A simple, rapid, precise, sensitive and, reproducible reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of Cefpodoxime in a pharmaceutical dosage form. Chromatographic separation of Cefpodoxime was achieved on Waters Alliance -2695, by using Luna Pheny Hexyl (250mm x 4.6mm, 5µm) column and the mobile phase containing 0.1% TEA adj pH-2.5 with OPA &amp; ACN in the ratio of 75:25% v/v. The flow rate was 1.0 ml/min, detection was carried out by absorption at 222nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Cefpodoxime were NLT 2000 and should not be more than 2 respectively. The linearity of the method was excellent over the concentration range 7-105 µg/ml for Cefpodoxime respectively. The correlation coefficient was 0.999. % Relative standard deviation of peak areas of all measurements always less than 2.0. The proposed method was validated according to ICH guidelines.&nbsp; The method was found to be a simple, economical, suitable, precise, accurate &amp; robust method for quantitative analysis of Cefpodoxime and study of its stability

BEGUM, S. K. SABEEHA

JYOTHI, N. BARATHA

SARALA, R.

SRAVANI, G.

TULASI, R.

English

Asian Journal of Advances in Medical Science

_____________________________________________________________________________________________________  *Corresponding author: Email: jyothikiran.81@gmail.com;   Original Research Article Asian Journal of Advances in Medical Science    3(1): 416-425, 2021      A STUDY ON DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE ESTIMATION OF CEFPODOXIME IN BULK AND A PHARMACEUTICAL DOSAGE FORMS  S. K. SABEEHA BEGUM1, N. BARATHA JYOTHI2*, R. TULASI3, R. SARALA4  AND G. SRAVANI5 1Department of Chemistry, Maris Stella College (A) Vijayawada 520008, Acharya Nagarjuna University, Nagarjuna Nagar 522510, Guntur, Andhra Pradesh, India. 2Department of Zoology, Maris Stella College (A) Vijayawada 520008, Acharya Nagarjuna University, Nagarjuna Nagar 522510, Guntur, Andhra Pradesh, India. 3Department of Mathematics, Maris Stella College (A) Vijayawada 520008, Acharya Nagarjuna University, Nagarjuna Nagar 522510, Guntur, Andhra Pradesh, India. 4Department of Electronics, Maris Stella College (A) Vijayawada 520008, Andhra Pradesh, India. 5Department of Zoology and Aquaculture, Acharya Nagarjuna University, Nagarjuna Nagar 522510,  Guntur, Andhra Pradesh, India.  AUTHORS’ CONTRIBUTIONS  This work was carried out in collaboration among all authors. Author SKSB did the conception and design. Authors NBJ, RT, RS and GS did the provision of study material or patients: All authors. Authors SKSB, NBJ, RT, RS and GS did the collection and assembly of data. Authors SKSB, NBJ, RT, RS and GS did the data analysis and interpretation. All authors did the manuscript writing, read and approved the final manuscript.    Received: 04 September 2021 Accepted: 08 November 2021 Published: 19 November 2021   ABSTRACT  A simple, rapid, precise, sensitive and, reproducible reverse phase high performance liquid chromatography (RP-HPLC) method has been developed for the quantitative analysis of Cefpodoxime in a pharmaceutical dosage form. Chromatographic separation of Cefpodoxime was achieved on Waters Alliance -2695, by using Luna Pheny Hexyl (250mm x 4.6mm, 5µm) column and the mobile phase containing 0.1% TEA adj pH-2.5 with OPA & ACN in the ratio of 75:25% v/v. The flow rate was 1.0 ml/min, detection was carried out by absorption at 222nm using a photodiode array detector at ambient temperature. The number of theoretical plates and tailing factor for Cefpodoxime were NLT 2000 and should not be more than 2 respectively. The linearity of the method was excellent over the concentration range 7-105 µg/ml for Cefpodoxime respectively. The correlation coefficient was 0.999. % Relative standard deviation of peak areas of all measurements always less than 2.0. The proposed method was validated according to ICH guidelines.  The method was found to be a simple, economical, suitable, precise, accurate & robust method for quantitative analysis of Cefpodoxime and study of its stability.  Keywords: HPLC; Cefpodoxime; stability; pharmaceutical dosage.     Begum et al.; AJOAIMS, 3(1): 416-425, 2021    417  1. INTRODUCTION  1.1 Drug  A drug may be defined as a substance meant for diagnosis, cure mitigation, prevention or treatment of diseases in human beings or animals or for altering any structure or any function of the body.  The development of human civilization plays a vital role in finding cures for diseases. Today the majority of drug users is of synthetic origin. These are produced in the bulk and used for their therapeutic effects in pharmaceutical formulations [1-2]. There are biologically active chemical substances generally formulated into convenient dosage forms such as tablets, capsules, ointments and, injectables, these formulations deliver the drug substance in stable, non-toxic and acceptable form, ensuring its bioavailability and,  therapeutic activity [3-4].  1.2 Methodology   Purpose and objectives of the study  In order to develop a simple, reliable and, accurate method for the assay of Cefpodoxime tablet dosage form by reverse-phase HPLC and validate the method for its repeatability and reproducibility [5-6].  The plan work includes:  ✔ Procurement of raw materials. ✔ Establishment of system suitability parameters. ✔ Trials for the method development for Cefpodoxime.  ✔ Setting the optimized method. ✔ Validation of optimized methods for Cefpodoxime.  Validation parameters like:  ❖ System suitability and system precision. ❖ Specificity ❖ Linearity and range. ❖ Accuracy. ❖ Precision.  ● Method precision ● System precision ● Intermediate precision.  ❖ Robustness ❖ Force degradation  2. MATERIALS AND EQUIPMENTS  1. Chemicals and Reagents:  Table 1. Chemicals and reagents  S. NO NAME of REAGENT MAKE GRADE 1 Water Rankem HPLC Grade 2 Triethyl amine Merck HPLC Grade 2 Ortho phospharic Acid Merck HPLC Grade 3 Acetonitrile Merck HPLC Grade 4 Hydrochloric acid Merck Pure 5 Sodium hydroxide Merck Pure 6 Hydrogen peroxide Merck Pure 7 0.45 µm nylon filter Zodiac life sciences # 100428036  2. Standards and samples:  Table 2. Standards and Samples  S. NO NAME of the DRUG MANUFACTURER  or SUPPLIER 1 Cefpodoxime working standard Cipla pvt .Ltd 2 Cefpodoxime drug substance Cipla pvt .Ltd 3 Cefpodoxime tablets 200 mg  Cipla pvt .Ltd       Begum et al.; AJOAIMS, 3(1): 416-425, 2021    418  3. Instruments or equipments details:  Table 3. Instruments or equipment’s  S. NO INSTRUMENT NAME MAKE  and MODEL 1 HPLC Waters(alliance 2695) 2 Ultra-sonicator Unichrome 3 pH meter GT Sonic 4 Electronic balance Ohaus 5 HPLC column Luna Pheny Hexyl, 250mm x 4.6mm, 5µm. 6 Centrifuge Remi 7 Refrigerator Whirlpool  2.1 Experimental Details  Analytical method development:  In the proposed project a successful attempt has been made to develop a simple, accurate for analysis of Cefpodoxime tablets (200mg) by RP-HPLC.  Method development parameters:  Selection of following parameters is very important in method development.  ⮚ Mode of chromatography. ⮚ Wave length. ⮚ Column. ⮚ Mobile phase composition and buffer PH. ⮚ Column temperature. ⮚ Solvent delivery system. ⮚ Flow rate. ⮚ Injection volume.  Selection of mode of chromatography:  Selected mode of chromatography:  Reverse phase  Basis of selection: polarity of the molecule  Reason for selection: As Cefpodoxime is a polar molecule it elutes at faster rates along with the mobile phase.  Selection of detector wavelength:  Selection of detector wavelength is a critical step in finalization of the analytical method. To determine exact wave length standard API is prepared and injected into a chromatographic system with a PDA detector and the wave length which gives higher response for the compound [7-9].  3. RESULTS AND DISCUSSION  PDA Spectrum    Fig. 1. PDA spectrum  S. NO WAVE LENGTH 1. 222 nm     Begum et al.; AJOAIMS, 3(1): 416-425, 2021    419  ANALYTICAL METHOD DEVELOPMENT OF FENOFIBRATE BY RP-HPLC METHOD TRAIL CHROMATOGRAMS  TRAIL -1    Fig. 2. Typical chromatogram trail 1   Name Retention Time Area % Area USP Tailing USP Plate Count 1  4.471 1937340 100    TRAIL - 2   Fig. 3.  Typical chromatogram trail 2   Name Retention Time Area % Area USP Tailing USP Plate Count 1  2.554 92149 100    TRAIL – 3    Fig. 4. Typical chromatogram trail 3     Begum et al.; AJOAIMS, 3(1): 416-425, 2021    420  Name Retention Time Area % Area USP Resolution USP Tailing USP Plate Count  2.088 9551426 100.12  1.14 3345  TRAIL – 4    Fig. 5. Typical chromatogram trail 4   Name Retention Time Area % Area USP Tailing USP Plate Count 1  2.715 8456322 100.48 1.14 2563  TRAIL – 5    Fig. 6. Typical chromatogram trail 6   Name Retention Time Area % Area USP Tailing USP Plate Count 1  3.589 18586274 100.82 3.56 1858  TRAIL – 6    Fig. 7. Typical chromatogram trail 6     Begum et al.; AJOAIMS, 3(1): 416-425, 2021    421   Name Retention Time Area % Area USP Tailing USP Plate Count 1 Fenofibrate 4.357 2062896 100 1.20 5666  ANALYTICAL METHOD VALIDATION OF FENOFIBRATE:-  System suitability:  Results for system suitability of Cefpodoxime:  Acceptance Criteria:  ⮚ The % RSD for the retention times of Cefpodoxime peaks from 6 replicate   injections of each standard solution should be not more than 2.0 %. ⮚ The % RSD for the peak area responses of Cefpodoxime peaks from 6 replicate injections of each standard solution should be not more than 2.0%.  ⮚  The number of theoretical plates (N) for the Cefpodoxime peaks is not less than 2000. ⮚ The Tailing factor (T) for the Cefpodoxime peaks is not more than 2.0.     Table 4. Results for system suitability of cefpodoxime  Injection Retention time (min)  Peak area  Theoretical plates (TP)  Tailing factor (TF)  1 3.179 1613079 5059 1.21 2 3.187 1608749 5035 1.21 3 3.211 1606510 4913 1.23 4 3.208 1610506 5135 1.21 5 3.222 1614302 5033 1.21 6 3.255 1608089 52250 1.20 Mean 3.210 1610206 5070 1.21 SD  3012.83 -- -- %RSD  0.1871 -- --  Specificity:  BLANK    Fig. 8. Typical chromatogram of blank  S. No. Name Rt (min) Peak Area Efficiency 1 Blank - - -      Begum et al.; AJOAIMS, 3(1): 416-425, 2021    422  Acceptance Criteria:-  Chromatograms of blank should not show any peak at the retention time of analytic peak.  SAMPLE    Fig. 9. Typical chromatogram of sample  S. No. Name Rt (min) Peak Area 1 Cefpodoxime 3.179 1608089  STANDARD    Fig. 10. Typical chromatogram of standard  S.No. Name Rt (min) Peak Area 1 Cefpodoxime 3.208 1606510  Linearity:-  Linearity of detector response for Cefpodoxime:  Table 5. Linearity of detector response for atrovastatin  S.No. Conc.(µg/ml) of Cefpodoxime Area Acceptance criteria Cefpodoxime 1 7.0 µg/ml 214633 Squared correlation coefficient should be not less than 0.999. 2 17.5 µg/ml 524156 3 35 µg/ml 1032584 4 52.5 µg/ml 1526321 5 70 µg/ml 2062896 6 87.5 µg/ml 2524821 7 105 µg/ml 3025963     Begum et al.; AJOAIMS, 3(1): 416-425, 2021    423  Linearity graphs of Atrovastatin:    Fig. 11. linearity of detector response graphs for Atrovastatin  Table 6. linearity of detector response graphs for Atrovastatin  S. No Linearity Parameters Atrovastatin 1. Linearity range 7.0-105 µg/ml 2. Correlation coefficient 0.999 3. Y intercept 13374x + 52695  Acceptance criteria:  1. Individual % recovery and mean % recovery of both drugs should be between 98.0 and 102.0 . 2. For replicate preparations the %RSD should not more than 2.0. 3. For linearity of test method, the squared correlation coefficient derived from least square fit of the data should not be less than 0.999.  Chromatographs of linearity:    Fig. 12. Typical chromatogram of linearity 7.0 µg/ml     Begum et al.; AJOAIMS, 3(1): 416-425, 2021    424    Fig. 13. Typical chromatogram of linearity 17.5 µg/ml  Summary of Results:  S. No Parameter Result 1. System suitability   Relative standard deviation for area of Cefpodoxime peak for six replicate injections of standard solution 0.82 Tailing factor is 1.12  Theoretical plates are 3525 2. Specificity Blank interferences  No peaks are observed in the blank chromatogram at the retention time of the Cefpodoxime peaks. 3 linearity The square of correlation coefficient value of Cefpodoxime is 0.999. 4 accuracy Individual % recovery for Cefpodoxime is found to be in the range of 95.0 to 105%. Mean % recovery for Cefpodoxime is found to be in the range of 95.0 to 105%. %RSD for of Cefpodoxime is found to be in the range of  0.10 to 1.52. For the linearity of the test method, the squared correlation coefficient derived from the least square fit of the data for Cefpodoxime is 0.99. 5. Precision  Method precision System precision Intermediate precision The % assay of each individual preparation is found to be in range of 95.0 to 105%.  The % RSD for assay of six replicate preparations of Cefpodoxime is 0.32 and 0.34. The %RSD of individual preparation of Cefpodoxime should be in the range of less than 2. i.e. 1.21  6. Robustness From rate variation organic phase variation  All the parameters are found to be within the acceptable limits for all the robustness parameters.  4. CONCLUSION  Development and validation of RP-HPLC method for the estimation of Cefpodoxime in bulk and Pharmaceutical dosage forms with the facilities and the results are incorporated in this paper. In conclusion a validated RP-HPLC method has been developed for the determination of Cefpodoxime the bulk and tablet dosage forms. The results show that the method was found to be specific, simple, accurate, precise and sensitive. The method was successfully applied for the determination of the Cefpodoxime tablet dosage form.  Several analytical procedures have been proposed for the quantitative estimation of Cefpodoxime separately and in combination with other drugs.         Begum et al.; AJOAIMS, 3(1): 416-425, 2021    425  So the attempt was taken to develop and validate a reversed-phase high performance liquid chromatographic method for the quality control of Cefpodoxime in pharmaceutical preparations with lower solvent consumption along with the short analytical run time that leads to an environmentally friendly chromatographic procedure and will allow the analysis of a large number of samples in a short period of time.  COMPETING INTERESTS  Authors have declared that no competing interests exist.  REFERENCES   1. Laurenc L Brunton, John S Lazo, Keith L. Parker. ‘Goodman and Gilman’s The pharmacological basis of Therapeutics; McGraw Hill, Newyork, U.S.A. 2006;11:1136-1142.  2. O’Neil MJ. The Merk Index- an encyclopedia of chemicals, Drugs and Biologicals, New Jersy, Merk and co., INC. 1917;13:159. 3. Indian Pharmacopoeia. Indian Pharmacopoeia commission, Ghaziabad. 2010;2:149,857,151, 1018. 4. British Pharmacopoeia. London, Medicines and Health care products Regulatory Agency (MHRA). 2005;3:192. 5. John HB, John MB. Wilson and gisvold’s textbook of organic medicinal & pharmaceutical chemistry, lippincott williams & wilkins, wolterskluner company. 2004;11:330. 6. Sean C. Sweetman, martindale the complete references; pharmaceutical press, 1 Lambeth High Street, London SEI 7 IN, UK. 2002;33:172.  7. Darji B H, NJ Shah, AT Patel, NM Patel. Development and validation of a HPTLC method for the estimation of cefpodoxime proxetil. Indian J. of Pharm. Sci. 2007;69(2):331-3. 8. Siddalinga Swamy MS, Sathish Kumar Shetty A, Anil kumar SM. UV-Visible spectrophotometric methods for the estimation of cefpodoxime proxetil in bulk drug and pharmaceutical dosage form. Int.J. PharmTech Res. 2012;4(2):750-6. 9. Narendra Nyola, Govindasamy Jeyabalan. Simultaneous estimation of Cefixime And Azithromycin in API’S and pharmaceutical dosage form by RP-HPLC. Indo American. J.of Pharm Res. 2013;2(12):1472-81. __________________________________________________________________________________________ © Copyright MB International Media and Publishing House. All rights reserved.  

A STUDY ON DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE ESTIMATION OF CEFPODOXIME IN BULK AND A PHARMACEUTICAL DOSAGE FORMS

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A STUDY ON DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR THE ESTIMATION OF CEFPODOXIME IN BULK AND A PHARMACEUTICAL DOSAGE FORMS

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