Development and Validation of HPLC Method for Diphenhydramine Hydrochloride

Acknowledgement The authors will like to acknowledge the Zakfas pharmaceuticals, Multan, especially the Quality control department for permitting some aspects of the work to be carried out in their laboratory space. Article info. Received: April 25, 2020 Accepted: October 28, 2020 Funding Source: Nil Conflict of Interest: Nil Cite this article: Javed H, Shah SNH, Javed N. Development and Validation of HPLC Method for Diphenhydramine Hydrochloride. RADS J Pharm Pharm Sci. 2020; 8(3):154160. *Address of Correspondence Author: j_hani2003@yahoo.com Aim: The aim of present method development for Diphenhydramine HCl performed on HPLC is to obtain specific, more accurate and precise results as compare to spectrophotometric method. Methods: HPLC analysis was performed according to USP method with wavelength detection at 220nm and 1.0ml/min flow rate. Wufeng thermo HPLC system UV -100detector was used having column C18(4.6mm*250mm) 5. Methanol and water (4:1) mixture was used as mobile phase and pH was adjusted at 7.4 with the help of triethanolamine. Validation parameters like linearity, accuracy, precision, solution stability, robustness, LOD, LOQ and system suitability were successfully evaluated. Results: The regression co-efficient for calibration curve was 0.991 and % recovery was in range (80-110%), whereas no robustness was observed in this reported method. Conclusion: In summary, the expected linearity, accuracy and % recovery indicating that HPLC is more precise method than spectrophotometry and suggested that present method qualifies the validation criteria.


I N T R O D U CTION
High performance liquid chromatography (HPLC) is one of the analytical method used for validation of drugs and chemical substances [1,2]. Diphenhydramine HCl (Figure 1) is antihistaminic drug substance, widely used in the treatment of allergic rhinitis, common cold and skin allergies. DPH is H1 receptor antagonist, white crystalline powder having melting point 168-172 o C. Diphenhydramine HCl is lipophilic in nature and well absorbed from nasal cavity. Its molecular weight is 291.82 g/mol (less than 1KDa), rapidly absorbed transcellularly across the nasal membrane. It can cross Blood Brain Barrier (BBB) having 40-60% bioavailability and metabolize in liver [3][4][5]. In present study, HPLC system using column C18 was used for the method validation of DPH HCl. Linearity, precision, reproducibility, limit of detection (LOD), limit of quantitation (LOQ), robustness and system suitability were determined. All of these RADS J. Pharm. Pharm. Sci. 155 parameters are the important indicators of system functionality, sample preparation and column [6,7].

E X P E R I M E N T A L Chemicals
Diphenhydramine hydrochloride was gifted from Pfizer pharmaceuticals, Karachi (Pakistan). Methanol of HPLC analytical grade was purchased from Merck, Germany. Double distilled water was used gifted from Zakfas Pharmaceuticals Multan (Pakistan). Membrane disc filters (0.45µm pore size)

Preparation of Mobile Phase
Mobile phase was prepared by mixing the 40ml methanol (HPLC-analytical grade) with 10 ml double distilled water (4:1, V/V) and pH was adjusted at 7.4 with the help of Triethanolamine (0.5ml). Then filtered the mobile phase through 0.45µm pore size filter paper under vacuum and degassed through ultrasonic bath before use.

Chromatographic Method for Validation
According to the ICH-Guidelines, parameters for chromatographic validation were determined such as linearity, precision, LOD, LOQ, solution stability and system suitability [8].

Linearity
It describes the relationship between response and analyte concentration over the range. For the evaluation, linearity range depends on purpose of analytical method [9]. ICH guideline specify the minimum, five concentration levels with minimum specified range. The linearity data is accepted by observing the values of Regression coefficient (r 2 ≤1), y-intercept (less than percent response obtained for analyte), slope and % relative standard deviation (% RS) [10]. In present study, linearity for DPH was determined over the concentration range of 10-50µg/mL (n=3).

Specificity of Assay
In order to detect any interference between reference standard solution of DPH, placebo and mobile phase, specificity was evaluated by comparing chromatograms of 3 replicate injections of reference standard solution and placebo [11].

Accuracy
Accuracy is the closeness of test values to the true values of method [11]. In this study, it was obtained by preparing the sample solution over range of 10-50µg/mL (n=3) and % recovery was calculated. According to USP for acceptance of accuracy criteria, the %recovery must be in range 80-110%.

Precision
Intra-day assay measures the degree of repeatability of analytical method under normal specific conditions. The intra-batch precision is determined under same condition over short interval of time. In present study, it was determined by assessing the replicating 10 injections of DPH at same concentration (10 µg/mL, n=3) during same day and under same experimental conditions.
Inter-day assay is performed on different days at different experimental conditions [12]. In present study, it was determined by selecting the three different concentrations (20,30 and 40 µg/mL, n=3) at three different consecutive days.
The mean, standard deviation and % RSD were calculated for both intra-day and inter-day precision.

Limit of Detection (LOD) and Limit of Quantitation (LOQ)
Limit of Detection (LOD) is the lowest amount of analyte, detected at three times the noise level (S/N=3) [6] and calculated by; Whereas SD is the standard deviation and m is the slope of calibration curve.
While LOQ is the lowest amount of analyte that reproducible quantified above baseline noise (S/N=10) [6] and calculated by; Whereas SD is the standard deviation and m is the slope of calibration curve.
In present study, 20µL injection of DPH (10 µg/mL, n=3) standard used to calculating the LOD and LOQ with % RSD.

Analytical Solutions Stability
It was measured by keeping the mobile phase and standard solutions in capped volumetric flask in laboratory for 48 hours under normal conditions and was assessed at 12 hours intervals and determined the %RSD of DPH standard solutions [13].

Robustness
It was determined by observing the changes in various experimental conditions. Three standard solutions were prepared and then analyzed by using established conditions and by make variation in some chromatographic conditions. The changes in mobile phase pH (±0.3) and composition (±3), wavelength (±1 nm) and experimental temperature (±2°C) were made and obtained the data that was then subjected for statistical analysis by using analysis of variance (ANOVA) test.

System Suitability Test
It is the most important step in HPLC analysis, used for the verification of accuracy and precision of system [14]. In present study, system suitability was performed on HPLC system by injecting 10 injections of same concentration of DPH (10 µg/mL), assessed with mobile phase. The different parameters such as theoretical plates per column, tailing factor, %RSD of peak area and %RSD of retention time were calculated.

R E S U L T S A N D D I S C U S S I O N Linearity
The calibration curve of Diphenhydramine HCl was linear over the concentration range of 10-50 µg/mL Three injections of each concentration were applied and regression equation (Y=180063X+222402) was obtained by plotting the injecting concentration (µg/mL) against obtained peak area (Y). The value of correlation coefficient (r 2 =0.991) has shown the significantly good relationship between injecting concentration (µg/mL) and obtained peak area (Y) as shown in Figure 2. The data for linearity was obtained for this experiment has been tabulated in Table 1 that showed %RSD was less than 1 for each concentration of standard solutions. This linearity data is in agreement with spectrophotometrically analytical method as previously reported in literature [15].

Specificity of Assay
The specificity of this method was analyzed for reference standard solution of DPH, Placebo and mobile phase by applying separate injections of each. There was no interfering peak was observed as in Figure 3.

Accuracy
The accuracy was determined by repeating three injections of each concentration (10, 20, 30, 40 and 50 µg/mL) of DPH in mobile phase at 220nm. The mean % recovery for each concentration was calculated as described in Table 2 that was in range (80-110%) according to USP with %RSD less than 1.

Repeatability (Intra-Day Assay)
The intra-day precision was evaluated by injecting the 10 injections of 10 µg/mL concentration at same experimental conditions. Mean % RSD for Retention time, peak Height, peak area and obtained concentration was less than 1 as tabulated in Table 3.

Intermediate Precision (Inter-Day Assay)
Inter-day precision was evaluated by injecting the three injections of selected three concentrations (20, 30 and 40 µg/mL) on different consecutive days. The retention time, peak area, obtained concentration and % recovery was observed and data showed mean % RSD for % recovery was less than 1 as tabulated in Table 4.

LOD and LOQ
Limit of Detection and Limit of Quantitation by injecting the known lowest concentration (10µg/mL) at three and ten times S/N response for DPH was 1.49×10 -5 µg/mL and 4.5×10 -5 µg/mL respectively. The graph of LOD and LOQ has shown in Figure 4 (A&B).

Analytical Solution Stability
The stability of Diphenhydramine hydrochloride and mobile phase was calculated by comparing area percent and area response two standards at 10µg/ml over specific time for 48 hours. The standard solution has shown no significant change in DPH concentration throughout this time period as described in Table 5. This was indicated by RSD less than 1% changes in peak area, obtained concentration and recovery between T=0 hours and T=48 hours. This data also showed no significant quantitative change in % recovery and as well retention time within 48 hours.

Robustness
Changes observed for pH (±0.3) and composition of mobile phase (±3 %), wavelength determination (±1 nm) and experimental temperature (±2°C), produced no affect in present developed method. This indicates developed analytical method has high level of robustness as no significant differences were observed by changing the chromatographic conditions.

System Suitability Test
In present study, this test was performed to determine the accuracy sand precision of HPLC system by injecting 10 injections of 10 µg/mL DPH. The results showed that mean % RSD for peak area and retention time was less than 1(0.0003 and 0.042 respectively). While the tailing factor was less than 2 (1.06) and theoretical plates were greater than 2000 (7076.7) as shown in Table 6.

C O N C L U S I O N
HPLC analysis of drug substances and validation is complexed and time consuming method. But in spite of all these, it is more precise and accurate analytical technique. This article is intended in providing the guidance to perform validation method for HPLC that generates useful data in order to meet all requirements of USP, ICH and FDA for the validation of DPH analysis.