The present work describes the development of a sensitive and economic stability indicating high performance liquid chromatographic (HPLC) method for the determination of cefpodoxime proxetil (CP) as bulk drug and as pharmaceutical formulation. Both R and S isomers of the drug were separated using Phenomenex (
Stability indicating methods are the quantitative analytical methods that are based on the characteristic structural, chemical, or biological properties of each active ingredient of a drug product and that will distinguish each active ingredient from its degradation products so that the active ingredient content can be accurately measured [
Stability indicating method is an analytical procedure that is capable of discriminating between the major active pharmaceutical ingredient (API) and any degradation (decomposition) product(s) formed under defined storage conditions during the stability evaluation period [
Structure of Cefpodoxime proxetil (CP).
In many of the HPLC methods, acetonitrile is used as the organic phase. It is a toxic chemical as it can cause environmental pollution and health hazards to human beings and animals [
Cefpodoxime proxetil API (with a purity of 99.30% which contains both R and S isomers) was available as a gift sample from See Gee Pharmaceuticals Pvt. Ltd., Puducherry. Methanol, disodium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from S.D Fine Chemicals (Mumbai, India) and were of HPLC grade. Hydrochloric acid, sodium hydroxide pellets, hydrogen peroxide, and orthophosphoric acid were purchased from S.D Fine Chemicals (Mumbai, India) and were of A.R grade. Purified water was prepared in-house by distillation of water in triplicate followed by filtration through filter paper of 0.45
The HPLC system consists of a Shimadzu LC 20 AD binary pump and SPD 20 A UV detector. The column consists of Phenomenex (250 mm length, 4.6 mm internal diameter, and 5
Chromatographic separation was achieved using Phenomenex ODS column. The LC system was operated isocratically using a mobile phase consisting of a mixture of methanol and phosphate buffer of pH 4.0 (prepared with 5.04 g of disodium hydrogen phosphate and 3.01 g potassium dihydrogen phosphate dissolved in 1000 mL of triple distilled water and the pH was adjusted to 4.0 using orthophosphoric acid) in the ratio of 65 : 35 as the mobile phase at a flow rate of 1.0 mL/min at room temperature. The injection volume was 20
The stock solution of CP was prepared by weighing 10 mg of the reference substance and transferring it to a 10 mL volumetric flask, diluting to 10 mL with methanol (1000
Two brands of cefpodoxime tablets (Cefoact—100 mg, Cepodem—100 mg) were purchased from a local pharmacy. Twenty tablets of each brand were accurately weighed and crushed to fine powder separately. A quantity of powder equivalent to 25 mg of CP was transferred to a 25 mL volumetric flask and added 10 mL of methanol, kept in an ultrasonic bath for 10 min and made up to the volume with methanol and filtered.
The developed method was validated as per ICH guidelines using CP with respect to the following parameters: accuracy, precision, LOD, LOQ, specificity, robustness, stability, and system suitability.
For testing linearity, seven calibration standards were prepared in the range of 5 to 100
Normally, limit of detection (LOD) and limit of quantitation (LOQ) are estimated at a signal to noise ratio of 3 : 1 and 10 : 1, respectively. LOD and LOQ were calculated using (
Consider
Specificity is the ability of a method to measure analytical response in presence of its potential impurities. Specificity of the method was carried out by the deliberate degradation of the drug by oxidation, heat, hydrolysis (acidic, alkaline, neutral), and photolysis, followed by its analysis using the developed method.
Experimental conditions were deliberately altered, and resolution of CP from its degradation products was noticed, in order to determine the robustness. From the different experimental conditions such as flow rate (1.0 mL/min), lambda max (252 nm), and percentage of methanol (65), each selected factor was changed at three levels (−1, 0, and +1). One factor was changed at a time to study the impact of the change in the experimental conditions on the assay results. Difference in the peak area and the retention time were noted at each change in the analytical parameters.
Sample solution was prepared and analysed by the HPLC instrument using fresh mobile phase at different time intervals (0 h, 8 h, and 24 h).
Accuracy of the developed method was assessed in triplicate at three concentrations (40, 60, and 80
The precision of the analytical method was evaluated by the determination of the repeatability of the method (intraday precision) and intermediate precision (interday precision) of the sample solutions. Repeatability was calculated by assaying six samples prepared on the same day. Intermediate precision was calculated by assaying 3 days. The relative standard deviation of the area of peaks was calculated.
The chromatographic conditions were optimized with a view to obtain symmetrical peaks with good resolution between the R and S optical isomers of cefpodoxime proxetil. At the same time, these peaks should be well separated from the degradation peaks so that they can be quantified even in the presence of degradants. Separation was achieved using a mobile phase consisting of methanol and phosphate buffer of pH 4.0 (pH was adjusted to 4.0 with o-phosphoric acid) in the ratio of 65 : 35 by an isocratic profile, pumped at a flow rate of 1 mL min−1. The eluent was monitored using UV detector at a wavelength of 252 nm. The column was maintained at ambient temperature with an injection volume of 20
Concentrations of LOD and LOQ were found to be 53 and 160 ng/mL, respectively.
The linearity graph is given in Figure
(a) Chromatogram of CP standard 100
The regression equation for the graph is
The drug was found to be stable in the mobile phase. This was proved by injecting the standard solution at 0, 8, and 24 h after preparation which showed the absence of any extra peaks due to degradation, and there was not much change in the drug peak area (% RSD = 1.19) (Table
Stability of sample solution.
Concentration of std. ( |
Postpreparation time in h | Peak area |
---|---|---|
100 | 0 | 5329310 |
100 | 8 | 5239298 |
100 | 24 | 5361394 |
| ||
% RSD | 1.19 |
The percentage relative standard deviation (% RSD) of the area of CP during intraday study was found to be less than 0.4 and for interday study was found to be less than 1.1, which indicated a good precision of the method (Table
Intra- and interday precision.
Con. (µg mL−1) | Intraday precisiona | Interday precisionb | ||||
---|---|---|---|---|---|---|
Mean con. | SD | % RSD | Mean con. | SD | % RSD | |
40.0 | 40.60 | 0.072 | 0.178 | 40.55 | 0.405 | 1.000 |
60.0 | 60.43 | 0.300 | 0.496 | 60.73 | 0.130 | 0.215 |
80.0 | 80.33 | 0.154 | 0.192 | 80.37 | 0.346 | 0.431 |
aMean concentration of six trials. bMean concentration of nine trials.
The quantitative recovery of CP achieved ranged from 100.24 to 102.25% with a low % RSD value. The results of the recovery experiments done at 3 concentration levels and the % RSD values are given in Table
Recovery studies.
Excess drug added to analyte (%) | Drug content in µg mL−1 | Recovery (%) | % RSD | |
---|---|---|---|---|
Theoretical | Practicala | |||
0 | 20 | 20.49 | 102.46 | 0.687 |
16 | 36 | 36.08 | 100.236 | 0.705 |
20 | 40 | 40.90 | 102.253 | 0.165 |
24 | 44 | 44.90 | 102.041 | 0.212 |
aMean concentration of six trials.
The validated method was applied for the assay of CP in 2 brands of cefpodoxime tablets (Cefoact—100 mg, Cepodem—100 mg) (Table
Analysis of the marketed formulation.
Formulation | Labelled amount | Amount found ± SDa | % RSD | % Assay |
---|---|---|---|---|
Tablet Cefoact | 100 mg per tablet | 101.04 ± 0.84 | 0.415 | 101.04 |
Tablet Cepodem | 100 mg per tablet | 101.78 ± 0.06 | 0.027 | 101.77 |
aMean concentration of three trials.
The results in the robustness study, as shown in Table
Robustness evaluation.a
Factor | Level | Retention time (Rt) for both isomers of CP (min) | Total area for both isomers | |
---|---|---|---|---|
(A) Flow rate (mL min−1) | ||||
0.95 | −1 | 7.212 | 8.122 | 5280908 |
1 | 0 | 7.066 | 7.927 | 5239656 |
1.05 | +1 | 6.99 | 7.836 | 5272832 |
Mean ± SD ( |
7.089 ± 0.113 | 7.962 ± 0.146 | 5264465 ± 21861 | |
| ||||
(B) Percentage of methanol in mobile phase | ||||
64 | −1 | 7.133 | 8.01 | 5339448 |
65 | 0 | 7.066 | 7.927 | 5239656 |
66 | +1 | 6.911 | 7.82 | 5311649 |
Mean ± SD ( |
7.037 ± 0.114 | 7.934 ± 0.099 | 5296917.67 ± 51501 | |
| ||||
(C) Wavelength of measurement | ||||
251 | −1 | 7.068 | 7.932 | 5256020 |
252 | 0 | 7.066 | 7.927 | 5239656 |
253 | +1 | 7.095 | 7.912 | 5369302 |
Mean ± SD ( |
7.0763 ± 0.016 | 7.924 ± 0.010 | 5288326 ± 70603 |
aMean values of six trials.
In order to check the specificity of the proposed method, degradation studies were carried out by using acidic, basic, photolytic, oxidative, and thermal conditions. Cefpodoxime proxetil active pharmaceutical ingredient (API) powder was stressed under various conditions to conduct forced degradation studies. Intentional degradation was performed by subjecting it to various stress conditions of acidic (1 N HCl), basic (0.1 N NaOH), neutral (water), oxidative (3% H2O2), thermal (heated at 105°C), and photolytic degradation to evaluate the ability of the proposed method to separate CP from its degradation products. Chromatogram of CP, along with the degradation products under various stress conditions, is shown in Figures
(a) Chromatogram for alkaline (0.1 N NaOH) degradation. (b) Chromatogram for dry heat degradation. (c) Chromatogram for hydrolytic degradation. (d) Chromatogram for photolytic degradation. (e) Chromatogram for oxidative degradation. (f) Chromatogram for acidic (1 N HCl) degradation.
The degradation kinetics of CP was performed by extending the stress degradation to various time intervals for acidic, alkaline, and oxidative stress, the data of which is given in Tables
Residual drug (%) after acidic degradation (
Time in h | Residual drug (%) ± SD |
---|---|
0 | 100 ± 0 |
24 | 84.21 ± 2.52 |
48 | 82.49 ± 1.83 |
72 | 77.25 ± 2.11 |
96 | 72.07 ± 0.20 |
Residual drug (%) after alkaline degradation (
Time in min | Residual drug (%) ± SD |
---|---|
0 | 100 ± 0 |
1 | 20.17 ± 0.29 |
2 | 14.90 ± 0.34 |
3 | 2.67 ± 0.15 |
4 | 1.87 ± 0.052 |
Residual drug (%) after oxidative degradation (
Time in h | Residual drug (%) ± SD |
---|---|
0 | 100 ± 0 |
24 | 62.34 ± 0.12 |
48 | 30.62 ± 2.33 |
72 | 12.84 ± 0.22 |
Degradation rate constant and half-life.
Sl. no. | Stress condition | Rate constant | Half-life |
---|---|---|---|
(1) | Alkaline (0.1 N) | 0.021 sec−1 | 33.10 sec |
(2) | Oxidative (3% v/v H2O2) | 0.023 h−1 | 29.96 h |
(3) | Acidic (1 N) | 0.0029 h−1 | 302.18 h |
Kinetics of degradation plotted as residual drug (%) ± SD versus time for (a) acidic degradation, (b) alkaline degradation, and (c) oxidative degradation.
An economic stability indicating HPLC method has been developed and validated for the determination of CP in API and different pharmaceutical formulations. The method is accurate, precise, and specific and also has the ability to analyse the drug in presence of its degradation products, and it can be employed as a stability indicating assay. The drug peak was well separated from degradation products’ peaks. The most interesting fact is that the method could separate the R and S isomers without the use of a chiral column or a chiral selector. On comparing the stability of the drug under different stresses, it had undergone faster degradation in alkaline condition, as the drug was almost completely degraded within 4 min and then in oxidative condition. In acidic media, the drug was comparatively stable.
The authors declare no conflict of interests.
The authors wish to thank the principal, Professor C. V. S. Subrahmanyam, and the management of Gokaraju Rangaraju College of Pharmacy, Bachupally, Hyderabad, for wholeheartedly supporting this work.