Stability Indicating RP-HPLC Method Development for Related Substances of Anti-histamine Promethazine hydrochloride and its Validation study

A smooth, specific, precise, and accurate high-performance reverse phase (RP-HPLC) method for determining related substances in antihistamine Promethazine Hydrochloride (PMZ∙HCI) has been recorded. The RP-HPLC system is developed and tested in compliance with recommendations Q2(R1) of the International Council for Harmonization (ICH). Column 5m and reverse step with linear gradient elution is accomplished in Symmetry Shield RP8 (4.6 mm x 150 mm). Mobile phase A is 3.4% in the 7.0 pH water, modified to dilute potassium hydroxide solution. In contrast, the 60:40 mixture of acetonitrile and methanol combination as mobile phase B. The handled phase with a continuous flow rate of 1.0 ml min-1 is provided by choosing the wavelength 254 nm using a PDA/UV detector. The temperature of the column oven and sampler is 25°C and 4°C, respectively. The injection amount chosen is 10.0 μL. The method is linear in the quantitation limit range (LOQ) to 150 percent concerning the specification impurity concentration limit. Both impurities and PMZ have a correlation coefficient of greater than 0.999. The LOQ for all known impurities and PMZs has a specification limit of between 10 to 30%. The relative response factor for all four known impurities is calculated. The unexplained peaks are very different; the effects obtained are similar to the original values. There are no significant changes improvements to the suitability parameters, such as tailing factor, Plates theoretical sheets, and % RSD in Robustness Studies. The forced degradation analysis of PMZ∙HCI was performed. This RP-HPLC is descriptive, accurate and precise. This further defines the parameters of linearity, consistency and robustness used for regular quality control analysis.


INTRODUCTION
Promethazine (PMZ), ), 10 H-Phenothiazine-10-ethanamine, N,N,a-trimethylmonohydrochlorid is a common antihistamine pharmaceutical drug that is sold as hydrochloride salt (PMZ•HCI). (see Fig.1). PMZ is an antihistamine agent of the first generation used to treat allergies, sleep disorders and nausea 1 . It was approved in the United States for medical use in 1951. PMZ is a derivative of phenothiazine and structurally different from the same but different effects 2 of phenothiazine. PMZ is a crystalline powder that is white to faint-yellow odorless. PMZ hydrochloride salt can be freely water soluble and slightly alcohol soluble. It is also a chiral compound, which occurs as an enantiomer mixture 3 . PMZ is molecular in C 17 H 20 N 2 S.HCI and weight 320.88 g/ mol. It is preserved under nitrogen at 25°C in tight, light-resistant containers.

Standard and test preparation
Weighed 100 mg standard/test sample of Promethazine in 100 mL volumetric flask was dissolved and diluted with diluent. 5.0 mL pipetted above solution in 100 mL flask and diluted with the diluent amount.

Method Development
The PMZ and its related impurities are polar molecules; hence reverse phase (RP) chromatography is used to establish the associated substances. The fixed phase of chromatography in the reversed-phase is non-polar as C4, C8, C18, and polar as the mobile phase is water, acetonitrile, methanol, or buffer solution. The document shows several HPLC methods for estimating promethazine hydrochloride in bulk medicinal products and formulations [4][5][6][7][8] . No specific method for determining related substances was reported in the literature.
Other parameters such as column temperature, diluents, wavelength, and pH are also crucial in developing an HPLC method and stationary and mobile phases. The stationary phase of the HPLC method screened from specific Hypersil BDS C8, and Symmetry Shield RP8 is both 4.6 mm internal diameter, 150 mm long, and 5μ particle size.

mettler toledo
The Promethazine (PMZ) sample and its impurities are taken from Emcure Pharmaceutical Limited, Analytical Research Center, Hinjewadi Center for Analytical Analysis, Hinjawadi, Pune. Potassium phosphate analytic grade, potassium hydroxide, and millet-Q grade purified water. For a mobile phase and diluent preparation, gradient methanol, acetonitrile, and triethylamine are used. The research equilibrium shall be used for the mettler toledo and the HPLC waters with UV/PDA detector using the empower two software of data acquisition and calculations.

Mobile phase A
3.4% of potassium dihydrogen phosphate solution homogeneously in water with 7 pH adjusted in the ratio of 40:40:20 by the dilute potassium hydroxide solution, acetonitrile, and methanol.

Mobile Phase B
The homogeneous mixture of acetonitrile and methanol in the ratio of 60:40. Also, the HPLC analysis was carried out with the gradient method, as at the beginning, the composition of step A and B was used at 70:30, which is gradually changed to 40:60 and up to 45 minutes. It is adjusted to a ratio of 35:65 up to 50 min. It lasts up to 52 min then it suddenly hits initial values of 70:30 and is sustained for up to 65 min throughout the entire course.

RESULTS AND DESCUSION
The analytical method validation study was carried out as per ICH guideline Q2(R1) 9-12 . The analytical method validation parameters are discussed below.

Specificity
The specificity study conducted by spiking with an adequate level of known impurities (up to 0.15% concentration) into pure substances and demonstrating findings is not affected by the presence of these spike impurities. A blank run shows no intervening PMZ peak and its associated impurities. The PMZ peak is well-differentiated from recognized impurities (Fig. 1). The peak purity angle of PMZ and its related impurities is less than the threshold for standard, non-spiked, and spike test solutions ( Table 2). The percent relative standard deviation (%RSD) of six standard solutions replicates with a mixture of impurities A to D and PMZ•HCI are 0.60, 0.87, 1.86, 0.69 and 4.58%. percent. The resolution between the near escape of impurities B and A is 5.72, which indicates a sharp break between these peaks.

Determination of limit of detection and limit of quantitation
The limit of detection (LOD) is the smallest concentration of an analyte in a solution that can only be detected but quantitatively measured. At the same time, the LOQ is the lowest amount of analyte in a sample solution, which can be quantitatively determined with precision and accuracy. In this analysis, LOD and LOQ levels are calculated by injecting the various concentrations in the standard solutions (between 10 and 120%) of PMZ•HCI (0.1%) and its maximum concentrations in impurity A to D (0.15%) and calculating the signal-to-noise ratio. The expected LOD and LOQ data in Table 3 are shown.

Linearity and Range
The linearity was determined by using the standard solutions of impurities and by PMZ•HCI at the level of LOQ at a 150 percent level (incl.50, 80, 100, and 120 %). The concentrations of LOD and LOQ, correlation coefficient, and linearity data intercept are shown in Table 4 and the corresponding linearity graph (Fig. 2 and 3). Linearity data are shown in Table 4. The concentration data for the peak areas versus concentration were treated with less square analysis of linear regression and the correlation coefficients obtained for all impurities and PMZ•HCI are higher than 0.999. The linearity graph reveals that the PMZ•HCI research sample has variations in A-D impurities. Uncleanness D has a lower reaction, whereas uncleanness A has more significant reaction overall known impurities found in PMZ•HCI.

Relative response factor
The RRF is a practical approach for the systematic study of impurities found in drug compounds and their components. The RRF is usually specific for each impurity and is independently calculated. It cannot be used to measure the concentration of respective ingredients by other detectors for one particular detector type. The RRF value depends on the response of PMZ•HCI and to RRF of PMZ.HCI 1.00 for unknown impurities measurement. The observed impurity D has lower response while for impurity A shown that higher response in PMZ.HCl.

Precision
The precision study is divided into repeatability, inter mediate precision, and reproducibility. Six replicates of PMZ.HCI study system precision with a regular mixed impurity solution. The repetitiveness of the relevant compounds was accomplished by injecting six individual test sample solutions with the concentration of identified impurities in the spiking definition stage. The percentage of impurities and the percentage RSD of the solution is determined.

Accuracy
The reliability of an analytical procedure is the continuity of the actual value and value observed. A three-fold accuracy impurity analysis was performed at the LOQ level of 50, 100, and 150% of the test solution's stated impurities concentration. The quality of known impurities has been found well below the admission criterion. The average accuracy of the percent and its standard deviation from its LOQ, 50%, 100%, and 150% of the specification level specified in Table 6.

Solution Stability
The consistency of the test solution was maintained on a routine basis for up to 3 days at room temperature. The percentage recovery of known impurities for the test solution was determined. All known impurities except impurity B have total %RSD values under the admission criterion. Cumulative level %RSD impurity B values are up to 1 day appropriate. This shows that the test preparation is stable until one day if it is kept at room temperature.

Robustness
In the robustness test, the experimental conditions such as flow rate of the mobile process are intentionally changed, oven column temperature adjustment, and the specific bunch of columns are used. Considering these significant fluid differences as well as the temperature of the column oven. It is determined that a robustness analysis carried out at a flow rate modified by ±10% of the real flow rate shown in the process. The maximum mobile phase flow rate is 1.0 mL/min between 0.9 mL/min between 1.1 mL/minute. The oven column temperature in the initial system is altered by 30°C to 20°C at a temperature between ±5°C to 25°C. The buffer pH is adjusted by 0.2. The average accuracy measurements are conducted on various column lots that display no differences in the perfor mance. In both cases, the retention periods differ by ±0.2 min in contrast with accurate retention. For these intentionally variable chromatographic conditions (flow rate, pH column oven temperature, and multiple column lots), significant improvements for system suitability parameters, such as tailing factor, theoretical plates, and the percent RSD, are not detectable. Such criteria's principles are far beyond reasonable limits. Table 7 gives the total mean and percent RSD of n=8 test preparations (6 from repeatability and 2 from robustness).

Forced degradation Study
The forced degradation study [13][14][15] is useful to pick an overview of stabilization showing conditions. The test of degradation was performed for aqueous, photolytic, thermal, temperature, acidic, necessary, and oxidative stress conditions. PMZ•HCI is subjected to thermal, photolytic, and humidity stress conditions, while 50 mg of PMZ•HCI is dissolved in the 5.0 mL dilution sample and is subject to similar stress conditions in acid, alkaline and oxidation degradation tests. Table 8  All the samples collected in 24 h are prepared using the current analytical process PMZ•HCI at a similar concentration (1000 ppm). Any physical and chemical modifications were not found during the heat, humidity, and photolytic stress analysis. The PMZ•HCI peak region was constant without a degradation point, which suggests that this molecule is stable at temperature, humidity, and photolytic stress.
The figure illustrates the chromatograms of the degradation from blank-aqueous, test-aqueous, blank-oxidation, and test-oxidation ( Fig. 4 to Fig. 7). Both known and decaying impurities are well isolated from the PMZ•HCI and have the maximum pure requirements. The 2.0 mL of 30 percent H 2 O 2 depletion has kept the solution at room temperature for 15 minute. The PMZ has been reduced by 11.99 percent, mainly to impurity D. The alkali degradation was achieved by the application of 5.0 mL of 5.0 NaOH to test the solution held for 30 min at room temperature, neutralized with HCI and diluted to the limit after the heat was finished. Yet there is no degradation. In acid oxidation, the 5.0 mL 5.0 N HCI sample was exposed to the solution for 30 min at room temperature. It neutralized with NaOH after time had done. However, no declining peak is detected.

CONCLUSION
A highly reliable and precise stability system of RP-HPLC is developed and successfully tested in compliance with the ICH Guidelines Q2(R1) for the PMZ•HCI based material analysis of an API. The characteristics demonstrate that the PMZ optimum is well-resolved from both known and unknown impurities. Concerning performance concentration, this approach is linear to the LOQ point of 150 percent and measured correlation coefficient of all identified impurities above 0.999. The robustness analysis showed no significant changes to suitability parameters, such as tailing factor theoretical plates and percent RSD.