1. INTRODUCTION:

Doxylamine succinate and pyridoxine hydrochloride are used in morning sickness during pregnancy in combined dosage regimen. This drug combination was approved in April 2013 by United States Food and Drug Administration (USFDA)¹. Both the drugs are unrelated but possess a synergistic effect when given in combination to minimize nausea vomiting during pregnancy^2-3.Doxylamine succinate chemically butanedioic acid; dimethyl ({2-[1-phenyl-1-(pyridin-2 yl)ethoxy]ethyl}) amine is white solid powder. It is an antihistaminic H1 receptor antagonist. This implies that it inhibits the proteins that perceive and react to histamine levels in the body.

Typically, histamine makes alerting responses. Patients taking doxylamine succinate are more prawn to drowsiness but it suppresses hypersensitive responses⁴. Whereas pyridoxine hydrochloride chemically called 4,5-bis(hydroxymethyl)-2-methylpyridin-3-ol hydrochloride or Vitamin B6 is a white solid powder. The major metabolites of pyridoxine hydrochloride are pyridoxal phosphate and a minor metabolite pyridoxamine phosphate in erythrocytes. This is a coenzyme for different metabolic functions affecting the utilization of carbohydrate, protein, fat. No mechanistic evidence has been proved for antiemetic action of pyridoxine⁵. The adverse reactions of the above combination tablets are somnolence, headache, dizziness, dry mouth, and hypersensitivity¹. Various literature surveys indicate that different kind of analytical methods has been developed for the estimation of the two drugs either in combination or in single or in combination with other drugs. Different UV spectrophotometric approaches, including zero order, first order, and second order derivative methods, were used to estimate both drugs individually and in combination or with other drugs^6-11. Using HPLC, HPTLC, and UPLC technologies, a few chromatographic techniques have been developed for the simultaneous measurement of both drugs, either alone or in combination with other drugs^12-18. Literature survey also reveals the estimation of different anti-histaminics and vitamins by UV spectroscopy and RP-HPLC^19-29. Validation of the method was performed according to the ICH guidelines³⁰. Forced degradation study of drugs and drug products are required for the determining the shelf life or expiry date of products (ICH)³¹. Degraded products are nothing but impurity which may be harmful for the users. The present scenario gives the idea of impurity profiling and impurity percentage which replaces the percentage concept. Degradation study helps in evaluating the effect of different environmental and storage condition on the drug or drug products. Our aim is to develop a simple, easy, reliable, robust, user-friendly, cost-effective and valid method for the stability indicating assay method for doxylamine succinate and pyridoxine hydrochloride from previously available methods.

Figure 1: A) Doxylamine succinate and B) Pyridoxine hydrochloride

2. MATERIALS AND METHODS:

2.1 Chemicals and Reagents:

Doxylamine succinate (Figure 1a) and pyridoxine hydrochloride (Figure 1b) were supplied by Harshada Corporation and Harika Drugs Pvt. Ltd respectively as a gift sample. Doxilar tablet (10mg/10mg) was obtained from Mercury Laboratories Ltd. All solvents were HPLC grade, such as acetonitrile, methanol, and water. All additional reagents, such as analytical-grade phosphate buffer, were also used.

2.2 Instruments and Apparatus:

The HPLC LC 10AT (Shimadzu, Kyoto, Japan) was monitored and connected with LC solution software using a C-18 column (250 × 4.6mm × 5µm). A Hamilton (Rheodyne-20 L) syringe and a Himedia Syringe-driven filters (0.22m) syringe filter were utilised. UV-Vis double beam spectrophotometer (model Shimadzu, Kyoto, Japan 1800) with 1cm. matched pair quartz cell coupled with UV-probe software were also employed in this work. Analytical balance, the pH meter and ultra-sonication employed were of Lab India and EIE Instrument Limited.

2.3 Development of analytical method:

2.3.1 Solubility study:

For the development of an analytical method solubility plays a vital role. Solubility study helps in deciding a common solvent in which both the drugs will be soluble. Solubility studies were performed for both the drugs in water, methanol, ethanol, chloroform, benzene, ether, acetonitrile, and 0.05 M Phosphate buffer (pH 4.0).

2.3.2 Selection of wavelength:

UV spectrophotometric study was performed to find out the maximum wavelengths (λ_max) of the drugs which are a characteristic property of a chemical entity. This helps in setting the UV-visible detector of HPLC to identify the components present in it. A working sample solution (50μg/ml) in 0.1 N hydrochloride as blank to determine the λ_max of these two drugs.

2.3.3 Selection of chromatographic condition:

The chromatographic condition which is most suitable for the drug depends on the various factors like nature of the sample, its ionic property whether it is a neutral or ionizable, un-ionizable molecule, its molecular weight, solubility, viscosity, affinity towards the stationary and mobile phase. HPLC (Shimadzu, Kyoto, Japan) was composed of an LC-20AT accurate solvent delivery system, a manual rheodyne injector with a 20μl fixed loop and a SPD-20A Prominence ultraviolet–visible detector. Separation was performed on C-18 column (250 × 4.6mm × 5µm) at ambient temperature. Data were acquired on a LC solution. Acetonitrile, phosphate buffer and acetate buffer of different pH in different combination ratio were tried as the mobile phase to find out the most suitable mobile phase for isolation. Finally, a 70:30 mixture of acetonitrile and phosphate buffer (pH 4.0) was used. In (V/V) as mobile phase which gave the best resolution, symmetry and the capacity factor was chosen as the optimal mobile phase for isolation. Mobile phase was filtered through 0.45-micron pore size followed by degassing and the mobile phase was allowed to run at a flow rate 1.0ml/min.

2.3.4 Preparation of standard stock solutions:

The solubility study indicates that phosphate buffer is the most suitable choice in which both the drugs are dissolved. For the stock solution of DOXY 100mg drug was taken in dissolved in 50ml of mobile phase in a 100 ml volumetric flask and made up to100ml mark with the same mobile phase to make a final concentration of 1000µg/ml. Stock solution of PYRI was prepared by taking the same amount of drug in another volumetric flask followed by dilutions as mentioned for DOXY previously to make the concentration of 1000µg/ml.

2.3.5 Preparation of calibration curve:

The two drugs were given concentrations in the range of 10-50g/ml for the calibration curve. From the stock solutions of DOXY and PYRI 1, 2, 3, 4 and 5ml volume were taken in the same 100ml volumetric flask and adjusted up to 100ml with the solvent to prepare standard solutions of 10, 20, 30, 40 and 50µg/ml respectively. Then the standard solutions were injected in HPLC at a flow rate of 1.0ml/min. From the chromatogram peak area vs. concentration of both the drugs were plotted to prepare the calibration curve.

2.4 Method Validation:

Analytical method validation is a way of establishing documented as well as practical evidence that an analytical procedure is appropriate for its intended use and is capable of establishing identity, quality, quantity, and purity of a chemical substance. For this analytical method development and validation in pharmaceuticals research, severalguidelines are followed like guidelines of International Conference on Harmonisation (ICH), USFDA, etc¹⁹. There are few common parameters are discussed for the validation of the method.

2.4.1 System suitability parameters for UV and HPLC:

System suitability is an essential and integral part and essential of chromatographic method validation. Those tests need to verify resolution and reproducibility is up to the mark for analysis operations. This include- Resolution {R_s = 2 (t_R2 - t_R1)/(W₁+ W₂)}, Number of theoretical plate {N=16(t_r/W)²}, Tailing factor {T_f = W/ 2d}, and HETP¹⁶.

2.4.2 Linearity and Range:

Linearity is nothing but the capability (within a given range) of an analytical procedure to get a proportional relationship between the response and concentration. The range for an analytical method is the window within the maximum and minimum amount of analyte which can be assessed with a suitable amount of precision, accuracy, and linearity. The range of concentrations for linearity plot was taken from 10.0-50.0μg/ml for both the drugs by diluting the stock solution. A calibration curve or standard curve was plotted by taking the concentration on X-axis and peak area in Y-axis. From calibration curve, the correlation coefficient (R) and equations of regression lines were determined to establish the linearity and range.

2.4.3 Accuracy:

Accuracy or trueness is the measurement that test results obtained by that method are how much closer to the true value. Accuracy is assessed as the recovery percentage in assay when a known amount of analyte added into the sample or the difference of the mean from the accepted true value. Recovery study was performed by incorporating the standard drug at various concentration levels 80%, 100% and 120% and then the deviation of concentration between the spiked value and the actual value was compared.

2.4.4 Precision:

Study of precision is the degree of agreement of an analytical method between the different test results, when the method is repeated for more than one sample of same concentration and same analyte. It is determined by using standard deviation or coefficient of variation.

SD (Standard deviation) =, x = value of each observation, = mean value, n = no of observations

Precision is measured either by the degree of reproducibility or repeatability of the analytical method. The precision of the above method was analyzed by means of repeatability, intraday and interday variations. The repeatability was ascertained by analyzing the peak area of the lower, middle and higher concentrations that is 10µg/ml, 30µg/ml and 50µg/ml for both drugs three times. Intraday and interday variations were determined by taking the same concentrations as per the repeatability study on three different days. The variations were calculated in terms of RSD.

2.4.7 Robustness:

Robustness of a procedure may be defined as a measure of the capacity of a method to remain intact by little but intentionally variation in method parameter which proves its reliability^25.Robustness was performed by changing the column of different brand names, little variation in mobile phase pH ±0.2 range, and variation in the flow rate (±0.2ml/min). The middle concentrations of 30µg/ml standard solution containing both the drugs were analyzed in the above mentioned conditions.

2.4.8 Limit of Detection and Limit of Quantitation:

LOD can be defined as the lowest concentration of the analyte in a sample which is detectable by an analytical procedure but it may not be quantified and LOQ is defined as the lowest concentration of analyte can be calculatedor quantitated with suitable accuracy and precision. LOD and LOQ were determined from the signal-to-noise ratio by comparing test with the known concentration¹⁹. LOD can be calculated by plotting the calibration curve (n=3) and the SD of the intercepts by taking 10, 30, and 50µg/ml concentrations of the drugs.

LOD= (3.3*SD)/Slope, LOQ= (10*SD)/Slope

Where, SD= standard deviation of Y-intercept of the calibration curves, Slope= mean slope of the calibration curves

2.4.9 Robustness:

Robustness of a procedure may be defined as a measure of the capacity of a method to remain intact by little but intentionally variation in method parameter which proves its reliability. Robustness was performed by changing the column of different brand names, little variation in mobile phase pH ±0.2 range, and variation in the flow rate (±0.2ml/min). The middle concentrations of 30µg/ml standard solution containing both the drugs were analyzed in the above mentioned conditions.

2.5. Determination of DOXY and PYRI from its Dosage form (DOXILAR TABLET):

2.5.1. Preparation of sample:

Ten Doxilar tablets were weighed and mean of the weight was noted. Tablets were triturated and powder equivalent to 100mg of DOXY and PYRI in a 100ml volumetric flask and dissolved in 50ml mobile phase. Volume was made up to 100ml with the same solvent to prepare a stock solution of 1000µg/ml of DOXY and PYRI. The solution is filtered through filter paper (0.45µm). From the stock solution, 3ml was taken and diluted up to 100ml with mobile phase to make the final concentration 30µg/ml for the analysis of combined dosage form.

2.5.2. Estimation of DOXY and PYRI in its dosage form:

The sample solution was injected in HPLC and the mobile phase was allowed to run at a flow rate of 1.0 ml/min. The peak area obtained from chromatogram was taken to determine the amount of DOXY and PYRI in its combined dosage form.

2.6. Forced degradation study:

For the proper establishment of an analytical method, the tablet and active pharmaceutical ingredient (API) of DOXY and PYRI were subjected to forced degradation ¹⁹ to know the conditions may be responsible for the drugs to degrade.

2.6.1 Hydrolysis:

The most common reasons for degradation of drug product in a wide range of pH are due to hydrolysis. This is simply due to reaction with water¹⁸. Hydrolytic studies are generally performed using acidic and basic conditions. In the 3ml of standard stock solution (1000 µg/ml) of the drugs, 5ml of 0.1N hydrochloric acid (HCl) was added in a 10ml of volumetric flask and the volume was made up to the mark with the mobile phase. Then, the volumetric flask was kept at room temperature for 4hours. After 4 hours’ time interval, 1ml of solution was taken from this flask, neutralized and diluted with mobile phase up to 10ml to achieve 30µg/ml concentration and the sample solution was injected in HPLC. Basic hydrolysis was carried out by taking 5ml of 1N sodium hydroxide (NaOH) with 3ml of stock solution (1000µg/ml) in 10ml volumetric flask and kept for 4 hours in room temperature and the procedure was repeated as per the acid hydrolysis. From the retention time and peaks in the standard chromatogram and chromatogram obtained after degradation, the percentage degradation was calculated.

2.6.2 Oxidative degradation:

The oxidative degradation involves the transfer of electron mechanism to form reactive anions or cations. Generally hydrogen peroxide (H₂O₂) was used as oxidizing agent¹⁸. Oxidative degradation was carried out by taking 3 ml of stock solution (1000µg/ml) and 2 ml of 8% H₂O₂as an oxidizing agent in a 10ml flask. The mobile phase was added up to 10ml and it was kept in room temperature for 4hours. After 4 hours sample solution was pipette out, followed by addition and dilution up to 10ml finally to prepare a concentration of 30µg/ml and injected in HPLC. From the peak area and retention time the % degradation was calculated.

2.6.3 Photolytic degradation:

Degradation may also occur due to the exposure to sunlight or exposure to other light. It results in the photolytic reaction. The photolytic study is generally performed in an exposed to a minimum of 1.2million lux h and 200Wh/m² light particular wavelength region 300-800nm¹⁸. Sample of the drugs were exposed in UV light for 4 hours. Sample was prepared from 100mg of drug sample was dissolved in 50ml of mobile phase in a 100 ml volumetric flask and diluted up to the mark to prepare a 1000µg/ml concentration stock solution. From the above stock solution, required amount taken and diluted to achieve 30µg/ml concentration. Then the sample was injected in HPLC for analysis.

2.6.4 Thermal degradation:

Thermal exposure to a drug is the most common type problem during storage. Therefore ICH suggests conducting thermal degradation study of the drug product. Many pharmaceutical products are heat sensitive or heat labile¹⁸. The effect of temperature on thermal degradation of a substance can be stated by the Arrhenius equation:

k=A e^-Ea/RT

Where, K= specific reaction rate, A= frequency factor, Ea = energy required for activation, R = gas constant For thermal degradation the sample drug was taken in a petridish and exposed to 60ºC temperature for 24hours. After 24hours 100mg of sample was dissolved in 50ml mobile phase and diluted up to 100ml to prepare 1000 µg/ml concentration. From this stock solution 30µg/ml concentration was prepared. Then the sample was injected in HPLC system and percentage degradation was estimated by comparing the chromatogram with standard.

3: RESULTS AND DISCUSSION:

3.1 Solubility study:

From the solubility study it was found that DOXY is freely soluble in water and phosphate buffer (pH 4.0) and slightly soluble in alcohol whereas PYRI was found to be very soluble in water and phosphate buffer (pH 4.0). Therefore phosphate buffer of pH 4.0 was used as the solvent of choice for the preparation of drug solution.

3.2 Selection of wavelength:

Both the drugs were scanned in the UV spectrophotometer. Absorbance maxima of DOXY and PYRI were at 290nm and 260nm respectively. From the overlay it was found that both drugs were showing significant absorbance at 260nm, therefore the working wavelength for HPLC was selected at 260nm.

3.3 Chromatographic condition:

After trying a number of mobile phases the most suitable chromatographic condition was decided for the estimation of both the drugs. The standard chromatogram (Figure 2a) was obtained in 0.5 M phosphate buffer (pH 4.0) and acetonitrile in 70:30 ratios.

Figure 2a: Chromatograms of the standards 2a) chromatogram of the standard DOXY and PYRI in most favorable conditions and suitable mobile phase and 2b) overlay chromatogram of different concentrations of DOXY and PYRI. 2c) Chromatogram of Doxilar tablet and 2d) chromatogram of DOXY and PYRI standard (30 µg/ml) before degradation.

3.4 System suitability parameters:

System suitability parameters were calculated as per ICH guidelines. Parameters of system suitability study such as retention, theoretical, resolution, theoretical plate, tailing factor, HETP of DOXY were 4.256, 8932.71, 11.30, 1.03, 0.028 respectively and PYRI were found to be 2.400, 3931.62, 0.00, 1.25, 0.063.

3.5 Calibration curve for DOXY and PYRI:

The calibration curve was plotted taking 10, 20, 30, 40 and 50 μg/ml concentration for DOXY and PYRI respectively. Using the peak areas that were obtained from the chromatograms (Figure 2b) of DOXY and PYRI calibration curve was plotted. The coefficient of determination (R²), slope and intercept for DOXY and PYRI were 0.9995 and 0.999, 15927 and 15237, 2383.6 and 4032 respectively. The Regression equation for DOXY and PYRI were found to be y = 15927x + 2383.6 and y = 15237x + 4032 respectively.

3.6 Analytical Method Validation:

3.6.1 Linearity and range:

The linearity ranges for both the drugs were found to be from 10-50µg/ml concentration. The coefficient of determination (R²) for DOXY and PYRI were 0.9995 and 0.999 respectively.

3.6.2 Accuracy:

The mean percentage recovery of doxylamine succinate and pyridoxine hydrochloride was found to be in the range of 100.079–100.086% and 100.051–100.074%, respectively, within the acceptable limits for 80%, 100% and 120% of the both drugs (Table 1`).

3.6.3 Precision

The precision in terms of repeatability, intraday and interday precision was expressed as percentage RSD. For this method the % RSD was found to be in the range as per the guideline which is less than 2 % (Table 2).

3.6.4 Limit of detection and limit of quantitation:

Mean slop (n=6) for DOXY and PYRI were found to be 15927 and 15237 respectively, SD (n=6) for DOXY and PYRI were found to be 0.9447 and 0.1211, LOD for DOXY and PYRI were found to be 0.9447µg/ml and 0.1211µg/ml respectively and the LOQ for the two drugs were 2.8629µg/ml and 0.3670µg/ml respectively.

3.6.5 Robustness:

There was no certain change in retention time though the chromatographic condition was deliberately changed. The amount recovery and the % RSD were within limits for both drugs (Table 3).

Table 1: Accuracy study of the two drugs

Recovery data of DOXY
% of Spike	Amt of drug spiked (μg/ml)	Peak Area of sample	Amount recovered (μg/ml)	% Recovery	Mean % recovery ± SD	% RSD
80	20	321150	20.0142	100.071	100.079 ± 0.007	0.00735
	20	321194	20.017	100.085
	20	321186	20.0165	100.082
100	30	480764	30.0358	100.119	100.086 ± 0.084	0.08394
	30	480905	30.0447	100.149
	30	480150	29.9973	99.9909
120	40	639832	40.0231	100.058	100.08 ± 0.021	0.02166
	40	640108	40.0405	100.101
	40	639972	40.0319	100.08
Mean recovery			100.082 %
Standard Deviation			0.00396
% RSD			0.00396
Recovery data of PYRI
% of Spike	Amt of drug spiked (μg/ml)	Peak Area of Sample	Recovered Amount (μg/ml)	% Recovery	Mean % recovery ± SD	% RSD
80	20	308980	20.0137	100.068	100.063 ± 0.011	0.01167
	20	308924	20.01	100.05
	20	308990	20.0143	100.072
100	30	461445	30.0199	100.066	100.051 ± 0.087	0.08732
	30	460948	30.0391	100.13
	30	460948	29.9873	99.9576
120	40	613920	40.0268	100.067	100.074 ± 0.011	0.01143
	40	614045	40.035	100.087
	40	613929	40.0274	100.068
Mean recovery			100.063 %
Standard Deviation			0.011
% RSD			0.011

Table 2: Precision study of DOXY and PYRI

Amount of drug taken (µg/ml)		Amount of drug found (µg/ml)			SD		% RSD
Repeatability (n=3)
DOXY	PYRI		DOXY	PYRI	DOXY	PYRI	DOXY	PYRI
10	10		10.05	9.913	0.0119	0.007	0.0118	0.079
30	30		29.992	30.049	0.0121	0.046	0.0403	0.156
50	50		49.983	50.007	0.0374	0.056	0.0748	0.112
Intraday precision (n=3)
DOXY	PYRI		DOXY	PYRI	DOXY	PYRI	DOXY	PYRI
10	10		10.085	10.081	0.008	0.006	0.081	0.063
30	30		30.016	29.941	0.024	0.023	0.081	0.078
50	50		49.969	50.006	0.052	0.009	0.104	0.019
Interday precision (n=3)
DOXY	PYRI		DOXY	PYRI	DOXY	PYRI	DOXY	PYRI
10	10		10.075	10.056	0.015	0.017	0.153	0.171
30	30		29.959	29.96	0.05	0.034	0.167	0.116
50	50		50.21	49.993	0.057	0.049	0.114	0.098

Above results provide confidence that the method is simple, easy, reliable, robust, user-friendly, cost-effective and valid. The system suitability factors such as resolution, theoretical plate, HETP, tailing factor, etc. indicates that method was suitable for doxylamine succinate and pyridoxine hydrochloride. The Linearity range was found to be 10-50µg/ml for both drugs. The %RSD for accuracy, precision, robustness was found to be less than 2% that indicates the method was validated as per ICH. The LOD and LOQ values of the two drugs were very less than the other methods available. The degradation study showed there was no certain change in retention time but the presence of extra peaks other than the two drugs indicated the presence of degradation product. Doxylamine succinate was found to be more degraded in basic condition that was 2.20% whereas the pyridoxine hydrochloride was found to be more degraded in oxidative degradation condition which was 11.542%. Literature study indicates that our method is much simpler, easy, reliable, robust, user-friendly, and cost-effective than the previous methods of HPLC and UPLC. When we compare our validation parameters with the reported UPLC method by Panchal V. J. Panchal et al.²² in Table 5 we found that the SD and %RSD values for accuracy and precision in our method are far better and % degradation can be determined more specifically.

4. CONCLUSION:

The above mentioned RP-HPLC method was very effective stability indicating assay method for the estimation of doxylamine succinate and pyridoxine hydrochloride drugs in bulk as well as in combined dosage form than the previously reported other methods. This method was also validated as per ICH guidelines using suitable parameters like linearity, range, specificity, precision, repeatability, robustness, LOD, LOQ, etc. The process was linear in the range of 10-50 µg/ml. for both drugs. This method was very much effective for determining LOQ very effectively at a very minimum concentration. The stability indicating assay reflects, DOXY was more susceptible and degraded in basic condition (12.20 %) whereas PYRI was more susceptible and degraded in oxidative condition (11.54 %). In photolytic condition both the drugs were most stable. From the forced degradation study it can be concluded that both the drugs should be protected from high temperature, light and are to be stored in a closed container. This method is a very simple, sensitive, reliable as well as economical and precise method which can be used in laboratory or industry purpose.

5. ACKNOWLEDGEMENTS:

The authors are thankful to Mr. Darshan Patel, QC Officer, and Mr. H. C. Roy, QC Executive, of Mercury Laboratories Pvt. Ltd., Vadodara. We are also thankful to the management team of the company for providing the Doxilar tablet, required chemicals, and equipment. Our sincere gratitude to the Harshada Corporation and Harika Drugs Pvt. Ltd. for providing the API of pyridoxine hydrochloride and doxylamine succinate as a gift sample.

6. CONFLICT OF INTEREST:

The authors declare no conflict of interest hereby.

7. REFERENCES:

1. Jin P. Xia L. Li Z. Che N. Zou D. Hu X. Rapid determination of thiamine, riboflavin, niacinamide, pantothenic acid, pyridoxine, folic acid and ascorbic acid in vitamins with minerals tablets by high-performance liquid chromatography with diode array detector. J. Pharm. Biomed. Anal. 2012; 1(70):151-7.doi.org/10.1016/j.jpba.2012.06.020

2. Nuangchamnong N. Niebyl J. Doxylamine succinate–pyridoxine hydrochloride (Diclegis) for the management of nausea and vomiting in pregnancy: an overview. Int J Womens Health. 2014; 6: 401. doi.10.2147/IJWH.S46653.

3. Badell ML. Ramin SM. Smith JA. Treatment options for nausea and vomiting during pregnancy. Pharmacotherapy: The Journal of Human Pharmacology and Drug Therapy. 2006; 26(9):1273-87. doi.org/10.1592/phco.26.9.1273.

4. Harde MT. Lakade SH. A stability-indicating HPLC method for estimation of doxylamine succinate in tablets and characterization of its major alkaline stress degradation product. Future J. Pharm. Sci. 2021; 7(1):1-3. doi.org/10.1186/s43094-021-00276-6

5. Habib NM. Abdelwhab NS. Abdelrahman MM.Ali NW. Spectrophotometric methods for analysis of different dosage forms containing pyridoxine hydrochloride. Eur. J. Chem. 2016; 7(1):30-6. doi.org/10.5155/eurjchem.7.1.30-36.1350

6. Pathak A. Rajput SJ. Simultaneous derivative spectrophotometric analysis of doxylamine succinate, pyridoxine hydrochloride and folic acid in combined dosage forms. Indian J. Pharm. Sci. 2008; 70(4):513. doi.10.4103/0250-474X.44607

7. Uslu B. Özkan SA. Aboul-Enein HY. Spectrophotometric determination of melatonin and pyridoxine HCL in binary mixture using first derivative of the ratio spectra method. Anal. Lett. 2002; 35(14):2305-17. doi.org/10.1081/AL-120016104

8. Bhamre PR. Pathak AS. Rajput S. Simultaneous determination of doxylamine succinate, pyridoxine hydrochloride and folic acid by chemometric spectrophotometry. Int. J. Pharm. Sci. 2013; 4(1):738-49.doi.10.4103/0250-474x.44607

9. Consigliere VO. Vals NR. Magalhãs JF. First-derivative spectrophotometric determination of pyridoxine hydrochloride in pharmaceutical preparations. Anal. Lett. 2001; 34(11):1875-88. doi.org/10.1081/AL-100106118

10. Malothu N. Areti AR. Araja S. Paidi M. Swathi S. Chamala VD. Srivani NN. UV-Spectroscopic Method development and Validation for simultaneous estimation of Doxylamine succinate and Pyridoxine hydrochloride in bulk and Pharmaceutical dosage form. Research J. Pharm. and Tech. 2020; 13(10):4613-20. doi.10.5958/0974-360x.2020.00812.4

11. Wate SP. Bhagwat GB. Mundhey AS. Bawankar PM. Absorption Ratio Method for Simultaneous Estimation of Prochlorperazine Maleate and Pyridoxine Hydrochloride in Tablet. Research J. Pharm. and Tech. 2012; 5(8):1112-3.doi.10.5958/0974-360x.

12. Ibrahim MM. Elzanfaly ES. El-Zeiny MB. Ramadan NK. Kelani KM. Spectrophotometric determination of meclizine hydrochloride and pyridoxine hydrochloride in laboratory prepared mixtures and in their pharmaceutical preparation. Spectrochimica Acta Part A: Spectrochim Acta A Mol Biomol Spectrosc. 2017; 178:234-8. doi.org/10.1016/j.saa.2017.02.013

13. Pathak A. Rajput SJ. Simultaneous derivative spectrophotometric analysis of doxylamine succinate, pyridoxine hydrochloride and folic acid in combined dosage forms. Indian J. Pharm. Sci. 2008; 70(4):513.doi.org/10.4103%2F0250-474X.44607

14. R. E. Tarigan. S. M. Sinaga. Muchlisyam. Z. Alfian.Simultaneous Determination of Dextromethorphan Hydrobromide, Doxylamine Succinate And Pseudoephedrine Hydrochloride In Cough And Cold Syrup By Area Under Curve Spectrophotometry Method. Rasayan J. Chem. 2021; 14(1): 629-634. doi.org/10.31788/ RJC.2021.1416068

15. Argekar AP, Sawant JG. Simultaneous determination of pyridoxine hydrochloride and doxylamine succinate in tablets by HPTLC. J. Liq. Chromatogr. Relat. 1999; 22(13): 2051-60. doi.org/10.1081/JLC-100101785

16. Giriraj P. Sivakkumar T. Development and validation of a rapid chemometrics assisted RP-HPLC with PDA detection method for the simultaneous estimation of pyridoxine HCl and doxylamine succinate in bulk and pharmaceutical dosage form. Chromatogr. Res. Int. 2014; 2014. doi.10.1155/2014/827895

17. Nawaz M. A new validated stability indicating RP-HPLC method for simultaneous estimation of pyridoxine hydrochloride and meclizine hydrochloride in pharmaceutical solid dosage forms. Chromatogr. Res. Int. 2013; 2013.doi.org/10.1155/2013/747060

18. Gil-Agustí M. Capella-Peiró ME. Monferrer-Pons L. García-Alvarez-Coque MC. Esteve-Romero J. Chromatographic analysis of phenethylamine–antihistamine combinations using C8, C18 or cyano columns and micellar sodium dodecyl sulfate–pentanol mixtures. Analyst. 2001; 126(4):457-64. doi.org/10.1039/b009546j

19. Almakaiev MS. Dvinskikh NV. Almakaieva LG. Kryvanych OV. Development of a combined solution of pyrimidine nucleotides with vitamin B6. Research J. Pharm. and Tech. 2021; 14(12):6228-34.doi.10.52711/0974-360x.2021.01078

20. Bhagwat GB. Jaybhaye M. Development and Validation of a RP-HPLC Method for the Simultaneous Estimation of Prochlorperazine Maleate and Pyridoxine Hydrochloride in Combined Dosage Forms. Research J. Pharm. and Tech. 2018; 11(4):1338-42. doi.10.5958/0974-360x.2018.00249.4

21. Nagappan KV. Meyyanathan SN. Raja RB, Reddy S. Jeyaprakash MR. Birajdar AS. Bhojraj S. A RP-HPLC method for Simultaneous Estimation of Ambroxol Hydrochloride and Loratidine in pharmaceutical formulation. Research J. Pharm. and Tech. 2008; 1(4):366-9.doi.10.5958/0974-360x.

22. Thorat Shashikant D. Ghodeswar B. C. Darode G. S. Shinde N. S. Chaudhari S. R. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Cholecalcererol (Vitamin D3) and Tocopherol Acetate (Vitamin E) in Powder Dosage Form. Research J. Pharm. and Tech. 2013; 6(10): 1107-1110. doi.10.5958/0974-360x.

23. Sulthoniyah D. Primaharinastiti R. Annuryanti F. Method validation of flame atomic absorption spectrophotometry (FAAS) for determination of iron (FE) in multivitamin mineral capsule dosage form. Research J. Pharm. and Tech. 2018; 11(6): 2569-74.doi.10.5958/0974-360x.2018.00475.4

24. Nalini CN. Method Development and Validation for the Simultaneous Estimation of Ascorbic acid, Phenylephrine HCl, Paracetamol and Levocetirizine HCl using RP-HPLC. Research J. Pharm. and Tech. 2020; 13(4):1911-6.doi.10.5958/0974-360x.2020.00344.3

25. Das P. Prabhu PP. Reddy J. Development and Validated Stability indicating RP-HPLC method for quantitative determination of Ascorbic acid and piperine in Dhathryaristam. Research J. Pharm. and Tech. 2020; 13(8): 3729-32. doi.10.5958/0974-360x.2020.00660.5

26. Choudhari VP. Kale AN. Polshettiwar SA. Sutar AS. Patel DM. Kuchekar BS. Derivative and Absorption Factor Spectrophotometric Estimation of Montelukast Sodium and Levocetirizine Dihydrochloride from Pharmaceutical Formulations. Research J. Pharm. and Tech. 2011; 4(3):389-92. doi.10.5958/0974-360x.

27. Bitar Y. Separation and assay of three anti-cough drugs pseudoephedrine, dextromethorphan and chlorpheniramine in pharmaceutical forms by using single RP-HPLC METHOD. Research J. Pharm. and Tech. 2020; 13(2):831-9.doi.10.5958/0974-360x.2020.00157.2.

28. Singh SK. Validation of isocratic RP-HPLC method and UV spectrophotometric method for the estimation of loratadine in pharmaceutical formulations. Research J. Pharm. and Tech. 2015; 8(4):452-61. doi.10.5958/0974-360x.2015.00076.1

29. Polawar PV. Shivhare UD. Bhusari KP. Mathur VB. Development and validation of spectrophotometric method of analysis for fexofenadine HCl. Research J. Pharm. and Tech. 2008; 1(4):539-40. doi.10.5958/0974-360x.

30. Guideline ICH. Validation of analytical procedures: text and methodology. Q2 (R1). 2005;1(20):05.

31. Guideline, ICH Harmonised Tripartite. Stability testing of new drug substances and products. Q1A (R2), current step 4 (2003): 1-24.

Received on 13.01.2022 Modified on 17.08.2022

Research J. Pharm. and Tech 2023; 16(10):4559-4567.

DOI: 10.52711/0974-360X.2023.00743

Parameters (n=3)	Variation	Average amount found (µg/ml)		% RSD
Parameters (n=3)	Variation	PYRI	DOXY	PYRI	DOXY
Flow rate	+ 0.2	29.71	29.65	0.8487	0.8312
Flow rate	- 0.2	29.63	29.74	0.8312	0.6171
Column	Phenomenex	29.79	29.61	0.4089	0.7109
Column	Grace Smart	29.66	29.66	0.6824	0.9217
Ph	+ 0.2	29.64	29.36	0.7991	0.8466
Ph	- 0.2	29.57	29.60	0.8843	1.0321

Drug	Amount as per marketed formulation	Concentration taken for assay (µg/ml)	Peak area of sample	Concentration found (µg/ml)	Assay
DOXY	10 mg	30	481147	30.0599	100.2 %
PYRI	10 mg	30	461253	30.0073	100.02%

Degradation	Drug	Time Interval	Peak Area	Percentage Undegraded (%)	Percentage Degraded (%)
Acid	DOXY	4 hours	441263	91.860	8.14
Acid	PYRI	4 hours	431451	93.510	6.49
Base	DOXY	4 hours	421865	87.800	12.20
Base	PYRI	4 hours	424068	91.890	8.11
Oxidation	DOXY	4 hours	449666	93.620	6.38
Oxidation	PYRI	4 hours	431451	88.460	11.54
Photolytic	DOXY	4 hours	467912	97.430	2.57
Photolytic	PYRI	4 hours	453831	98.401	1.599
Thermal	DOXY	24 hours	464071	96.626	3.374
Thermal	PYRI	24 hours	451755	97.947	2.053