Development and validation of High-Performance Liquid Chromatographic Technique to Estimate Pure Form of Loperamide Hydrochloride

Introduction

Loperamide is a medication used to treat diarrhea. The chemical name of loperamide is 4-[4-(4-chlorophenyl)- 4-hydroxypiperidin-1-yl]-N, N-dimethyl-2, 2-diphenyl butanamide hydrochloride.¹ It is prescribed for indications such as inflammatory bowel disease and gastrointestinal disorders. It is one of the long-acting antidiarrheal drugs. It is not sufficiently absorbed through the gut and has no proper action on the adrenergic system or central nervous system, but demonstrates affinity towards histamine receptors and interaction with acetylcholine release locally¹ . The chemical structure of loperamide is presented in Figure 1.

Analytical chemistry is a discipline of chemistry with a broad mission to understand the chemical composition of all matter and develop tools to elucidate compositions. Analytical chemistry is concerned with chemical characterization of matter, both qualitative and quantitative.²

Analytical techniques play important role in maintaining and assuring the quality of drugs and is a critical component of quality assurance (QA) or quality control (QC). The reliability, utility, accuracy, interception, and specificity of the measurement are responsibilities of the analytical chemist. In general terms pharmaceutical analysis comprises of procedures necessary to determine the identity, strength, quality and purity of drugs and chemicals under a specified set of conditions. It only assesses the physiological availability of the drug substance, and does not measure the safety or efficacy of the drug. It is an vitro method to minimize variation among different batches of manufactured tablets. The dissolution medium must be aqueous, and the pH media should be controlled and should simulate in vivo conditions.³

Chromatography was devised and named by the Russian botanist M. Tswett. In all types of chromatographic techniques and procedures, two phases are selected to enable distribution of components between the mobile and stationary phases. Components based on their affinity to the stationary phase move along with the mobile phase. As a result of differences in the migration rates, sample components separate into discrete bands or zones and aid in identification and quantification.^4,5

The working principle behind the HPLC is that the analyte is passed through a column of stationary phase by pumping a liquid (mobile phase) at a specific rate and high pressure through the column. The final chromatogram with a good resolution can be achieved by applying high pressure which results in less time for the components to diffuse into the column because of increased linear velocity.^6,7

HPLC system has a high efficiency to analyze samples. For example, if the sample consists of polar analytes then reverse phase HPLC achieves adequate retention and resolution, when compared to normal phase HPLC. Impurities may be formed during the manufacture of active pharmaceutical ingredient (API), and these impurities may be related to the raw material, process-related impurities, synthetic intermediates, or degradation products and suitable methods need to be developed to remove these impurities.⁸

The validation elements addressed may vary, depending on the phase of development or the application of data obtained. The acceptance criteria are presented as guidelines and may differ between products. Firms should develop standard operating procedures to documents appropriate acceptance criteria for all their products and important specifications for special dosage forms. The degree of validation depends on the phase of the product development. Full validation takes place at the time of Phase III clinical trials. Validation studies should address the variations factors related to different profile time points. In products containing more than one active ingredient, the dissolution method should be validated for each active ingredient.⁹

Validation Parameters^10,11,12

Typical analytical parameters used in dissolution validation include specificity/placebo interference, linearity and range, accuracy/recovery, precision, ruggedness, robustness, solution stability and system suitability.

In HPLC analysis, it is essential to determine the compatibility of dissolution media and mobile phase when large volumes (≥ 100 µL) are injected. Samples are analyzed by HPLC using a spectrophotometric detector and an auto injector. Single injections of each vessel time point with standards throughout the run constitute a typical run design. The minimum system suitability tests include retention window and injection precision. Typically, the reproducibility of an HPLC analysis should be ≤2% RSD for five or six standard determinations. The standard level is typically at 100% label claim level, especially for a single-point analysis.

Materials And Methods

Chemicals and reagents

Analytical grade pure sample of loperamide was procured from IPCA laboratory, Mumbai, India. All reagents and chemicals used were of HPLC and analytical grade procured from Merck Chemicals and Spectrochem Chemicals, India.

Instrumentation and chromatographic conditions

Water alliance data system, water 2695 separation module with Empower software (UV-visible detector). Nova-Pak C18 column with 150 mm x 4.6 mm, 5 μm specification was used as stationary phase. pH measurement was performed using pH tutor (Eutech Instuments 510). Mobile phase was filtered using 0.45 μm Acrodisc vaccum filter and sonication was done using Life-care ultrasonicator. Typical operating conditions include flow rate 1.5 mL/min, injection volume 20 μL, wavelength 226 nm, column compartment temperature 35o C and at room temperature.

Method development

Selection and Preparation of mobile phase

Buffer for Mobile Phase: Add 1.09 g sodium octane sulphonic acid and 1 ml of 13.5 M ammonia (NH3 ) and 0.5 ml triethylamine in 800 ml millipore water. Adjust pH to 3.2 with orthophosphoric acid and dilute to 1000 ml with millipore water (diluent:0.01 N HCL).

During the selection of mobile phase, multiple trials using various solvents such as 0.1 M potassium dihydrogen orthophosphate, buffer: acetonitrile (90:10%v/v), tetrahydrofuran: acetonitrile (80:20%v/v), buffer: acetonitrile (70:30%v/v), buffer: acetonitrile (50:50%v/v) were performed. Acetonitrile (55:45% v/v), was used as the mobile phase because good peaks were obtained.

Preparation of standard stock and working standard solutions

A stock solution of loperamide hydrochloride was prepared by accurately weighing 20 mg of the drug, and transferred to a 200 ml volumetric flask. To the flask, approximately 100 ml of diluents were added and sonicated for 2 minutes to dissolve the drug completely and then the volume was made up to the mark with diluents (100 μg/ml). Accurately weighed 20 mg of loperamide hydrochloride was transferred into 200 mL volumetric flask and dissolved in small amount of mobile phase and sonicated. Finally, volume was adjusted up to the marked level using mobile phase (100 μg/ml). Working Standard solutions containing loperamide hydrochloride were prepared by diluting the standard stock solution. Mobile phase was used as solvent for the dilutions.

Recording the chromatogram and retention time

Aliquot of the standard stock solution was further diluted with mobile phase to obtain 2 μg/ml of loperamide hydrochloride. The samples were injected into high performance liquid chromatography system and chromatogram was recorded. To achieve desirable resolution in peaks, various combinations of mobile phase components, column temperature, and columns were tried.

Method validation

Various validation parameters such as system suitability, linearity and range, sensitivity precision ruggedness, robustness and accuracy the HPLC method were developed and validated as per ICH Q2A and Q2B guidelines.^13,14

System suitability

A chromatographic system suitability test was performed by injecting six repeated injections of standard solution (2 µg/ml) into the chromatographic system. Relative standard deviation (RSD) and column efficiency for the six suitability injections were determined.

Precision

Repeatability (Method Precision)

The dissolution assay was conducted as mentioned in the methodology on six sample vials from dissolution media. The % release was calculated.

The mean, relative standard deviation and 95% confidence interval of the result were calculated.

Intermediate precision

The analytical method was conducted as described in the repeatability exercise. A different analyst carried out this analysis on a different day using a different HPLC system as well as columns.

Accuracy: Accuracy was carried out in triplicates after spiking pure drug equivalent to 80%, 100%, and 120% of the standard concentration of loperamide hydrochloride (2 μg/ml). The results obtained (Table: 30) reported that the recovery was significant, and within 100% ± 2.

Stock solution for Accuracy

Accurately weighed 20 mg of compound standard was transferred to a 250 ml volumetric flask, 100 ml water was added, dissolved, and them made up to 250 ml with water.

Linearity and Range: To evaluate the linearity, serial dilutions of analyte were prepared from the stock solution by diluting with the mobile phase, to get a series of concentrations ranging from 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2, 3.6 and 4.0 μg/ml. From these solutions, 50 μl injections of each concentration were injected into the HPLC system and observed under regulated conditions. The resultant calibration curve was framed by plotting the mean peak area (Y-axis) versus the concentration (X-axis).

Specificity

Methods to determine specificity of dissolution of hydroxyzine in hydroxyzine tablets was established by preparing the control blend of all the excipients in compliance with the quantitative composition and testing samples of the blend as per the dissolution method and calculating the % interference.

Robustness: The method robustness was determined by making slight changes in the chromatographic conditions, such as change in mobile phase pH and column. There were no significant changes in the chromatograms, which demonstrated that the RP-HPLC method developed was robust.

Ruggedness: The degree of reproducibility of assay results obtained by the successful application of the developed assay over time and across different laboratories and performed by different analysts. Ruggedness of the method was determined by carrying out the analysis by two different analysts and the respective peak areas were noted. The result was presented as % RSD which was <2% indicating that the method was rugged.

Results

Method development and optimization of chromatographic conditions

The chromatographic conditions were adjusted in order to achieve good performance (mobile phase buffer: acetonitrile (55:45% v/v), Novapak C18 (150 mm x 4.6 mm, 5 μm) and at pH < 5). After sonication mobile phase was filtered using 0.45 μm filter paper. Typical operating conditions applied were flow rate 0.5 mL/ min, injection volume 2 μL,wavelength 226 nm, column compartment temperature 35o C, and room temperature. The λ max was determined using Shimadzu UV – visible spectrophotometer (Model UV) in the range of 200 – 400 nm and it was found to be 226 nm (Figure 2). The retention time of loperamide hydrochloride was 1.926 min and the chromatogram is presented in Figure 3.

Method validation

System suitability

Every day, system suitability parameters were calculated by injecting six replicate solutions of the analyte. The %RSD values for parameters such as retention time, theoretical plates and tailing factor area were within the expected limit, i.e., 2% which indicates low variation in observed values and asymmetry factors for all the peaks were <2. The results of system suitability are presented in Table 1

Precision

Method Precision (Intermediate precision)

Analysis was conducted as described in the repeatability exercise. A different analyst carried out this analysis on a different day using a different HPLC system and different column. The obtained results for % assay of compound is presented in the Table 2.

The % RSD of the % release of six individual samples in intermediate precision for compound was 1.03%. So, the absolute difference of the mean assay result obtained in repeatability and intermediate precision for compound was 4.67, which is well within the acceptance criteria of not more than 5.0%. Based on the above results it is observed that the proposed method for assay of compound is precise.

Accuracy

Accuracy for dissolution was performed by spiking compound at 3 levels, viz. 80%, 100% and 120% of working concentration i.e. 5.6 ppm and each level had 3 replicates. The results of accuracy are presented in Table 3. The accuracy experiments were carried out by the standard addition method.

Linearity and range

Linear correlation attained between peak area and concentration range of loperamide was between 0.2 ppm and 4 ppm. The linearity of the calibration curve was validated using the highest value of correlation coefficient of regression (Figure 4) and the results were presented in Table 4.

Specificity

Specificity study to determine dissolution of loperamide hydrochloride in loperamide hydrochloride tablets was established by preparing the control blend of all the excipients in compliance with the quantitative composition and testing samples of the blend as per the dissolution method and calculating the % interference.

Robustness

Robustness of the method was tested by injecting the system suitability solution by deliberately changing the chromatographic and dissolution parameters and monitoring the system suitability parameters under different conditions. The parameters that were modified were pH of mobile phase ±2 (3.0-3.2), column (column Novapak C18RP, column WATERS Symmetry C18).

Ruggedness: The degree of reproducibility of assay results obtained by the successful applications of the assay over time and among multiple laboratories and by different analysts. The results of ruggedness are presented in Table 7.

Conclusions

The developed analytical methods to carry out dissolution studies of loperamide hydrochloride were simple, precise, specific, accurate, quick, reliable and reproducible. The method was validated showing desirable data for all the method validation parameters assessed. The results showed that the method developed can be used for quantitative analysis of the compound. The amount found in formulations well agreed with the label claim. Thus, the reported method is of significant importance and has great industrial applicability for QC and analysis of Loperamide HCL tablet dosage form. This methodology can be used for the further routine analysis of loperamide hydrochloride in pharmaceutical formulations.

Conflicts of interest

None

Acknowledgements

Authors are thankful to IPCA laboratory, Mumbai, Maharashtra for providing the drug sample. Authors are also thankful to the Principal and staff members of KLE College of Pharmacy, Nipani for the constant support and guidance. Authors are also thankful to the Principal and staff members of HKE College of Pharmacy, Gulbarga, Karnataka