Validation of Lamotrigine in Pharmaceutical Dosage by Reverse Phase HPLC with Internal Standard Method B.VENKATA KIRAN, BATTULA SREENIVASA RAO* and SOM SHANKAR DUBEY Department of Chemistry, GITAM Institute of Technology, GITAM University, Visakhapatnam –530045, Andhra Pradesh, India. e-mail :
[email protected] . Abstract A rapid, specific and accurate isocratic HPLC method was developed and validated for the assay of lamotrigine in pharmaceutical dosage forms. The assay involved an isocratic – elution of lamotrigine in Grace C18 column using mobile phase composition consists of (50:50 v/v) of methanol and 10ml of potassium dihydrogen orthophosphate. The wavelength of detection is 308nm.The method showed good linearit y in the range of 2.01 -50.2 mg/mL. The runtime of the method is 6 mins. The proposed method can be used for routine quality control samples in industry in bulk and in finished dosage forms. In present study, a rapid specific precise and validated HPLC method for the quantitative estimation of lamotrigine in pharmaceutical dosage forms has been reported. The developed method can be applied to directly and easily to the analysis of the pharmaceutical tablet preparations. The percentage recoveries were near 100% for given methods. The method was completely validated and proven to be rugged. The excipients did not interfere in the analysis. The results showed that this method can be used for rapid determination of lamotrigine in pharmaceutical tablet with precision, accuracy and specificity. Key words: Lamotrigine, hydrochlorthiazide, Assay, reverse phase, HPLC.
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Corresponding author 1
Introduction Lamotrigine [6-(2,3-Dichlorophenyl)-1,2,4-triazine-3,5-diamine] Fig-1 is a
broad
spectrum
antiepileptic drug, chemically different from other anti-convulsants. It is used for the treatment of epilepsy, bipolar disorders, partial seizures, primary and secondary tonic-clonic seizures and seizures associated with Lennox-Gastaut syndrome. It also acts as mood stabilizer. It is first medicament after Lithium approved by Food and drug Administration (FDA) for the treatment of bipolar disorder type-I. Lamotrigine is thought to exert its anticonvulsant effect by stabilizing pre-synaptic neuronal membranes. The invitro pharmacological studies suggest that lamotrigine inhibits voltage-sensitive sodium channels, thereby stabilizing neuronal membranes and consecutively modulating pre-synaptic transmitter release of excitary amino acids (e.g. glutamate and aspartate).Lamotrigine molecular weight is 256.091, its chemical formula C9H7Cl2N5.Plasma binding capacity of lamotrigine is 55%. The drug has half life capacity of approx 25±10 hrs in healthy volunteers; it is available in tablets form in dosage level of 5 mg , 25 mg ,100
mg ,150 mg ,200 mg. It is white to pale cream colored powder and pKa value of 5.7. It is very slightly soluble in water and slightly soluble in 0.1N HCl. Lamotrigine is rapidly and completely absorbed after oral administration with negligible first-pass metabolism (absolute bioavailability is 98%). The bioavailability is not affected by food. Peak plasma concentrations occur anywhere from 1.4 to 4.8 hours following drug administration. The lamotrigine chewable/dispersible tablets were found to be equivalent, whether they were administered as dispersed in water, chewed and swallowed or swallowed as whole, to the lamotrigine compressed tablets in terms of rate and the extent of absorption.
2
Several methods have been reported for the quantitative determination of lamotrigine in bulk and pharmaceutical and biological samples. These methods include HPLC1-20, HPLC-MS21-23, HPTLC24, UV-Visiblespectrophotometric25-27, GC28, Capillary electrophoresis29. Three reliable, rapid and selective methods have been reported by Nadia Fayek Yossef and Elham Anwer Taha12, for the determination of lamotrigine in the presence of its impurity, 2,3-dichlorobenzoic acid by using HPLC and TLC. K.M Matar et.al19, has reported a rapid liquid chromatographic method for determination of lamotrigine in plasma using chloramphenicol as an internal standard.Murali Subramanium.et.al22,has reported simultaneous determination of nine epileptic drugs using liquid chromatography-mass spectrometry. K.M Patel.et.al
23,
has reported a validated HPTLC method for
determination of Lamotrigine in tablets.Talekar.et.al24, have reported the development of UVspectrophotometric method for the determination of lamotrigine in Bulk drugs and in tablets. S.J.Rajput et.at
25,
has reported two analytical methods for estimation of lamotrigine and nicorandil by difference
spectroscopy.
Literature survey revealed that there is no internal standard method has been reported for the quantification of lamotrigine in bulk and pharmaceutical formulations using hydrochlorthiazide as internal standard. So, the authors have developed a new internal standard HPLC method using hydrochlorthiazide as internal standard which is more rugged, precise and accurate. Experimental Chemicals and Reagents Lamotrigine (99.95%) pure was gift sample from corpuscle research solutions and hydrochlorthiazide (99.89% pure, internal standard was procured from corpuscle research solutions. methanol (HPLC grade) was obtained from qualigens fine chemicals. Milli-Q water 3
was purchased from Ranbaxy fine chemicals limited (RFCL). All chemicals used were of analytical grade. Instrumentation The HPLC system consisted of a Shimadzu Class VP Binary pump LC-10Atvp, SIL-10Dvp Auto sampler, CTO-10Avp column temperature Oven, PDA-UV Detector. All the components of the system are controlled using SCL-10Avp System Controller. Data acquisitions was done using LC-solution software. Mobile phase composition consists of (50:50 v/v) of methanol and 10mM of potassium dihydrogen orthophosphate operated on isocratic mode. Analysis was carried out at 308nm. The chromatographic separation of lamotrigine (drug) and hydrochlorthiazide (ISTD) was carried out using GRACE Genesis C18 column (50x4.6 mm ID,3 um). The flow rate is 0.6 ml/min .The injection volume is 10µL. Diluents consist of 50:50 (v/v) methanol and 0.1% orthophosphoric acid. Preparation of Solutions Drug stock Solution and Internal Standard Two different Stock solutions of lamotrigine working standard and hydrochlorthiazide (internal standard) was prepared by dissolving accurately weighed 10mg of drug in 10 ml of acetonitrile, so that final concentration is 1mg/1ml.The prepared stock solution is stored in 40C protected from light. Suitable dilutions of drug and internal standard were prepared by using 50:50 5 v/v methanol and 0.1% orthophosphoric acid as diluents solution. Dilution of internal standard is prepared to obtain a final concentration of 600 µg/ml. Calibration Standards and Quality Control Samples
4
An eight point linear calibration curve standards were prepared using diluents solutions in the concentration range of 2.01 to 50.20 µg/ml Calibration standards were prepared at the concentration of 2.008, 5.120, 10.040, 15.060, 25.100, 40.160, 45.180, 50.200 µg/ml from first standard stock solution 950 µL of the linear calibration standard and 50 µL of internal standard dilution and transferred into the auto sampler for analysis. Three quality control samples were at the concentrations of 10.04 µg/ml, 25.10µg/ml and 40.160 µg/ml representing low, medium and high concentration respectively .The quality control samples were prepare from second standard stock solution. For the preparation of linearity curve calibration standards 950µg/ml is mixed with 50 µg/ml of internal standard and transferred into auto sampler for analysis. Sample Preparation Commercially available tablets of lamotrigine are taken from two different brands and tested for assay. Twenty tablets of each brand are taken and crushed to powder. A powder equivalent to 50mg of lamotrigine is taken and transferred into a stoppered conical flask to which 25ml of methanol is added. The contents are transferred into a stoppered flask and shaken for 20 mins to extract the drug. Contents are carefully transferred into a centrifuge tube and centrifuged for 3000 rpm for 20mins. The supernatant liquid is taken and diluted with diluents, to obtain approximately final concentration of 50µg/ml. This sample is analyzed in triplicate. The accuracy and concentration is determined using regression equation. Method Validation System Suitability
5
The system suitability was assessed by six replicate analysis of the drug at a concentration of 25.10 µg/ml. The acceptance criterion is ± 1% for the per cent coefficient of the variation for the peak area and retention times for the both drug and internal standard. Detection and Quantization Limits (Sensitivity) Limits of detection (LOD) (Fig-2) and quantization (LOQ) (Fig-3) were estimated from both linearity calibration curve method and signal to noise ratio method. The detection limit was defined as the lowest concentration level resulting in a peak area of three times the baseline noise. The quantization limit was defined as the lowest concentration level that provided a peak area with signal to noise ratio higher than 10, with precision (%CV) and accuracy with (±) 10% Linearity (Calibration Curve) The calibration curve was constructed with eight concentrations ranging from 2.01 to 50.20 µg/ml. The peak area ratio of the drug to the internal standard was evaluated by linearity graph. The linearity was evaluated by linear regression analysis, which was calculated by least square method. It is depicted in (Fig-4). Accuracy and Precision Accuracy of assay method was determined for both intra-day and inter-day variations using triplicate analysis of the QC samples. Precision of the assay was determined by repeatability (intra-day) and intermediate precision (inter-day). Repeatability refers to the use of the analytical procedure within the laboratory over the shorter period of the time that was evaluated by assaying the QC samples during the same day. Intermediate precision was assessed by comparing the assays on different days (3 days). Specificity 6
Specificity of the method was determined by injecting 3 samples 1) Blank sample. (Fig-5). 2) Sample with Internal Standard and No Drug (Zero Blank) (Fig-6). 3) Sample containing both internal standard and drug. (Fig-7). A less than 20% interference of the peak area at the retention time of the drug in the blank sample and zero blank samples are taken as acceptance criteria for the analyte. The interference of the internal standard the peak area at the retention time of the internal standard must the less than 5% in the blank sample. Specificity is also observed in the degradation study of the drug. None of the degraded products must interfere with the quantification of the drug. Stability The stability of the drug is determined by using QC samples for the short term stability by keeping at room temperature up to 12 hours and then analyzing them. Further, auto-sampler stability for up to 24 hrs and long-term stability unto 30 days were also established. Results and Discussion Method Development and Validation The HPLC procedure was optimized with a view to develop a stability indicating assay method. Different permutations and combinations, at different pH values ranging from pH 3.0 to pH 11.0 using various columns like Hypersil-BDS-C18, Symmetry C18, Ymc-pack C18, Ymc-pack pro, Sperisorb C18, Phenomenox C18 have been tried with different buffer salts such ammonium acetate, orthophosphoric acid, di-potassium hydrogen orthophosphate, in combination with acetonitrile, methanol and tetrahydrofuran. However good resolution, less 7
tailing and high theoretical plates are obtained with Genesis column C18 50 X 4.6 cm. The mobile consists of 50:50 v/v methanol and 10mM of potassium dihydrogen orthophosphate. The flow rate of the method is 0.6 ml/min. Diluents is prepared in the same way as mobile phase which consist of (50:50) methanol and 0.1% orthophosphoric acid. The wavelength of detection is 308 nm. The column temperature is maintained at 250 C. At the reported flow rate peak shape was excellent, however increasing or decreasing the flow rate increased the tailing factor and resulting in poor peak shape and also resolution between the drug and internal standard also decreased. Hence 0.6 ml/min was optimized flow rate decreasing the consumption of the mobile phase, which in turn proves to be cost effective for long term routine quality control analysis. There was no interference in the drug and internal standard, from the blank. The peak shape and symmetry were found to be good when the mobile phase composition of 50:50 v/v was used with better resolution of the drug and internal standard. Method Validation System Suitability The % RSD of the peak area and the retention time for both drug and internal standard are within the acceptable the range (Table-1). The efficiency of the column was expressed as the number of theoretical plates for the six replicate injections was around 21795 ± 261 and the USP tailing factor was 1.22 ± 0.008 and the resolution between the Internal standard and drug is 6.0 ± 0.046.
.
Determination and Quantization Limits (Sensitivity)
8
(Fig-2) and (Fig-3) represents the six replicate injections of the limit of detection and limit of quantification. The method is found to be sensitive which can be determined from the data obtained from the (Table-2) and (Table-3). Linearity The calibration curve constructed was evaluated by its correlation coefficient. The peak area ratio of the drug and internal standard was linear, and the range, is 2.01 and 50.20 µg/ml. The linearity was determined in three sets, the correlation coefficient (R2) was consistently greater then 0.999 (Table-4). From the data in (Fig-4 and Table-4) regression equation, limit of quantification and limit of detection was determined from the calibration curve method. Regression equation: y = 31.68x-0.696 (Equation:1) Accuracy and Precision Accuracy and precision calculated for the QC samples during the intra- and inter –day run are given the (Table-5). The intra-day (day-1) and inter-day accuracy ranged from 97.95 to 101.06. The results obtained from intermediate precision (inter-day) also indicated a good method precision .All the data were within the acceptance criteria. Specificity Specificity was determined from Blank (Fig-5),zero Blank(Fig-6) and sample containing both internal standard and Drug (Fig-7). Stability
9
Stability studies were done for short term stability up to 12 hrs, auto sampler stability up to 24hrs and long term stability up to 30 days at three different concentrations of low QC, medium QC, High QC levels conditions and the mobile phase is stable up to 72 hrs.(Table-6). Robustness study Robustness is the measure of method capacity to remain unaffected by deliberate small changes in the chromatographic conditions. The experimental conditions were deliberately altered to test evaluate the robustness of the method. The impact of flow-rate(0.6±0.1), column temperature (250C±50C)changes and effect of mobile-phase composition(±10%) was evaluated on the important system suitability factors such as retention time, theoretical plates, tailing factor, and resolution were studied. The experimental results were presented in the (Table-7). Application of the method to dosage forms The HPLC method developed is sensitive and specific for the quantitative determination of lamotrogine. Also the method is validated for different parameters, hence has been applied for the estimation of drug in pharmaceutical dosage forms. Lamotrigine tablets of 25mg, 100mg strength from two different manufacturers were evaluated for the amount of lamotrigine .The amount of lamotrigine in tablet 1 is 98.49 ± 0.36 and tablet 2 is 98.59 ± 0.35 (Table-8) .None of the tablets ingredients interfere with the analytic peak. The spectrum of lamotrigine is extracted from the tablets was matching with that of standard lamotrigine showing the purity of peak of lamotrigine in the tablets. Conclusions
10
The method gave accurate and precise results in the concentration range of 2.01 to 50.20µg/mL. The mobile phase composition is (50:50 V/V) methanol:10mM potassium dihydrogen orthophosphate, at the flow rate of 0.6 ml/min. The retention times of internal standard and the drug are 2.99 ± 0.01 and 4.14 ± 0.01 respectively. The column is a 50 X 4.6mm C18 column with the particle size of 3 µm.A rapid sensitive and specific method for the determination of Lamotrigine in the pharmaceutical formulations has been developed using hydrochlorothiazide as the internal standard. Nomenclature : 1) P/A ratio : Peak-Area ratio. 2) mV : mill volts. 3) nm : Wave length. 4) min : minutes. 5) Fig : Figure. 6) Inj : Injection.
11
Figure-1 :Molecular structure of Lamotrigine
Figure-2:Representative chromatogram of LOD Injection
12
Figure-3: Representative chromatogram of LOQ Injection
13
Figure-4:Linearity Data
Figure-5: Blank
Fig-6- Zero Blank
14
Fig-7 - Typical Chromatogram containing internal standard and drug
15
Table-1 System Suitability Study ISTD
Drug
T.P
Tailing
Resolution
P/A ratio
R.T(ISTD) RT(Drug)
Inj-01
120814
89991
21849
1.21
6.03
0.745
2.994
4.151
Inj-02
123370
92422
21916
1.22
6.01
0.749
2.997
4.153
Inj-03
122482
91806
21991
1.21
6.07
0.750
2.994
4.157
Inj-04
123779
92913
21300
1.22
5.95
0.751
2.991
4.14
Inj-05
124334
93002
21599
1.21
5.98
0.748
2.986
4.134
Inj-06
123599
92895
21573
1.23
5.96
0.752
2.986
4.131
Mean
123063
92172
21705
1.22
6.00
0.75
2.99
4.14
S.D
1257.39
1158.79
260.67
0.008
0.046
0.002
0.005
0.011
RSD
1.02
1.257
1.201
0.671
0.760
0.314
0.152
0.261
16
Table-2 Limit of detection Injection. No 01
Drug(Area) 1025
P/A ratio 0.009103
T.P 23725
T.F 1.07
Resolution 6.62
02
1151
0.010605
27202
1.14
6.09
03
1113
0.010108
22575
1.11
6.33
04
1233
0.011142
20000
1.12
5.98
05
1095
0.009899
22190
1.2
6.37
06
1199
0.010845
23340
1.08
6.16
1136.00
0.0103
23172
1.12
6.258333
75.01
0.0007
2364.625
0.046904
0.229732
6.60
7.1818
10.20466
4.187871
3.670813
Mean S.D RSD
17
Table-3 Limit of Quantification Injection.No
Drug(Area)
P/A ratio
T.P
T.F
Resolution
01
4109
0.041062
20529
1.19
5.99
02
4137
0.041342
20524
1.18
6.05
03
4291
0.042881
20038
1.19
5.79
04
4217
0.042142
20204
1.19
5.86
05
4291
0.042881
20038
1.19
5.79
06
4182
0.041792
20347
1.18
5.95
Mean
4204.5
0.042017
20280
1.186667
5.905
S.D
76.55521
0.000765
223.1278
0.005164
0.108397
RSD
1.820792
3.112017
1.696398
4.623959
0.306078
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Table-4 Results and regression analysis of linearity data of lamotrigine Mean ± S.D(n=3) 31.69 ± 0.02
Slope
0.702 ± 0.009
Intercept Correlation coefficient(R2)
0.9965 ± 0.0012
Each mean value is a result of triplicate analysis (n=3)
Table-5 Intra-day and Inter-day precision and accuracy of HPLC assay of lamotrigine Nominal concentration 10.04µg/ml
25.10 µg/ml
40.16 µg/ml
Day=1 Mean (n=3)
44371
104895
163051
S.D
259.71
3953.78
4112.70
0.59
3.77
2.52
101.0635
97.95
9
Mean(n=3)
43950
105029
164958
S.D
606.47
3727.55
4492.67
R.S.D
1.38
3.55
2.72
Recovery(%)
99.77
101.00
99.40
R.S.D Recovery(%) Day=2
Day-3
19
Mean (n=3)
44205
105006
162749
S.D
321.20
3098.16
3961.55
0.73
2.95
2.43
100.80
98.08
98.59
R.S.D Recovery(%)
Each mean value is a result of triplicate analysis (n=3)
Table-6 Short-term, long term and auto-sampler stability of lamotrigine Nominal concentration 10.04µg/ml
25.10 µg/ml
40.16 µg/ml
Short term stability (12 hrs) Mean (n=3) S.D R.S.D Recovery(%)
43804.67
104471.3
164357
587.17
0.013
6159.57
1.34
0.001
3.74
101.43
98.95
98.68
44071
105779
164084
571.0425
3126.25
5503.06
1.30
2.96
3.35
101.82
98.30
99.93
Auto sampler stability(24 hrs) Mean(n=3) S.D R.S.D Recovery(%)
20
Table-7 Effect of Various parameters in assessment of method Variation Parameters 0.5ml/min
R.T 4.247
T.P 20786
Tailing 1.24
Resolution 5.92
0.7ml/min
4.091
21453
1.19
6.02
200C
4.21
22345
1.18
6.09
300C
4.13
21564
1.2
6.12
90% organic
4.25
20456
1.18
6.06
110% organic
4.14
21453
1.23
6.02
Flow rate
Column temperature
Observed values
Mobile phase
Table-8 Results of Lamotrigine in marketed product Marketed formulation
Drug
% Amount obtained
% RSD
Brand-1
lamotrigine -25 mg
98.31± 0.18
0.18
Brand-2
lamotrigine -100 mg
98.86 ±0.41
0.42
Each value is a result of triplicate analysis.
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References 1. Papadoyannis, I.N.; Zotou ,A.C and Samanidou.V.F, Journal of Liquid Chromatography and related techniques.1995,18(13),2593-2609. 2. Emami, J; Ghassami, N and Ahmadi F, Journal of pharmaceutical and Biomedical medical analysis. 2006,40,999-1005. 3. Stoforidis,P. A;Morgan, D. J; O’Brien ,T. J and Vajda F J E, Journal of Chromatography B,1999727(1-2),113-118. 4. Vidal.E;Pascual C and Pou L, Journal of Chromatography B, Biomedical Science Applications,1999,736(1-2),295-298. 5. Yamashita. S,; Furuno .K;Kawasaki .H;Gomita. Y; Yoshinaga .H; Yamatogi .Y and Ohtahara S.Journal of chromatography B,Biomedical science applications,1995,670,354-357. 6. Danilo.C; Andrea. S; Ugo. D. G and Gaetano .B, Ther Drug Monit, 2001,23, ,665. 7. Hart A.P;Mazarr-Proo .S; Blackwell .W and Dasgupta .A, Ther Drug Monit., 1997,19, 431435. 8. Manuela.C;Monica .B;Erica C;Carmina C;Fiorenzo .A; Roberto .R and Agostino. B, Journal of Chromatography B, 2005,828,113-117. 9. Merce.T;Miquel.R;Santiago .A and Jacint C, Ther Drug Monit., 2000,22,621. 10. Khoschsorur .G A;Fruhwirth .F and Baumann G .H, Chromatogr., 2001,54, 345-349. 11. Dumortier. G, Pons .D, Zerrouk .A, Januel D, Saba G and Degrassat K,Journal of Liquid chromatography and related techniques, 2001,24, 3171. 12. Youssef .N. F and Taha E A, Chem Pharm Bull, 2007,55(4), 541-545. 13. Patil. K. M and Bodhankar, S. L, Journal of Pharmaceutical and Biomedical Analysis,2005,39,181-186. 14. Lensmeyer,G.L;Gidal,B.E.,Wiebe .D. A.,Ther. Drug Monit., 1997,19, 292-300. 15. Angelis-Stoforidis P., Morgan D. J., O’Brien T. J.,Vajda F. J.E., Journal of Chromatography.B, Biomedical. Appl., 727, 1999, 113—118. 16. Croci D., Salmaggi A., de Grazia U., Bernardi G., Ther. Drug Monit.,23,2001,665-668. 17. Castel-Branco ,M.M.,Almeida ,A.M;Falcao,A.C;Macedo,T. A;Caramona,M. M;Lopez,F.G;J. Chromatogr. B,Biomed. Appl.,2001.755,119-127. 22
18. Cheng, C. L; Chou C. H;Hu,O.Y.P,J. Chromatogr. B, Anal. Technol.Biomed. Life Sci.,817, 2005, 199-206. 19. Matar K. M., Nicholls P. J., Bawazir S. A., Al-Hassan M. I., Tekle A., J. Pharm. Biomed. Anal, 1998,17, 525-531. 20. Jimenez .G .M;Moreno ,A .C.P.A; Morales .L.M;Espinosa .L.R and Martínez, M M,Bioanalysis, 2009, 1(1), 47-55. 21. Olof,B;Inger .O and Helena .N, Ther Drug Monit, 2006, 28(5), 603-607. 22. Murali,S; Angela,B and Rory,R, Ther Drug Monit, 2008,30(3), 347-356. 23. Patil,K .M; Agarwal,A .K and Bodhankar,S.L, Indian J Pharm Sci, 2004,66(3), 283-286. 24. Talekar,R.S;Dhake,A.S; Sonaje ,D. B and Mourya,V.K, Indian J Pharm Sci, 2000,62, 51-52. 25. Rajput,S. J and Patel,A.K, Indian J Pharm Sci,2004, 66(3) ,342. 26. Alizadeh,N,Khakinahad,R and Jabbari,A,Pharmazie, 2008,63(11), 791-795. 27. Hallbach J;Vogel,H;Guder,W.G;Eur.J. Clin.Chem.Clin.Biochem,1997,35, 755-759 28. Shihabi Z.K; Oles,K.S, J. Chromatogr. B, Biomed Appl,1996,683, 119-123 29. Theurillat,R;Kuhn,M,Thormann,W,J. Chromatogr. A, 2002,979,353-368. 30. B.V.Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey.Validation of Venlafaxacine in Pharmaceutical Dosage by Reverse Phase HPLC Method. Journal of Pharmacy Research, 2012, 5(5), 2683-2687. 31. B.V.Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey.Development and validation of a reversed-phase HPLC method for the determination of Efavirenz in pharmaceutical dosage forms. Journal of Pharmacy Research, 2012, 5(1), 94-99. 32. B.V.Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey. Validation of Quetiapine fumarate in pharmaceutical dosage forms by reverse phase HPLC with Internal standard method. Journal of Chemistry, Hindawi publications, Volume 2013, ID : 578537. 33. B.V.Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey. Validation of Abacavir Sulphate in Pharmaceutical Dosage by Reverse Phase HPLC with Internal Standard Method. International Journal of pharmaceutical sciences and research, 2012, Vol. 3(8) 2590-2598. 34. B.V.Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey.Simultaneous determination and validation of Amalodipine and Metoprolol in Pharmaceutical Dosage forms by Reverse Phase HPLC Method, International Journal of Pharmacy and Pharmaceutical Sciences, Vol 4, Supplement 5, 2012. 35. B.V.Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey.Kinetic-Spectrophotometric Determination of Co (II) in Vegetable Samples by Using Indigo-Caramine. International Journal of Pharmaceutical Research, 2012, 4(3), 64-68. 36. K. Nagendar Rao, B.Venkata Kiran, Battula Sreenivasa Rao* and Som Shankar Dubey. Development and Validation of a Reverse Phase HPLC method for the Determination of Metformin HCL in Pharmaceutical Dosage forms. Asian Journal of Chemistry, 2012, Vol 24(12), 5460-5462.
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37. B.S.A Andrews*, B.Sreenivas Rao, Som Shankar Dubey and B.Venkata Kiran.Titrimetric Determination of Ascorbic acid and Isonicitonic Acid Hydrazide in Drug Formulations with Chloramine-T as Oxidant. International Journal of Pharmacy and Technology, Dec-2010, Vol 2(4), 886-889. 38. B.S.A Andrews*, B.Sreenivas Rao, Som Shankar Dubey and B.Venkata Kiran.O-Anisidine as Indicator in Titrimetric Determination of Ascorbic Acid and Isonicotinic Acid Hydrazide in Pharmaceutical Formulations. International Journal of Applied Biology and Pharmaceutical Technology. Aug-2010, Vol 1(2), Page: XXX. 39. B.S.A.Andrews, B.Venkat Kiran, Battula Sreenivas Rao,*, Som Shankar Dubey and D. Malleswara Rao.Kinetic Spectrophotometric Determination of Trace Amounts of Vanadium (V) based on its Catalytic Effects on the Reaction of o-Anisidine and Potassium Bromate. Asian Journal of Chemistry, June-2011, Vol 23 (10), 4419-4424. 40. Som Shankar Dubey, Battula Sreenivasa Rao, B. S. A. Andrews and B.Venkata Kiran. Efficient Removal of Ce(III) and Eu (III) Ions from Aqueous Solutions by Local Clay-A Radiotracer Study. 2011, Journal of Chemistry, Vol 8(2), 917-923. 41. Battula Sreenivasa Rao, Som Shankar Dubey and B.Venkata Kiran. New Analytical Technique for the Determination of Mercury (II) by Extraction Spectrophotometric Method with Isonitriso p-Isopropyl Acetophenone Phenyl Hydrazone In Sewage Wastes and Spiked Water Samples. International Journal of Life Sciences and Pharma Research, 2011, Vol 1(1), 75-79. 42. Battula Sreenivasa Rao*, Som Shankar Dubey and Kiran BV. Determination of Molybdenum (VI) in Amaranthus and Potato by New Extractive - Spectrophotometric Method with Isonitriso p-Isopropyl Acetophenone Phenyl Hydrazone. Research Journal of Pharmaceutical Biological and Chemical Sciences, 2012, 3(1), 580-4. 43. Som Shankar Dubey, Battula Sreenivasa Rao and B.V. Kiran. Equilibrium and Thermodynamic Studies of Cesium Adsorption on Illite Clay. Asian Journal of Research in Chemistry. 2013, 6(2), 139-43. 44. Som Shankar Dubey*, Battula Sreenivasa Rao and B.V.Kiran. Equilibrium and Thermodynamic Studies of Cesium Adsorption on Bentonite Clay. International Journal of Pharmacy and Technology, 2013, Vol 5(1), 5204-5211. 45. K.Siva Nagaraju, B.S.Rao and B.V.Kiran. Development and Validation of stress degradation studies of Tenofovir Disoproxil fumarate and Emtricitabine by HPLC. Asian Journal of Research in chemistry. 2013. 6(10), 936-944. 46. K.Siva Nagaraju, B.S.Rao, B.V.Kiran and G.Srinivas Rao. Effect of Surfactant on Spectrophotometric determination of selenium with Isonitriso Isopropyl Acetophenone phenyl Hydrazone. Journal of Applicable Chemistry.2012. 1(2), 232-238.
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