The present paper describes the development of quick stability indicating RP-HPLC method for the simultaneous estimation of codeine phosphate and chlorpheniramine maleate in the presence of its degradation products, generated from forced degradation studies. The developed method separates codeine phosphate and chlorpheniramine maleate in impurities/degradation products. Codeine phosphate and chlorpheniramine maleate and their combination drug product were exposed to acid, base, oxidation, dry heat, and photolytic stress conditions, and the stressed samples were analysed by proposed method. The proposed HPLC method utilizes the Shimadzu HPLC system on a Phenomenex C18 column (
Codeine phosphate (CP) chemically is 7,8-didehydro-4,5-epoxy-3-methoxy-17-methylmorphinan-6-ol dihydrogen phosphate hemihydrate [
Chemical structure of (a) codeine phosphate and (b) chlorpheniramine maleate.
Chlorpheniramine maleate (CPM) chemically is (
The literature survey reveals that many analytical methods are reported for the determination of CP and CPM individually and in combination. The literature survey revealed spectroscopic [
The stability of a drug dosage form refers to the ability of a particular form to maintain its physical, chemical, therapeutic, and toxicological specification presented in the monograph on identity, strength, quality, and purity. The stability of a drug product should ordinarily be demonstrated by its manufacturer by methods appropriate for the purpose. Obviously, a stability testing problem is never simple. Stability testing is an important part of the process of drug product development. The purpose of the stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors, such as temperature, humidity, and sun light, and enables recommendation of storage conditions, retest periods, and shelf lives to be established.
Two main aspects of a drug product that play an important role in shelf life determination are the assay of the active drug and the degradation products generated during the stability study. The drug product in a stability test sample needs to be determined using a stability indicating method, as recommended by the International Conference on Harmonization (ICH) guidelines [
The literature survey reveals that many analytical methods are reported for the determination of CP and CPM individually and in combination. The combination of CP and CPM in liquid dosage form has not been reported in any pharmacopoeia. So far, no RP-HPLC stability indicating method has been reported for the simultaneous estimation of CP and CPM in liquid pharmaceutical formulation. Therefore, it is necessary to develop a new rapid and stability indicating method for simultaneous estimation of two compounds (CP and CPM) in liquid pharmaceutical formulation. The proposed method is able to separate CP and CPM with each other and from its impurities/degradation products. Thereafter, this method was validated as per ICH guidelines and successfully applied for separation and quantification of all compounds of interest in the liquid pharmaceutical formulation.
Codeine phosphate impurities (CP-IMPs); A, B, C, and D, are official in British Pharmacopoeia [
The aim of this work was to develop a quick stability indicating RP-HPLC method for the simultaneous estimation of codeine phosphate and chlorpheniramine maleate in the presence of its degradation products, generated from forced degradation studies. The stability indicating power of the method was established by comparing the chromatograms obtained under optimized conditions before forced degradation with those after forced degradation via acidic, basic, oxidative, thermal, and photolytic stress conditions.
Methanol, water, acetonitrile, and
Standard Stock Solutions of CP and CPM were prepared in mobile phase (1 mg/mL).
From CP solution, 10 mL was taken and diluted to 100 mL with mobile phase (100
An accurately weighed 2 gm of placebo solution was taken into the 100 mL volumetric flak. About 70 mL of diluent was added to this volumetric flask and sonicated in an ultrasonic bath for 5 min. This solution was then diluted up to the mark with diluent and mixed well. It is then filtered through 0.22
Syrup equivalent to 10 mg and 4 mg of CP and CPM was taken in a 100 mL volumetric flask. Volume was made to the mark with mobile phase. The flask was sonicated for 5 min. The above solution was filtered using 0.22
Assay of formulation.
Formulation | Drug | Label claim | Amount found, (mg) AM ± SD* | % Assay | % RSD |
---|---|---|---|---|---|
Coscodin | CP | 10 mg | 10.01 ± 0.05 | 100.1 | 0.55 |
CPM | 4 mg | 3.99 ± 0.006 | 99.75 | 0.16 | |
Mit’s Linctus Codeinae Co. | CP | 10 mg | 10.05 ± 0.05 | 100.5 | 0.53 |
CPM | 4 mg | 4.11 ± 0.006 | 102.75 | 0.16 |
The selectivity of the method was evaluated with regard to interference due to the presence of any other excipients. This shows that drugs were clearly separated from its excipients. Thus, the HPLC method presented in this study was found to be selective, depicted in Figure
Overlay chromatograms of blank, placebo, and sample preparation.
The linearity of calibration curves (peak area ratio versus concentration) in pure solution was checked over the concentration ranges of about 50–150
Three injections of same concentration were given on the same day, and these studies were also repeated on different days to determine interday precision.
The accuracy of the method was validated by recovery studies and was found to be significant under specification limits.
Limit of detection was calculated by using the formula:
Limit of quantification was calculated by using the formula:
To determine robustness of the method the experimental conditions were deliberately changed. The flow rate of the mobile phase, pH of the mobile phase, was varied parameters. The study was performed on same day. The area obtained from each variation was compared with that obtained under optimized conditions.
Sample compatibility was performed for nylon 0.22
Filter compatibility results (assay % w/w).
Compound | Centrifuged | PVDF filter 0.22 |
Nylon filter 0.22 |
---|---|---|---|
CP | 99.5 | 99.3 | 99.3 |
CPM | 100.3 | 100.5 | 100.4 |
Stability of sample solution was established by the storage of sample solution at 25°C for 24 hr. Sample solution was reanalyzed after 12 and 24 hr time intervals, and assay was determined for the compounds (CP and CPM) and compared against fresh sample. Sample solution did not show any appreciable change in assay value when stored at ambient temperature up to 24 hr. The results from solution stability experiments confirmed that sample was stable up to 24 hr during assay determination.
Stress studies were carried by using 50
The oxidative study was carried out in 10% hydrogen peroxide (H2O2) for 24 hr.
Photolytic studies on the drug were carried out by the exposure to UV lamp in UV cabinet at 254 nm.
For thermal stress testing, both the drugs were spread in petridish and placed in the oven at 105°C. Samples were withdrawn periodically and subjected to analysis after suitable dilution.
The main objective of the RP-HPLC method was to determine a validated stability indicating method for the estimation of CP and CPM simultaneously in liquid pharmaceutical formulation and to obtain well-resolved peaks of CP, CPM, and impurities/degradants. The method should be able to determine degradants/impurities and assay of two compounds in single run and should be accurate, reproducible, robust, stabile indicating, filter compatible, linear, free of interference from blank/placebo/impurities/degradation products, and straightforward enough for routine use in quality control laboratory.
Different chromatographic conditions were experimented to achieve better efficiency of the chromatographic system. Parameters such as mobile phase composition, wavelength of detection, column, column temperature and pH of mobile phase were optimized. Several proportions of buffer and solvents were evaluated in order to obtain suitable composition of mobile phase. Different composition of mobile phases containing a mixture (v/v) of 1%
Optimized chromatographic conditions.
Parameter | Optimized condition |
---|---|
Chromatograph | Shimadzu HPLC with UV detector |
Column | Phenomenex C18 column (250 × 4.6 mm, 5 |
Mobile phase | 1% |
Flow rate | 1 mL/min |
Detection | UV at 254 nm |
Injection volume | 20 |
Column temperature | 23°C |
All forced degradation studies were analysed at 50
(a) Acid degradation, (b) base degradation, (c) oxidative degradation, (d) photodegradation, and (e) thermal degradation.
Drug stability in pharmaceutical formulations is a function of storage conditions and chemical properties of the drug and its impurities. Condition used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. Stability data is required to show that the concentration and purity of analyte in the sample at the analysis corresponds to the concentration and purity of analyte at the time of sampling. Stability of sample solution was established by storage of sample solution at ambient temperature (25°C) for 24 hr. Sample solution was reanalyzed after 12 and 24 hr time intervals, and assay was determined for the compounds (CP and CPM) and compared against fresh sample. Sample solution did not show any appreciable change in assay value when stored at ambient temperature up to 24 hr, which are presented in Table
Solution stability results.
Time intervals | CP | CPM |
---|---|---|
% assay initial | 100.3 | 99.5 |
% assay after 12 hrs | 100.7 | 99.6 |
% assay after 24 hrs | 100.4 | 99.3 |
Calibration curves were obtained for CP and CPM from which the linear regression equation was computed and found to be
The percent standard error which is an indicator of accuracy is ≤1.0 and is indicative of high accuracy. The calculated percent relative standard deviation (%RSD) can be considered to be satisfactory. The percentage RSD values were <1%. The results obtained for the evaluation of accuracy and precision of the method are compiled in Tables
Result of accuracy studies.
Name of drug | Amount in sample (mg) | Amount of drug spiked in mg, (% recovery level) | Amount found, (mg) AM ± SD* | % SE | % RSD |
---|---|---|---|---|---|
20 | 16, (80%) | 36.37 ± 0.06 | 0.034 | 0.18 | |
CP | 20 | 20, (100%) | 40.36 ± 0.10 | 0.057 | 0.25 |
20 | 24, (120%) | 44.55 ± 0.11 | 0.063 | 0.25 | |
8 | 6.4, (80%) | 14.38 ± 0.01 | 0.005 | 0.13 | |
CPM | 8 | 8, (100%) | 15.98 ± 0.06 | 0.034 | 0.42 |
8 | 9.6, (120%) | 17.59 ± 0.06 | 0.034 | 0.38 |
Results of precision studies.
Drug | Concentration ( |
Intraday ( |
Interday ( |
||
---|---|---|---|---|---|
Measured concentration ( |
% CV | Measured concentration ( |
% CV | ||
CP | 100 | 99.33 ± 0.45 | 0.45 | 99 ± 0.45 | 0.46 |
CPM | 20 | 19.46 ± 0.12 | 0.64 | 19.58 ± 0.1 | 0.53 |
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters, and provides an indication of its reliability during normal usage. The results are summarized in Table
Robustness studies.
Sl. no | Parameter | Modification | Retention time (min) |
---|---|---|---|
1 | Flow rate | 0.8 mL/min | 3.2, 9.13 |
1.2 mL/min | 3.31, 9.32 | ||
2 | pH | 2.8 | 3.63, 9.7 |
3.2 | 3.3, 8.9 |
Stability of sample solution was established by the storage of sample solution at 25°C for 24 hr. Sample solution was reanalyzed after 12 and 24 hr time intervals, and assay was determined for the compounds (CP and CPM) and compared against fresh sample. Sample solution did not show any appreciable change in assay value when stored at ambient temperature up to 24 hr. The results from solution stability experiments confirmed that sample was stable up to 24 hr during the assay determination.
The system suitability was carried out after the method development and validation have been completed. For this, parameters like theoretical plates, resolution, retention time, and peak symmetry of samples were measured and shown in Table
System suitability parameters.
S. no. | Parameter | CP | CPM |
---|---|---|---|
1 | Retention time (min) | 3.47 | 9.45 |
2 | Peak asymmetry | 1.0 | 1.0 |
3 | Theoretical plates | 11100 | 17692 |
4 | Resolution | 3.05 | 7.78 |
5 | Limit of detection (LOD), |
2.263 | 0.756 |
6 | Limit of quantification (LOQ), |
6.859 | 2.293 |
The reported RP-HPLC method was proved to be simple, rapid, and reproducible. The validation data indicate good precision, accuracy, and reliability of the method. The developed method offers several advantages in terms of simplicity in mobile phase, isocratic mode of elution, easy sample preparation steps, and comparative short run time which makes the method specific and reliable for its intended use in simultaneous determination of codeine phosphate and chlorpheniramine maleate in liquid oral dosage form. Quick stability indicating RP-HPLC method was developed for the simultaneous estimation of codeine phosphate and chlorpheniramine maleate in the presence of its degradation products, generated from forced degradation studies. The developed method separates codeine phosphate chlorpheniramine maleate in impurities/degradation products. There were no reported stability indicating methods for this combination of drugs in liquid dosage form; hence, this method has an advantage of being unique and novel.
The authors declared that they have no conflict of interests or financial gains in mentioning the companies names or trademarks.
The authors acknowledge the management for providing necessary facilities.