International Journal of Innovative Pharmaceutical Sciences and Research

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1 International Journal of Innovative Pharmaceutical Sciences and Research METHOD DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ATENOLOL AND ALPRAZOLAM IN COMBINED TABLET DOSAGE FORM 1 D.Nirupama *, 2 P.Venkateswara Rao, 3 B.Thangabalan, 4 S.Manohar Babu Department of Pharmaceutical Analysis, Sims College of Pharmacy, Mangaldas Nagar, Opp Best Prize Vijayawada road, Guntur, INDIA Abstract A simple reverse phase HPLC method was developed for estimation of Atenolol and Alprazolam in their pharmaceutical formulation. A Waters symmetry shield Rp18,(250x4.6x5µ column, along with Di Potassium Hydrogen Phosphate and methanol in the ratio of 60:40 as mobile phase. The flow rate was 1.0 ml/min and effluent was monitored at 285 nm. The retention times were min & min for Atenolol and Alprazolam respectively. The proposed methods are simple, selective, reproducible, sensitive and accurate with good precision. Some of the methods were proved to be superior to most of the reported methods. All these proposed methods for estimation of Atenolol and Alprazolam were successfully applied in pharmaceutical formulations. Key words: Alprazolam, Atenolol, RP-HPLC, Retention time, ICH guideline. Corresponding author D.Nirupama Department of Pharmaceutical Analysis, Sims College of Pharmacy, Mangaldas Nagar, Opp Bestprize, Guntur, INDIA dnreddy.niru@gmail.com Phone: Available online: July Issue 1438

2 INTRODUCTION Atenolol 2-(4-(2-hydroxy-3-(isoprropylamino) propoxy) phenyl) acetamide [1, 2] is used for the management of hypertension and long-term management of patients with angina pectoris. Like metoprolol, atenolol competes with sympathomimetic neurotransmitters such as catecholamines for binding at beta (1)-adrenergic receptors in the heart and vascular smooth muscle, inhibiting sympathetic stimulation [3, 4]. This results in a reduction in resting heart rate, cardiac output, systolic and diastolic blood pressure, and reflex orthostatic hypotension. Higher doses of atenolol also competitively block beta (2)-adrenergic responses in the bronchial and vascular smooth muscles. Approximately 50% of an oral dose is absorbed from the gastrointestinal tract, the remainder being excreted unchanged in the feces [11, 12] unlike propranolol or metoprolol, but like nadolol, atenolol undergoes little or no metabolism by the liver, and the absorbed portion is eliminated primarily by renal excretion. Alprazolam 12-chloro-3-methyl-9-phenyl-2,4,5,8 tetraazatricyclo[ ^{2,6}]tetradeca- 1(10),3,5,8,11,13-hexaene is an Anti-anxiety and sedative-hypnotic [3,4,5] Benzodiazepines bind nonspecifically to benzodiazepine receptors BNZ1, which mediates sleep, and BNZ2, which affects muscle relaxation, anticonvulsant activity, motor coordination, and memory. As benzodiazepine receptors are thought to be coupled to gamma-aminobutyric acid-a (GABA A ) receptors, this enhances the effects of GABA by increasing GABA affinity for the GABA receptor [12]. Binding of the inhibitory neurotransmitter GABA to the site opens the chloride channel, resulting in a hyperpolarized cell membrane that prevents further excitation of the cell [14]. MATERIALS & METHODS METHOD DEVELOPMENT Instruments\equipments\Apparatus used for the method development S.No Instruments/Equipments/Apparatus 1. High performance liquid chromatography (Waters e 2695) 2. Electronic Balance (sartorius) 3. Ultra Sonicator (Fast-Clean) 4. Thermal Oven (newtech lab equipments) 5. A Waters symmetry shield Rp18,(150x4.6x5µ) column Available online: July Issue 1439

3 OPTIMIZED METHOD Reference standards Atenolol Alprazolam Strengths of dosage forms Brand name: AA-50 (NIRAVAM) Atenolol -25mg Alprazolam-4mg Preparation of mobile phase: Mix a Potassium di Hydrogen Phosphate (70%) and methanol (HPLC grade) (30%) and degas in ultrasonic water bath for 15 minutes. Filter through 0.45 µ filter under vaccum filtration. Standard stock solution preparation: Accurately weigh and transfer 25mg of Atenolol and 4mg of Alprazolam working standard into two 100 ml volumetric flask add about 50mL of Diluent(Mobile phase and sonicate to dissolve it completely and make volume up to the mark with the same solvent. (Stock solution). Standard solution preparation From the above stock solution take 5ml in 25 ml volumetric flask and make up the volume with diluent. Sample stock solution preparation Accurately weigh and transfer equivalently 275 mg of sample to clean dry 100 ml volumetric flask and add the 50ml volume to the mark with diluents (Mobile phase) and sonicate to dissolve it completely and make volume up to the mark with the same solvent Sample solution preparation From the above stock solution take 5ml in 25 ml volumetric flask and make up the volume with diluent. Evaluation of System Suitability: Inject 10 µl of the diluted standard solution in five replicate injections, into the chromatograph and record the chromatograms. The column efficiency as determined from Atenolol and Alprazolam peaks is not less than 3000 USP plate count and the tailing factor for Atenolol and Alprazolam peaks is not more than 2.0. Available online: July Issue 1440

4 The relative standard deviation for the peak areas of the five replicate injections is not more than 2.0%. Procedure: Separately inject 10µl of the blank, Standard (five injections) and sample solution in duplicate into the liquid chromatography, record the chromatographs and measure the peak areas. Calculation: Calculate the amount of each drug by using the following formula A L D S P (Mg/tablet) = x x A SL D T 100 Where, A L = Average area counts of injections for analyte peak in the chromatogram of sample solution. A SL peak in the chromatogram of standard solution. D S D T P = Average area count of five replicate injections for analyte = Dilution factor of standard solution (weight dilution). = Dilution factor of sample solution. = Percentage purity of working standard used. Content of each drug (mg/tablet) % Labeled Amount = x 100 Label claim, in mg Table 1 : Stationary phase (column) Mobile phase A Waters symmetry shield Rp C8,(150x4.6x5µ) column Potassium di hydrogen phosphate:methanol. ( 70:30 ) PH 9.2 Flow rate (ml/min) Run time (minutes) 1ml/min 7 mins Available online: July Issue 1441

5 Column temperature (0c) 300c Volume of injection loop (µl) 10 Detection wavelength(nm) Drug 230nm Atenolol and Alprazolam Drug RT (min) & RESULTS AND DISCUSSION SYSTEM SUITABILITY Results System suitability tests are used to verify the reproducibility of the chromatographic system. To ascertain its effectiveness, system suitability tests were carried out on freshly prepared stock solutions. Table 2: Results of system suitability for Atenolol S.NO RETENTION TIME PEAK AREA S No Table 3: System suitability Parameters for Atenolol System Suitability Parameter Result obtained Acceptance Criteria % RSD for six replicate injections of analyte peak in 0.2 NMT 2.0 Tailing factor for analyte peak in standard solution NMT 2 USP Plate count of Analyte 7950 NLT 3000 peak of standard solution Available online: July Issue 1442

6 Table 4: Results of System suitability for Alprazolam SNO RETENTION TIME PEAK AREA Table 5: System suitability Parameters for Alprazolam System S No Suitability Parameter Result obtained Acceptance Criteria 1 % RSD for six replicate injections of analyte peak in standard solution 0.1 NMT Tailing factor for analyte peak in standard solution NMT 2 3 USP Plate count of Analyte peak of standard solution NLT 3000 CONCLUSION: It was observed from the data tabulated above that the method complies with system suitability parameters. Hence, it was concluded that the system suitability parameter met the requirement of method validation. Available online: July Issue 1443

7 PRECISION Table 6: Results of Atenolol for precision studies S.NO RETENTION TIME PEAK AREA ASSAY % OBSERVATION The RSD of six replicate injections of Atenolol standard preparation are within the specified acceptance criteria. Table 7: Results of Alprazolam for precision studies S.NO RETENTION TIME PEAK AREA ASSAY % OBSERVATION: The RSD Of six replicate injections of Alprazolam standard preparation are within the specified acceptance criteria. ACCURACY Available online: July Issue 1444

8 The accuracy of an analytical method is the closeness of test results obtained by that method to the true value. Table 8: Results for Accuracy of Alprazolam Conc Retention time Peak area %recovery Mean recovery Table 9: Results for Accuracy of Atenolol Conc Retention time Peak area %recovery Mean recovery Available online: July Issue 1445

9 OBSERVATION The %Recovery at each level is between 98.0% to 102.0%. LINEARITY Table 10: Linearity Studies of Atenolol Sampel Name Inj Name Rt Area 1 LINEARITY-50% 1 Atenolol LINEARITY-75% 1 Atenolol LINEARITY-100% 1 Atenolol LINEARITY-125% 1 Atenolol LINEARITY-150% 1 Atenolol Table 11: Linearity Studies of Alprazolam Sampel Name Inj Name Rt Area 1 LINEARITY-50% 1 Alprazolam LINEARITY-75% 1 Alprazolam LINEARITY-100% 1 Alprazolam LINEARITY-125% 1 5 LINEARITY-150% 1 Alprazolam Alprazolam Available online: July Issue 1446

10 Table 12: Results of linearity for Atenolol S.No CONC(µg/ml ) AREA Fig. 1: Linearity plot of Atenolol OBSERVATION: The correlation coefficient is not less than 0.99 Table 13: Results of linearity for Alprazolam SNO CONC(µg/ml ) AREA Available online: July Issue 1447

11 Fig 2: Linearity plot of Alprazolam OBSERVATION: The correlation coefficient is not less than 0.99 Table 14: Results of LOD and LOQ for Atenolol S.No. SAMPEL NAME INJ NAME Rt AREA 1 LOD 1 Atenolol LOQ 1 Atenolol Table 15: Results of LOD and LOQ for Alprazolam S.No. SAMPLE NANE INJ NAME Rt AREA 1 LOD 1 Alprazolam LOQ 2 Alprazolam OBSERVATION The LOD and LOQ values from the above demonstrate that the method is sensitive for the determination of Atenolol and Alprazolam. ROBUSTNESS Table 16: Results of Robustness for Atenolol and Alprazolam S.NO. SAMPEL NAME INJ NAME RT AREA USP RESOLUTION USP TAILING USP PLATE COUNT 1 2 Std 2(flow1) Std 2(flow2) 1 Atenolol Atenolol Available online: July Issue 1448

12 3 4 Std 2(Temp1) Std 2(Temp2) 1 Atenolol Atenolol Table17: Summary Report of HPLC validation. Validation Parameters Acceptance Criteria HPLC Results Precision Accuracy Linearity Robustness The %RSD of peaks obtained from the 6 replicate injections should be NMT 2.0% The % recovery at each level shall be NLT 98.0% and NMT 102.0% of the added amount. The Correlation coefficient shall be NLT All the system suitability parameters should pass for all the conditions. Atenolol Alprazolam Atenolol Alprazolam Atenolol Alprazolam The system suitability parameters passed for all the Conditions. For 6 replicate injections Atenolol Alprazolam System Suitability The %RSD NMT 2.0% Tailing factor NMT 2.0% Plate Count NLT REFERENCES 1. Skoog, West, Holler. Fundamental of Analytical Chemistry. 7th ed. USA: Saunders college Publishing,USA, Available online: July Issue 1449

13 2. H.H.Williard, L.L. Merit, F.A. Dean and F.A. Settle, Instrumental methods of analysis, 7 th Edn, C.B.S. Publishers, New Delhi, Instrumental methods of analysis, 7 th Edn., CBS Publishers, New Delhi. 4. R.J. Hemilton and Swell, Introduction to HPLC, 2 nd Edn., Sharma. B.K. Instrumental Methods of Chemical Analysis. 6. Craig S.9Young and Raymond. J. Weigand, An efficient approach to column selection in HPLC Method Development, web.com 7. Lloyd R. Synder, Joseph J. Kirkland, Joseph L. Glajesh, Practical HPLC Method Development, 2 nd Edn., 1997, G.A. Shabir, "Validation of HPLC Chromatography Methods for Pharmaceutical Analysis. Understanding the Differences and Similarities Between Validation Requirements of FDA, the US Pharmacopeia and the ICH," J. Chromatogr. A. 2003;987(1-2), Williams B, Poulter NR, Brown MJ. "Guidelines for management of hypertension: report of the fourth working party of the British Hypertension Society, J Hum Hypertens ;2004;18 (3): O Neil MJ. The Merck index- an encyclopedia of chemicals, drugs and biologicals, 13 th ed. New Jersy, Merck and Co., 491, SC.Sweetman. Martindale; the complete drug reference. Pharmaceutical press, 33 rd ed. London; 2002; M. V. Kumudhavalli*, K. Anand Babu, B. Jayakar, Rp-Hplc Method Development And validation For The Simultaneous Estimationof Atenolol And Nitrendipin in Tablet dosage form. Der Pharma Chemica, 2011, 3 (4): S. K. Shah*, S. C. Dangre, N. B. Charbe; Rp-Hplc Method Development And validation For The Simultaneous Estimationof Atenolol And Fluoxetine Hydrochloride in Tablets.RJPT, Vol:5, Issue 07, July Belal F, Sharaf El-Din M1, Aly F, Hefnawy M and El-Awady M*: Development Of Simple RP-HPLC Method For Determination Of Atenolol In Bulk dosage form. E- Journal of Chemistry.2010, 7(3), Available online: July Issue 1450

METHOD DEVELOPMENT AND VALIDATION BY RP-HPLC FOR ESTIMATION OF ZOLPIDEM TARTARATE

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Ramalakshmi et al. SJIF Impact Factor 6.647 Volume 7, Issue 2, 1010-1018 Research Article ISSN 2278 4357 METHOD DEVELOPMENT AND VALIDATION BY RP-HPLC