A rapid and simple Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed for the quantification of Amoxicillin in tablet dosage form. Separation was achieved on Chromatopak-C18 (250mm×4.6×5micron) column in isocratic mode with mobile phase consisting of Acetonitrile: 0.2M Potassium dihydrogen phosphate buffer (pH 3) (22:78v/v) and conditions optimized with flow rate of 1 ml/minute and wavelength of detection at 283 nm. The retention time of Amoxicillin was found to be 6.4 min. Linearity was established for Amoxicillin in the range 10 100 μ g / ml with R2 value 0.999. This method was validated in accordance with ICH guidelines, the linearity, accuracy, precision, specificity, robustness, ruggedness, and system suitability results were within the acceptance criteria. Validation studies demonstrated that the proposed RP-HPLC method is simple, specific, rapid, reliable and reproducible for the determination of Amoxicillin for Quality Control level

Aktar, Kareem

B, Sivagami

C, Krishna Ekanth

G, Nagendra Babu

L.M, Sharmil Kumar

M, Bhaskar

M., Niranjan Babu

English

International Journal of Advances in Pharmaceutical Analysis

Sivagami B et al / International Journal of Advances in Pharmaceutical Analysis 2018; 08(01): 09-17.                                                     9 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com International Journal of Advances in Pharmaceutical Analysis ISSN: 2277-9353 (Online) Journal DOI: https://doi.org/10.7439/ijapa                                              Research Article A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms  Sivagami, B*1, Sharmil Kumar L.M, Bhaskar M, Kareem Aktar, Nagendra Babu G, Krishna Ekanth C, and M. Niranjan Babu  Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy, Venkataramapuram, Tirupati, Chitoor Dist, 517561, A. P., India   QR Code     *Correspondence Info: B. Sivagami, Associate Professor, Department of Pharmaceutical Analysis, Seven Hills College of Pharmacy, Venkataramapuram, Ramachandrapuram Mandal, Tirupati, Chitoor - 517561 Andhrapradesh, India.  *Article History: Received: 30/03/2018 Revised: 07/04/2018 Accepted: 07/04/2018 DOI: https://doi.org/10.7439/ijapa.v8i1.4727 Abstract A rapid and simple Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method has been developed for the quantification of Amoxicillin in tablet dosage form. Separation was achieved on Chromatopak-C18 (250mm×4.6×5micron) column in isocratic mode with mobile phase consisting of Acetonitrile: 0.2M Potassium dihydrogen phosphate buffer (pH 3) (22:78v/v) and conditions optimized with flow rate of 1 ml/minute and wavelength of detection at 283 nm. The retention time of Amoxicillin was found to be 6.4 min. Linearity was established for Amoxicillin in the range 10 – 100 μg / ml with R2 value 0.999. This method was validated in accordance with ICH guidelines, the linearity, accuracy, precision, specificity, robustness, ruggedness, and system suitability results were within the acceptance criteria. Validation studies demonstrated that the proposed RP-HPLC method is simple, specific, rapid, reliable and reproducible for the determination of Amoxicillin for Quality Control level. Keywords: RP-HPLC, Amoxicillin, Method Development, Validation.  1. Introduction 1.1 Amoxicillin Amoxicillin is an extended spectrum penicillin group of antibiotic. Amoxicillin is active against many gram positive and gram negative bacteria. Chemical structure The molecular formula is C16H19N3O5S•3H2O and the molecular weight is 419.45. The chemical structure is:   Figure 1: Structure of Amoxicillin 1.2 Mechanism of action Amoxicillin acts by inhibiting bacterial cell wall synthesis. Lack of bacterial cell wall results in death due to lysis of bacteria. So amoxicillin is useful only for actively growing and cell wall synthesizing bacteria. Amoxicillin is an extended spectrum, penicillinase-susceptible, semi-synthetic amino- penicillin. It is a close chemical and pharmacological congener of ampicillin (amino-p-hydroxy-benzyl penicillin). Amoxicillin like other penicillins, inhibit the penicillin binding proteins (PBPs {specifically PBP-1A}), which are transmembrane surface enzymes that catalyse the cross linking (transpeptidation) between the peptidoglycans in the bacterial cell wall. Amoxicillin is able to bind to the PBPs by geometrically mimicking the natural D-alanyl-D-alanine substrate that is generally bound to the enzyme. By substituting itself for D-alanyl-D-alanine, the appropriate Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         10 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com strands needed for cross-linking are not available and the cell wall biosynthesis process is left incomplete. It also acylates the penicillin-sensitive transpeptidase C-terminal domain by opening the lactam ring. This inactivation of the enzyme prevents the formation of a cross-link of two linear peptidoglycan strands, inhibiting bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins or murein hydrolases; it is possible that amoxicillin interferes with an autolysin inhibitor. [1-6]  2. Materials and Methods 2.1 Experimental  2.1.1 Materials  Tablets containing 125 mg of Amoxicillin was obtained from Apollo Pharmaceuticals Pvt. Ltd, Tirupati, India and used within their shelf life period. Acetonitrile and water (HPLC-grade) were purchased from SD Fine Chem. Pvt. Ltd, India. All other chemicals and reagents employed were of analytical grade, and purchased from SD Fine Chem. Pvt. Ltd, India.   2.1.2 Instrumentation  Ultrasonicator,0.45μm membrane filter, (PCI Analytical, Model Number 1.5L50) Analytical balance (Wenser), Analytical Technologies HPLC system, LC Solution soft ware having the configurations, Solvent degasser DCU-20A3 Solvent degasser, UV-VIS detector with class VP software, Columns of Chromatopak-C18 (250mm×4.6×5micron) were used in this study. A (Eutect) digital pH meter was used for pH adjustment.  2.1.3 Chromatographic conditions  The selected and optimized mobile phase composed of Acetonitrile: Potassium dihydrogen phosphate buffer (pH 3) (22:78v/v) and conditions optimized were with flow rate of 1 ml/minute, wavelength at 283 nm and Run time of 20 min. Here the peaks were separated and showed better resolution, appreciable theoretical plate counts and good peak symmetry. The proposed chromatographic conditions were found appropriate for the quantitative determination of the present drug   2.1.4 Preparation of mobile phase  Mobile phase was prepared by taking Acetonitrile: 0.1 M Potassium di hydrogen phosphate buffer (pH 3) (22:78v/v). Mobile phase was filtered through 0.45 μm membrane filter and degassed under ultrasonic bath prior to use. The mobile phase was pumped through the column at a flow rate of 1 ml/min.   2.1.5 Preparation of standard solutions  Dissolve 30 mg of amoxicillin working standard in mobile phase and dilute to 50 ml with the same mobile phase. One ml was diluted to 20ml with mobile. Diluted to 1ml of this solution to 50ml with mobile phase. Finally this gave 0.6 ppm solution, then the solution was filtered through the 0.45 μm membrane filter and degassed under ultrasonic bath prior to use. The solution was injected into the HPLC system.  2.1.6 Sample Preparation Amoxicillin is available as tablets containing 125mg of Amoxicillin. Amoxicillin is available in the local market with brand names AMOX 125.  Twenty tablets of Amoxicillin were taken into a fine powder of the tablets and the powder equivalent to 100mg of Amoxicillin was weighed accurately and transferred into a 100ml standard volumetric flask. The contents were dissolved in buffer and sonicated for 30 Minutes. This entire solution was filtered through 0.45 micron Whatmann filter paper (No. 41) and the final solution was made with mobile phase to get the solution of 1000μg/ ml. 1ml of this solution was transferred to 10ml volumetric flask, volume was made with acetonitrile. It gives 100 µg/ml. 5ml of the solution were pipetted out separately into 10ml volumetric flask to give 10μg/ml concentration. The sample solution 20µl was injected and chromatographed.   3. Results and Discussions The goal of the present study is to develop and validate a simple, accurate and precise ultraviolet spectrophotometric method and reversed-phase high-performance liquid chromatographic (RP-HPLC) for the estimation of Amoxicillin in bulk and pharmaceutical dosage form.          Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         11 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com 3.1 Experimental trails  Figure 2: Chromatogram of Trial – 1  Trial – 1 was performed on LC, Chromatopak-C18 (250mm×4.6×5micron) column using mobile phase of methanol: water in the ratio of 50:50v/v at U.V detection at 267ηm.  Result: Broad peaks with some extra peaks were appeared so this was not satisfactory.  Figure 3: Chromatogram of Trial – 2   Trial – 2 was performed on Chromatopak-C18 (250mm×4.6×5micron) column using mobile phase of Acetonitrile: phosphate buffer in the ratio of 95:05 v/v at U.V detection at 283ηm.  Fronting with broad peaks and some extra peaks were appeared so this was not satisfactory. 0.403'2.098'2.827'273645546372819099108mV1 2 3 4 5 6 7 8 9 10 11 12 min1.913'2.238'3.312'10.058'11.617'-20020406080100120140160mV2 4 6 8 10 12 14 16 18 minSivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         12 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com  Figure 4: Chromatogram of Trial – 3  Mobile phase of Acetonitrile: Potassium dihydrogen phosphate buffer adjusted to pH 3 with orthophosphoric acid in the ratio of 22:78 v/v at U.V detectection at 283ηm  A sharp peak with a retension time of 6.461. The trail 3 was selected for the futher work in HPLC as the result was satisfactory with a sharp peak with the retension time of 6.461mins  3.2 Method Validation  The method was validated in accordance with ICH guidelines [13]. The parameters assessed were linearity, accuracy, and precision, reproducibility, robustness and system suitability.  3.2.1 Accuracy  Accuracy was best determined by the standard addition method. Previously analyzed samples of Amoxicillin API were added with standard drug solutions and are analyzed by the proposed method. Recovery (%), RSD (%) and bias (%) were calculated for each concentration.  Accuracy is reported as percentage bias, which is calculated from the expression  %Bias = (measured value - true value) true value X 100  3.2.2 Precision  System precision:  Standard solution prepared as per test method and injected six times and the %RSD value was calculated.  Method precision:  Six preparations individually using single batch of Amoxicillin drug substance were prepared as per test method and injected each solution induplicate on the same day in to HPLC. % RSD value was calculated to determine intra-day precision.  3.3 Repeatability Table 1: Precision Concentration [µg/ml] Peak area 50 6381711 50 6272822 50 6363603 50 6384644 50 6385755 50 6490823 Area Mean 6379893 S.D 69419.4 %R.S.D 1.088 3.4 Linearity  Various standards in the range 10-100μg/ml of Amoxicillin were injected onto the chromatographic system and the peak area was measured. A graph of peak area (on Y-axis) versus concentration (on X-axis) is plotted and the correlation coefficient was calculated.Table19 gives information about the linearity data. From the results, the correlation coefficient for Amoxicillin is 0.999 and, it can be concluded that the linearity is well within the limit. Hence the method is linear. Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         13 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com  Figure 5: Linearity graph of Amoxicillin  Table 2: Summary of Linearity Concentration (μg/ml) Peak Area 10 735698 20 1301541 30 1952312 40 2603083 50 3253854 60 3904625 80 5206167 100 6490823  3.5 Accuracy studies Table 3: Summary of Accuracy studies Spiked level (%) Formulation Conc. (µg/ml) Amount Found % Recovery % Mean recovery SD %RSD  50 50 79.55 99.45 99.66 0.599 0.601 50 79.41 99.2 50 80.27 100.34  100 50 81.14 101.4 101.3 0.602 0.594 50 81.57 101.9 50 80.62 100.7  150  50 79.94 99.92 100.17 0.296 0.296 50 80.1 100.1 50 80.42 100.5  The accuracy of the method, recovery studies were carried out by adding different amounts (50%, 100% and 150%) of bulk samples of Amoxicillin within the linearity range were taken and added to the pre-analyzed formulation of concentration 20µg/ml. From that percentage recovery values were calculated. The results were within the range and were found to be highly accurate which was shown in table 3.  y = 64436x + 39737R² = 0.999010000002000000300000040000005000000600000070000000 20 40 60 80 100 120Peak AreaConcentrationY-ValuesLinear (Y-Values)Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         14 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com  Figure 6: Chromatogram of 50%   Figure 7: Chromatogram of 100%  3. 6 Limit of Detection and Limit of Quantification The LOD and LOQ were calculated based on the standard deviation of the response and the slope of the constructed calibration curve, as described in International Conference on Harmonization guidelines Q2 (R1) was shown in table 4. Table 4: Summaries of LOD and LOQ Parameter Concentration (μg/ml) LOD 0.168 LOQ 0.512   3.7 Robustness The Robustness of the method was determined by making slight changes in the experimental conditions such as change in the flow rate, mobile phase and wave length. Table 5: Summary of Robustness Parameters Condition  %R.S.D RT Flow rate  0.9ml/min 0.379 6.450 Actual flow rate  1ml/min 0.236 6.461            Flow rate  1.1ml/min 0.22 6.470 Wavelength  267nm 0.218 6.421 Wavelength  283nm 0.236 6.461            Wavelength  276nm 0.258 6.455 Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         15 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com  Figure 8: Chromatogram of Flow rate: 0.9ml/min  Figure 9: Chromatogram of Flow rate: 1.1ml/min  Figure 10: Chromatogram of Flow rate: 1ml/min Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         16 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com 3.8 System Suitability The result of system suitability parameters for Amoxicillin is represented in Table 6 which indicates that all the system suitability parameters pass the criteria.  Figure 11: Chromatogram for system suitability  Table 6: Summary of system suitability Parameters Amoxicillin Retention time (min) 6.461            Theoretical plates 1199   HETP 0.014 Asymmetry 1.1  4. Conclusion A simple reverse phase HPLC method was developed for the determination of Amoxicillin present in pharmaceutical dosage forms. An LC Chromatopak-C18 (250mm×4.6×5micron) column UV Detector in isocratic mode, with mobile phase Acetonitrile: phosphate buffer, pH adjusted to 3 with orthophosphoric acid (22:78 v/v) was used. The flow rate was 1.0ml/min and effluent was monitored at 283ηm. The retention times were 6.4 min for Amoxicillin pharmaceutical dosage form. The linearity range was found to be 10-100g/ml. The proposed method was also validated. The proposed study describes a new RP-HPLC method for the Estimation of Amoxicillin in pharmaceutical dosage form. The method gave good results within a short analysis time. The developed method was validated in accordance with ICH guidelines and all of the results were within the limits. The HPLC method for the Estimation of Amoxicillin in tablet dosage form was also found to be simple, rapid, precise, accurate and sensitive. A good agreement was observed with HPLC method. The validated HPLC method can be used for the routine analysis of quality control samples. Since the developed method has been applied only to a single brand (Amoxicillin 125) the same method is applicable to different brands. References [1]. Bojaraju V., Sireesha D., Prasad V.V.L.N. and Diwan Prakash. V. Reverse Phase High Performance Liquid Chromatographic Method for the Simultaneous Estimation of Amoxicillin Trihydrate and Tinidazole in the Tablet Dosage Form. World Journal of Chemistry. 2012; 7 (2): 47-52.  [2]. Sahoo NK, Sahu M, Algarsamy V, Srividya B and Sahoo CK. Validation of Assay Indicating Method Development of Amoxicillin in Bulk and One of Its Marketed Dosage Form by RP-HPLC. Ann Chromatogr Sep Tech. 2016; 2(1): 1014. [3]. Nidal Batrawi, Shorouq Wahdan and Fuad Al-Rimawi A Validated Stability-Indicating HPLC Method for Simultaneous Determination of Amoxicillin and Enrofloxacin Combination in an Injectable Suspension. Sci. Pharm. 2017; 85 (6): 1-8. [4]. Ahmed, Manzoor; Suresh Babu, G.; Shetty, A. Sathish Kumar Development and Validation of Amoxicillin by RP-HPLC Method in Bulk drug and Pharmaceutical dosage forms. International Journal of Chem Tech Research; 2011; 3 (3): 1037 [5]. Basavaiah K., Kalsang T, Rajendra Prasad N, Hiriyanna S G & Vinay K B, Determination of amoxicillin in pharmaceutical forms by Visible spectrophotometry and HPLC. IJCT, 2009; 16: 265-271. [6]. S. Beg, M. S. Hasnain, S. Swain, and K. Kholi validated stability indicating LC method for estimation of amoxicillin trihydrate in pharmaceutical Sivagami B et al / A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms                         17 IJAPA|VOL 08|ISSUE 01|2018                                                 www.ssjournals.com dosage forms and time dependant release formulations. IJPSN, 2011; 4(2): 1423-1427. [7]. ICH harmonized triplicate guide line text of validation of analytical procedures recommended for adaptation at September 4th of the ICH process on 27th October 1994.  [8]. ICH: Validation of analytical procedure: Methodology Q2B; 1996. [9]. ICH Stability Testing of New Drug Substances and Products Q1A (R2), International Conference on Harmonization, 2003.  [10]. ICH Q2A, Validation of Analytical Procedures: Definitions and Terminology, Geneva1995, in 2005 incorporated in Q2 (R1).  

A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms

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A Validated RP-HPLC Method Development for Amoxicillin in Pharmaceutical Dosage Forms

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International Journal of Advances in Pharmaceutical Analysis