. K. Hemamalini,Effect of solvents on Spectrophotometric Estimation of Tinidazole in bulk and dosage forms, World Journal of Pharmaceutical Research, 2018; 7(9): 1742-1754. [11] JadhavPB, Vivek Shejwal, Development and Validation of an RP-HPLC Method for Crizotinib, International Journal of Pharmacy and Pharmaceutical Research. Human, 2017; 9(2):100-106. Sl. No Linearity Level (%) Concentration (ppm) Area 1 0 0 0 2 25 25 400600 3 50 50 808783 4 75 75 1203917 5 100 100 1624787 6 125 125 2009776 7 150 150 2409818