Estimation of Cyproheptadine Hydrochloride and Tricholine Citrate Simultaneously in Syrup Dose by Applying Stability Indicating HPLC Methodology

An stability indicating HPLC methodology for the concurrent estimation of Tricholine citrate (TRC) and Cyproheptadine hydrochloride (CYH) in syrup dose and bulk using Waters column reverse phase C18 (5 μm, 250 mm and 4.6 mm) as stationary phase and 0.1M Na2HPO4 of pH 4.5 and acetonitrile in proportion of 60:40 (v/v) at low of 1.0 ml/min rate as mobile phase was reported. The linear scales were 275-825 μg/ml for TRC and 2-6 μg/ml for CYH with correlation coef icients of 0.9999 for TRC and 0.9997 for CYH. Followed ICH Q2(R1) strategies for validating the suggested method for precision, sensitivity, robustness, speci icity, selectivity and accuracy. The measures of LODandLOQare 0.023μg/ml and0.079μg/ml for CYH,while for TRC it was 0.565 μg/ml and 1.885 μg/ml, respectively. The precision measures for CYH and TRC were 0.073 and 0.212 relative measured deviation percentage, respectively. The accuracy measures for CYH and TRC were 99.40% and 99.09% mean assay percentiles, respectively. Recovery percentiles measures of CYH and TRC were ranged between 99.48% to 100.35% and 100.38% and 100.41%, respectively. While in degradation investigation, peaks of degraded products are very well differentiated from TRC and CYH peaks suggesting the speci icity and stability of suggested methodology. The results permit the application of the proposed stability indicating HPLC methodology in syrup dose forms.


INTRODUCTION
Cyproheptadine hydrochloride (CYH) is an antiserotonergic, antihistaminic and histamine H1 blocking agent (Kapur et al., 1997;Feng et al., 2015). CYH relieves allergy symptoms like redness in eye, itching in eyes, watery eyes, itching in nose, runny nose, sneezing, hives caused because of cold temperature exposure, and itching because of allergy in skin. CYH acts by preventing a certain natural substance termed histamine which is made by the body in an allergic response. This CYH also prevents one other natural substance named, serotonin, in the body. Tricholine citrate (TRC) is a lipotropic representative (Columbo and Rohr, 2016;Mehedint and Zeisel, 2013;NCBI, 2020). TRC is utilized in the therapy of elevated cholesterol levels. TRC is a binding agent of bile acid. Bile acids were expelled from the body by TRC. The liver at that point produce more bile acids utilizing cholesterol, accordingly, the choles-terol levels are brought down in the body.
CYH and TRC combination increases appetite, gives effective hepato-protective act, enhances protein production and assures weight gain (Kiran et al., 2018). CYH and TRC combination also used in anorexia-related hepatobiliary complications that may lead to weight losing. The CYH and TRC combination is accessible as formulation of syrup (Anuj, 2020), accessible in the markets of pharmacy as Cyprosal, Cyprohar, Cyprolac -T, Cypro -T and Clopex TC. The labelled claim of CYH and TRC in all syrup formulations was 2 mg of CYH and 275 mg of TRC per ive ml.
Analysis of pharmaceutically important compounds by reverse-phase liquid chromatography provides larger sensitivity, speci icity and consumes less time. It's the irst time, to our information from internet sources, that a stability indicating RP-HPLC dependent method for analysing the combination of CYH and TRC has been developed for syrup formulation.

Systems and Conditions
The HPLC system employed for CYH and TRC combined analysis comprised of Waters 2695 chromatography system, Waters 2998 photodiode detector and Waters column reverse phase C18 (5 µm, 250 mm and 4.6 mm). System management and processing of chromatography data of CYH and TRC were executed using version 2 of the Empower program. Mobile phase contained 0.1M Na 2 HPO 4 (Sd Fine Chemicals Ltd, India) of pH 4.5 and acetonitrile (Merck India Ltd, India) in proportion of 60:40 (v/v) at low of 1.0 ml/min rate. Injector sample volume, column's temperature and quanti ication wavelength was optimized at 10 µl, 25 o C and 265 nm, respectively.

CYH and TRC Calibration Curves
The linear correlation connecting area response of CYH and TRC with concentration of CYH and TRC was appraised by making measurements using proposed method at 5 concentration levels covering the range of 275-825 µg/ml for TRC and 2-6 µg/ml for CYH.

CYH and TRC Content Assay in Cypro -T Syrup
Volume of Cypro -T syrup (Nexkem Pharmaceutical, Indore, India; labelled strength 2 mg of CYH and 275 mg of TRC per ive ml) equal to 2 mg of CYH and 275 mg of TRC was mixed through 20 min sonication with 25 ml of 0.1M Na 2 HPO 4 (pH 4.5) and acetonitrile (60:40, v/v) solvent combination. Filtered the solution by 0.45 µm ilters and diluted to 50 ml volume with 0.1M Na 2 HPO 4 (pH 4.5) and acetonitrile (60:40, v/v) solvent combination. This is stock syrup solution of concentration 40 µg/ml CYH and 5500 µg/ml TRC. Working syrup solution of concentration 4 µg/ml CYH and 550 µg/ml TRC was made ready through diluting stock syrup solution with 0.1M Na 2 HPO 4 (pH 4.5) and acetonitrile (60:40, v/v) solvent combination. The area response of CYH and TRC peaks in working syrup solution were evaluated using recommended method. The contents of CYH and TRC in Cypro -T syrup was assessed from obtained area response of CYH and TRC peaks using their matching calibration graphs.

RESULTS AND DISCUSSION
The intent of this research was to create a stability indicating RP-HPLC approach for the measurement of CYH and TRC in the Cypro-T syrup dose type.

Figure 2: [I] Working syrup sample [II] Working CYH and TRC solution [III] Blank solvent combination
Chromatography was tested on C18 stationary phase columns like YMC, Aligent and Waters. The wavelength assignment was based on the highest area response for optimal sensitivity. Several trials were made with distinct solvents (K 2 HPO 4 : Methanol, K 2 HPO 4 : Methanol, Na 2 HPO 4 : Acetonitrile) with varying buffer type and polarity in different proportions to get sharp peaks with best resolution. Finally, Waters C18 stationary phase columns and mobile phase contained 0.1M Na 2 HPO 4 of pH 4.5 and acetonitrile in proportion of 60:40 (v/v) at low of 1.0 ml/min rate was opted. Column's temperature and quanti ication wavelength was optimized at 25 o C and 265 nm, respectively. Figure 1 shows the chromatogram for CYH and TRC with optimum chromatography conditions. The system appropriateness parameters (tailing factor, resolution and counts of plate) determined with optimum chromatography conditions are also acceptable.

Linearity
Linearity was found over the scope of 275-825 µg/ml for TRC and 2-6 µg/ml for CYH. The area response of CYH and TRC peaks were calculated for every concentration level. The graph of calibration was plotted for CYH and TRC concentrations against the area response of CYH and TRC peaks.

LOD & LOQ
The values of LOD & LOQ for CYH and TRC were accomplished dependent on the area response standard deviation and calibration graph slope. The LOD was expressed as: 3 × area response standard deviation/calibration graph slope and LOQ was expressed as: 10 × area response standard deviation/calibration graph slope. The value of LOD for CYH and TRC were 0.023 µg/ml and 0.565 µg/ml, respectively. The value of LOQ for CYH and TRC were 0.079 µg/ml and 1.885 µg/ml, respectively.

Precision
The precision valuation was conducted by analysing six independent replicates of the working CYH and TRC solution (CYH 4 µg/ml and TRC 550 µg/ml). Precision was stated as relative measured deviation percentage for CYH and TRC area responses. Precision was 0.073 and 0.212 relative measured deviation percentage for CYH and TRC, respectively.

Accuracy
The accuracy valuation was conducted by analysing six independent replicates of the working CYH and

Degradation Studies on CYH and TRC
The chromatogram for syrup solution degraded with 0.1N HCl showed 4 additional peaks at retention periods 0.945 min, 1.676 min, 1.823 min and 6.443 min (Figure 3.I). 10.98% of TRC and 11.34% of CYH are degraded with 0.1N HCl. Syrup solution degraded at 60 o C showed the occurrence of 5 additional peaks at retention periods 0.810 min, 1.675 min, 2.162 min, 5.971 min and 6.279 min in the chromatogram (Figure 3.II). In thermal heat induced stress, degradation of TRC and CYH are 12.38% and 9.71%, respectively. In degradation using sunlight, TRC and CYH degradation was evidenced at 6.55% and 8.36%, respectively. In response to degradation using sunlight, three additional peaks at 0.764 min, 1.822 min and 4.106 min (Figure 3.III) were observed. 0.1 N NaOH induced stress degraded 6.27% of TRC and 5.25% of CYH, and respective chromatogram presented three additional peaks at 1.452 min, 1.822 min and 5.681 min (Figure 3.IV). Under 30% peroxide stress, 4.46% of TRC and 7.96% of CYH are degraded. The chromatogram obtained displayed 4 additional peaks at retention periods 1.046 min, 1.673 min, 4.275 min and 5.196 min (Figure 3.V). The extra peaks in stress studies denotes the development of deterioration products. These experiments have shown the speciicity/stability of suggested methodology, because the peaks of degraded products are very well differentiated from the peaks of TRC and CYH (Figure 3.I, Figure 3.II, Figure 3.III, Figure 3.IV and Figure 3.V).
The system appropriateness parameters (tailing factor, resolution and counts of plate) are determined with variation parameters for TRC and CYH (Table 2). We got acceptable results.

CONCLUSION
An HPLC methodology for the estimation of TRC and CYH in formulations of syrup was herein reported. Validation speci ications were evaluated as per ICH guidance. This HPLC methodology enabled selective, sensitive, robust, precise, speci ic, accurate and high throughput quanti ication of TRC and CYH simultaneously in formulations of syrup and also in bulk materials.