International Journal of Innovative Pharmaceutical Sciences and Research www.ijipsr.com 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 3.779 min & 5.545 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 Email: dnreddy.niru@gmail.com Phone: +91 9603210830 Available online: www.ijipsr.com July Issue 1438
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[8.4.0.0^{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: www.ijipsr.com July Issue 1439
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: www.ijipsr.com July Issue 1440
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: www.ijipsr.com July Issue 1441
Column temperature (0c) 300c Volume of injection loop (µl) 10 Detection wavelength(nm) Drug 230nm Atenolol and Alprazolam Drug RT (min) 3.779 & 5.545 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 1 3.779 3505990 2 3.781 3453520 3 3.782 3451648 4 3.781 3454381 5 3.781 3463164 6 3.781 3464008 S No 1 2 3 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 1.101 NMT 2 USP Plate count of Analyte 7950 NLT 3000 peak of standard solution Available online: www.ijipsr.com July Issue 1442
Table 4: Results of System suitability for Alprazolam SNO RETENTION TIME PEAK AREA 1 5.455 4620541 2 5.548 4569380 3 5.549 4571277 4 5.550 4575402 5 5.551 4570003 6 5.549 4582807 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 2.0 2 Tailing factor for analyte peak in standard solution 1.091 NMT 2 3 USP Plate count of Analyte peak of standard solution 10149 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: www.ijipsr.com July Issue 1443
PRECISION Table 6: Results of Atenolol for precision studies S.NO RETENTION TIME PEAK AREA ASSAY % 1 3.783 3457098 99 2 3.783 3458534 99 3 3.784 3455049 99 4 3.783 3453832 99 5 3.783 3456866 99 6 3.782 3456871 99 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 % 1 5.550 4578648 100 2 5.551 4574845 100 3 5.553 4573148 100 4 5.551 4575517 100 5 5.553 4579782 100 6 5.551 4571828 100 OBSERVATION: The RSD Of six replicate injections of Alprazolam standard preparation are within the specified acceptance criteria. ACCURACY Available online: www.ijipsr.com July Issue 1444
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 50 5.553 2284097 100 50 5.550 2285246 100 50 5.553 2289948 100 50 5.549 2287691 100 50 5.547 2281134 100 50 5.544 2287538 100 100 5.544 4574401 100 100 5.544 4577882 100 100 5.545 477186 100 150 5.540 6864243 100 150 5.538 6862424 100 150 5.540 6866177 100 150 5.537 6868222 100 150 5.534 6865482 100 150 5.535 6864694 100 100 100 100 Table 9: Results for Accuracy of Atenolol Conc Retention time Peak area %recovery Mean recovery 50 3.781 1721678 100 50 3.783 1724156 100 50 3.783 1726115 100 50 3.783 1705916 99 50 3.782 1728781 100 50 3.781 1726972 100 100 Available online: www.ijipsr.com July Issue 1445
100 3.782 3453370 100 100 3.783 3458515 100 100 3.784 3456575 100 150 3.783 5189235 100 150 3.784 5189141 100 150 3.783 5188022 100 150 3.784 5188993 100 150 3.784 5187045 100 150 3.785 5182362 100 100 100 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 3.781 1721250 2 LINEARITY-75% 1 Atenolol 3.782 2598407 3 LINEARITY-100% 1 Atenolol 3.782 3456452 4 LINEARITY-125% 1 Atenolol 3.783 4322769 5 LINEARITY-150% 1 Atenolol 3.783 5182472 Table 11: Linearity Studies of Alprazolam Sampel Name Inj Name Rt Area 1 LINEARITY-50% 1 Alprazolam 5.533 2281842 2 LINEARITY-75% 1 Alprazolam 5.532 3431878 3 LINEARITY-100% 1 Alprazolam 5.529 4573695 4 LINEARITY-125% 1 5 LINEARITY-150% 1 Alprazolam Alprazolam 5.526 5716339 5.522 6862968 Available online: www.ijipsr.com July Issue 1446
Table 12: Results of linearity for Atenolol S.No CONC(µg/ml ) AREA 1 50 1721250 2 75 2598407 3 100 3456452 4 125 4322769 5 150 5182472 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 1 50 2281842 2 75 3431878 3 100 4573695 4 125 5716339 5 150 6862968 Available online: www.ijipsr.com July Issue 1447
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 3.781 887502 2 LOQ 1 Atenolol 3.781 1861034 Table 15: Results of LOD and LOQ for Alprazolam S.No. SAMPLE NANE INJ NAME Rt AREA 1 LOD 1 Alprazolam 5.521 1182603 2 LOQ 2 Alprazolam 5.517 2456550 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 5.029 4649714 8.307 1.141 8623 1 Atenolol 3.032 2699890 7.537 1.073 6703 Available online: www.ijipsr.com July Issue 1448
3 4 Std 2(Temp1) Std 2(Temp2) 1 Atenolol 5.026 4643108 8.339 1.140 8824 1 Atenolol 3.039 2711288 7.577 1.080 6688 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 0.999 All the system suitability parameters should pass for all the conditions. Atenolol Alprazolam 0.05 0.07 Atenolol Alprazolam 102 99 Atenolol Alprazolam 0.99 0.99 The system suitability parameters passed for all the Conditions. For 6 replicate injections Atenolol Alprazolam System Suitability The %RSD NMT 2.0% 0.2 0.1 Tailing factor NMT 2.0% 1.101 1.091 Plate Count NLT 3000 7950 10149 REFERENCES 1. Skoog, West, Holler. Fundamental of Analytical Chemistry. 7th ed. USA: Saunders college Publishing,USA, 1992. Available online: www.ijipsr.com July Issue 1449
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, 2002. 3. Instrumental methods of analysis, 7 th Edn., CBS Publishers, New Delhi. 4. R.J. Hemilton and Swell, Introduction to HPLC, 2 nd Edn., 2-94. 5. 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, www.alltech web.com 7. Lloyd R. Synder, Joseph J. Kirkland, Joseph L. Glajesh, Practical HPLC Method Development, 2 nd Edn., 1997, 1-14. 8. 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), 57-66. 9. 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): 139 85 10. O Neil MJ. The Merck index- an encyclopedia of chemicals, drugs and biologicals, 13 th ed. New Jersy, Merck and Co., 491, 5541. 11. SC.Sweetman. Martindale; the complete drug reference. Pharmaceutical press, 33 rd ed. London; 2002; 200. 12. 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): 63-68. 13. 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 2012. 14. 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), 962-966. Available online: www.ijipsr.com July Issue 1450