UV-Spectroscopic Method development and Validation for simultaneous estimation of Doxylamine succinate and Pyridoxine hydrochloride in bulk and Pharmaceutical dosage form

 

Narender Malothu^1*, Anka Rao Areti¹, Sreehasa Araja², Mounika Paidi ², Sonekar Swathi ², Vijaya Durga Chamala², Navya Srivani N²

¹KL College of Pharmacy, Koneru Lakshmaiah Education Foundation, Vaddeswaram, Guntur, AP, India.

²Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu, Vijayawada, India.

 

ABSTRACT:

Simple and precise UV-spectrophotometric methods were developed and validated for the simultaneous estimation of combined dosage form containing Doxylamine succinate and Pyridoxine HCl. A combination of these drugs is being used in the treatment of morning sickness associated with pregnancy. In the current study, both the drugs estimated by simultaneous equation and Q-absorbance ratio methods with the help of 260 and 290 nm as maximum absorption wavelengths, respectively for Doxylamine succinate and Pyridoxine HCl. Linearity was achieved at the concentration of 10-35 μg/mL for both drugs with correlation coefficient (r²) of 0.9949 and 0.9935, respectively. The % RSD for accuracy and precision were lies within the acceptance criteria. The percentage (%) recoveries found for both drugs were in the potency range (98.14-100.5 %). There was good agreement in results obtained for detection and quantitation limits studies which revealed the presence of sensitivity of the methods. The assay of the commercial sample was in well fit with the standards and the developed methods can allow for usual analysis of both drugs in combined dosage forms.

 

 

 

INTRODUCTION:

Doxylamine (DOX) is a first-generation monoethanolamine analog with anti-inflammatory, sedative and antihistamine properties. The drug works by blocking the action of endogenously secreted histamine and competes the intrinsic binding of histamine (H1) at receptor site thereby stops the subsequent release of pro-inflammatory mediators from basophils and mast cells.

 

Pyridoxine (PYD) is also termed as Pyridoxal, it is a vitamin B6 in the family of vitamin B. It is essentially required by the body to make amino acids, carbohydrates, and lipids. It is also a crucial component for maintaining the normal health conditions of nerves, skin and red blood cells.

 

Combination of DOX and PYD is available in the tablet dosage form (Doxinate tablets). It is commonly prescribed in oral route for sleep with or without food (Fig 1), and with other medications in the form of liquid and/or liquid-filled capsule to relieve the symptoms of the common cold. The combined form acts as sedating antihistamine, which is being prescribed for short-term treatment of insomnia. The same drugs also administered to relieve the runny nose, sneezing and nasal congestion in the combination with other medications such as decongestants.

 

Fig 1: Chemical structures of DOX succinate and PYD hydrochloride.

 

As per literature survey some analytical methods are reported for quantification of DOX succinate and PYD hydrochloride (HCl) individually and in combination with other drugs using UV-Visible spectroscopy^[1-4], HPLC^[5-17], HPTLC^[18] and Mass spectroscopy^[19] etc. Few methods^2-4 have been published for the determination of DOX succinate and PYD HCl in combine dosage form using UV-spectrophotometry. The UV-spectrophotometric analysis is frequently used for the control of quality of pharmaceuticals in ordinary laboratories due to its broader availability, suitability, and ease of use. Henceforth, an attempt has been made to estimate both the drugs in a combined dosage form with the help of simple quantitative methods i.e., Simultaneous Equation Method (SEM) and Q-Absorbance Ratio Method (Q-ARM). The methods were optimized by appropriate measures and validated as per ICH guidelines; in this trial the methods were expressed the simplicity and sensitivity towards determination of drugs in formulations.

 

METHODS AND MATERIALS:

Reagents and apparatus:

The DOX succinate and PYD HCl were obtained as gift samples from Granules India Limited, Hyderabad. All the chemicals used such as ortho-phosphoric acid and potassium dihydrogen ortho-phosphate were Analytical Grade (AR) and they were purchased from Merck India Limited, Mumbai and SD Fine Chemicals, Mumbai. The analysis was performed in 10mm quartz cells using a UV-Visible spectrophotometer (LAB India) with a fixed 2nm spectral bandwidth and UV-Win 5 software v5.1.1 was used for all absorbance measurements.

 

Determination of absorption maxima:

Based upon solubility and compatibility in absorption for both drugs in the UV region, 0.05M phosphate buffer (pH 5.0) was selected as solvent diluent. Standard solutions containing 10µg/mL of each DOX succinate and PYD HCl was prepared in the diluent (0.05 M phosphate buffer pH 5.0), approximately 3.0mL of solution in 10mm cuvette scanned under UV region 400-200nm using UV-Visible spectrophotometer (LAB India) with fixed slit width of 2.0nm.

 

Preparation of standard stock solution (100 μg/ mL):

The standard stock solution of DOX succinate and PYD HCl was prepared in two separate 100mL volumetric flasks by dissolving 10mg of each drug in 70mL of 0.05 M phosphate buffer (pH 5.0). The resulted solution was sonicated to dissolve for 20 min and the volume was made up to the mark with the diluent.

 

Preparation of standard solutions (10-60μg/mL):

From the above stock solution 1, 2, 3, 4, 5 and 6mL, was pipette out into separate 10mL volumetric flasks and the volume was made up to the mark with 0.05 M phosphate buffer pH 5.0 to get final concentrations of 10, 20, 30, 40, 50 and 60μg/mL. Resulted solutions were scanned under the UV region (200-400nm) to get absorbance and wavelength maxima (λ_max) of both the analytes.

 

Method development:

Preparation of sample solution:

Weighed accurately about 20 tablets of Doxinate and the tablet powder equivalent to 10mg of each DOX succinate and PYD HCl into 100mL was transferred into standard volumetric flask containing 70mL of 0.05M phosphate buffer (pH 5.0). The solution was sonicated for 10 minutes and the volume was made up to the mark to get the final concentration of 100µg/mL of each analyte. The resulted solution was filtered through 0.45 µ Millipore nylon membrane filter and scanned under UV- spectrophotometer.

 

Method Ⅰ (SEM):

From the two absorbing species in the sample (X = DOX succinate and Y = PYD HCl) each of which absorbs at the maximum wavelength (λ_max) of different from the other, henceforth the amount or concentration of both drugs was determined by the technique of SEM (Vierodt's method). The following data is required for the estimation,

·       aX₁ and aY₁ are the absorptivities of DOX succinate and PYD HCl at 260 nm.

·       aX₂ and aY₂ are the absorptivities of DOX succinate and PYD HCl at 290 nm.

·       A₁ and A₂ are the absorbances of the sample at 260 and 290 nm.

 

As the sample contains DOX succinate and PYD HCl and their concentration would be taken as C_DOX and C_PYD, respectively. Following equations were considered based upon the data obtained that at 260 and 290nm, the absorbance of formulation is the sum of the individual absorbances of DOX succinate and PYD HCl.

 

Method Ⅱ (Q-ARM):

As per the method, the quantitative assay of both the analytes in a mixture is determined by measuring the absorbance’s at two wavelengths, one being the λ_max of DOX succinate (260nm) and other being a wavelength of equal absorptivity of two drugs (268nm). The individual concentration of each drug determined by following equations,

 

Where,

·       QM =A₂/A₁; QX = aX₂/aX₁; QY = aY₂/aY₁;

·       A₁ and A₂ are the absorbances of the sample at 260 and 268 nm.

·       aX₁ and aY₁ are the absorptivities of DOX succinate and PYD HCl at 260 nm.

·       aX₂ and aY₂ are the absorptivities of DOX succinate and PYD HCl at 268 nm.

 

By considering the individual concentrations, the amount of DOX succinate (C_DOX) and PYD HCl (C_PYD) were determined.

 

Method validation:

After achieving the method development the validation studies was conducted for parameters such as linearity, precision, accuracy, robustness, ruggedness, limit of detection (LOD) and limit of quantification (LOQ) as per ICH Q2 (R1) guidelines²⁰.

 

Specificity:

Standard and sample solutions of DOX succinate and PYD HCl in 0.05 M phosphate buffer (pH 5.0) were scanned in UV-region, to know the presence of interference absorption in the spectrum.

 

Linearity:

Six concentrations of DOX succinate and PYD HCl were freshly prepared in the concentrations ranging from 10-35μg/mL for linearity studies. Solutions of each concentration were then filtered through a 0.45μ Millipore nylon membrane filter and scanned in triplicates. Finally, the linearity was assessed with the aid of linear regression analysis.

 

Accuracy:

The accuracy was assessed by spiking known amounts of standards of DOX succinate and PYD HCl to the sample at three concentrations levels (50, 100 and 150% of the label claim). Three determinations were made at each concentration level and the results were recorded in terms of percent (%) analyte recovered.

 

Precision:

Precision of the developed analytical procedure expressed from the various sampling of the same homogenous sample under the prescribed conditions and it was determined as repeatability. Both system precision and method precision were established in accordance with ICH guidelines.

 

Robustness:

Changes in detection wavelength and pH of the buffer were made in the spectroscopic system to determine the effect of deliberate variations in the optimized spectroscopic conditions.

 

Ruggedness:

To assess the ruggedness of an analytical method a variety of test conditions was applied such that by varying the test analysts, laboratory conditions and instrument (UV-Visible spectrophotometer by Lab India and PG instruments), to know the degree of reproducibility of test results to that of optimized conditions. The sample solution (10μg/mL) at the set concentration was prepared and analyzed in triplicate and their relative absorbances were measured at variable conditions.

 

LOD and LOQ:

As the limit of detection (LOD) and quantification (LOQ) are commonly associated with the signal-to-noise ratio (S/N), hence the LOD determined with (S/N) ratio of 2:1 or 3:1, while LOQ with (S/N) ratio of 10:1. The standard solutions were prepared by lowering the concentration over the linearity range until the signal-to-noise ratio 3:1 for LOQ and 10:1 for LOD could be reached. The detection limit (LOQ = 10 σ / S) and quantification limit (LOD = 3.3 σ / S) can be calculated by the prescribed formula [in which the ‘σ’ is standard deviation of y-intercept regression line (response/absorbance) and ‘S’ is the slope from calibration curves] with the help of measured triplicate absorbance values for each concentration.

 

RESULTS AND DISCUSSION:

This work deals with the development of the UV-Spectrophotometric method for the simultaneous estimation of DOX succinate and PYD HCl in the tablet dosage forms.

 

Selection of detection wavelength:

The absorption maximum for DOX succinate was found at 260nm whereas 290nm for PYD HCl. The iso-absorptive point was observed at 268nm (Fig 2A). At these wavelengths, it gave the maximum compatibility to the proposed methods. The blank spectrum indicated that the solvent system is not interfering the absorbance of analyte (Fig 2B). Further method development protocols for the assay of DOX succinate and PYD HCl (Doxinate tablets) were initiated based on general method development guidelines and literature.

 

Fig 2: A. Overlain spectrum of DOX succinate and PYD HCl in 0.05 M phosphate

buffer (pH 5.0). B. Blank spectrum.

 

Method development:

Method-I (SEM):

The standard solutions of drugs were prepared separately and measured absorbances at respective wavelength maxima. Absorptivities of both drugs aX₁, aX₂ (DOX succinate) and aY₁, aY₂ (PYD HCl) were calculated using standard solution at the concentration of 0.002g /100 mL. The sample solution containing nearly equivalent to the standard amount (as per label claim) was prepared and scanned to measure absorbance's i.e., A₁ and A₂ at 260 nm and 290 nm, respectively (Table 1).

 

Method-II (Q-ARM):

The standard solutions of drugs were prepared separately and measured absorbances at ʎ_max of DOX succinate and iso-absorptive point. Absorptivities of both drugs aX₁, aX₂ (DOX succinate) and aY₁, aY₂ (PYD HCl) were calculated using similar protocols of SEM. The sample solution containing nearly equivalent to the standard amount (as per label claim) was prepared and scanned to measure absorbance’s i.e., A₁ and A₂ at λ_max of DOX succinate (260 nm) and iso-absorptive point (268 nm), respectively. The absorbance of standard and sample at 260 nm and 268 nm were presented in Table 1.

 

Table 1: The absorbance of standard and samples at 260, 290 and 268 nm.

S. No.	Analyte	λmax of DOX succinate 260 nm	λmax of PYD HCl 290 nm	Iso-absorptive point 268 nm
1	DOX succinate	0.290	0.075	0.219
2	PYD HCl	0.198	0.321	0.227
3	Sample	0.379	0.386	0.322

 

Analysis of sample formulation:

DOX succinate and PYD HCl content in marketed formulations (Doxinate tablets) were determined as per the developed methods. The percentage of analyte present in the tablet formulation was determined individually. It was found to be 98.10 ± 0.00184 % of DOX succinate and 98.8 ± 0.00124 % of PYD HCl as per SEM. While in Q-ARM the amount of analyte drugs present was found to be 100.5 ±0.0215 % of DOX succinate and 99.05 ± 0.0134 % of PYD HCl, respectively. The amount of both the analytes was found as per the label claim amount in both methods.

 

Method validation:

Specificity:

The absorption spectrum showed for both standard and sample were similar and there was no interference of excipients (Fig 3). Besides, blank also determined using diluent under UV-region as per optimized conditions and there was no interference of solvent in observed absorbance.

 

Fig 3: Overlain spectrum of DOX succinate (260 nm) and PYD HCl (290 nm) Standard (A), the absorption spectrum of formulation (Doxinate tablets) (B).

 

 

Linearity:

The overlain spectrum was drawn for six concentrations individually (Fig 4A and 4B). The absorbances were recorded and a calibration curve was plotted against the concentration of drug v/s absorbances (Fig 5 and 6). Both the drugs showed a good linear relationship in the calibration curve plot with a regression equation of y = 0.0015x-0.0097 for DOX succinate and y = 0.0314x+0.0484 for PYD HCl (Table 2). The correlation coefficient (r²) (DOX succinate = 0.9949 and PYD HCl = 0.9935) was found within the prescribed range. Therefore, the methods would be clearly indicating the linear relationship at measured concentration range (10-35µg/mL).

 

Fig 4: Overlain spectrum of DOX succinate (A), and PYD HCl (B) at linearity levels (10-35 µg/mL).

 

Table 2: Linearity data of DOX succinate and PYD HCl

Analyte	Concentration (µg/mL)	Absorbance	Linear regression equation
DOX succinate	10	0.092	y = 0.0105x - 0.0097 r² = 0.9949
	15	0.154
	20	0.200
	25	0.248
	30	0.318
	35	0.353
PYD HCl	10	0.391	y = 0.0314x - 0.0484 r² = 0.9935
	15	0.505
	20	0.645
	25	0.821
	30	1.016
	35	1.147

 

Accuracy:

As per SEM the mean recovery achieved was 98.14-98.94 % for DOX succinate and PYD HCl, whereas in Q-ARM the mean recovery was found to be 99.51- 100.5 % (Table 3 and 4). As the % RSD was lies within 2.0 %, which revealed the methods are more accurate.

 

Fig 5: Calibration curve for DOX succinate (10-35 µg/mL).

 

 

Fig 6: Calibration curve for PYD HCl (10-35 µg/mL).

 

Table 3: Accuracy data of DOX succinate (standard addition method) (n=3)

S. No.	Spiked level	Absorbance			Mean% recovery ±SD
		260 nm	290 nm	268 nm	SEM	Q-ARM
1	50 % 50 % 50 %	0.324 0.324 0.321	0.355 0.354 0.355	0.355 0.354 0.355	98.1 ±0.0021	100.5 ±0.0012
2	100 % 100 % 100 %	0.453 0.453 0.451	0.389 0.389 0.386	0.480 0.481 0.481	98.2 ±0.0031	100.4 ±0.001
3	150 % 150 % 150 %	0.554 0.550 0.554	0.375 0.372 0.375	0.576 0.573 0.576	98.12 ±0.00213	100.5 ±0.0011

 

Table 3: Accuracy data of PYR HCl (standard addition method) (n=3)

S. No.	Spiked level	Absorbance			Mean % recovery ±SD
		260 nm	290 nm	268 nm	SEM	Q-ARM
1	50 % 50 % 50 %	0.306 0.305 0.305	0.510 0.511 0.510	0.364 0.363 0.365	98.99± 0.001	99.51± 0.002
2	100 % 100 % 100 %	0.30 0.30 0.30	0.756 0.752 0.756	0.398 0.398 0.396	98.98± 0.0032	99.52± 0.0013
3	150 % 150 % 150 %	0.314 0.314 0.315	0.880 0.880 0.879	0.432 0.431 0.432	99.85± 0.014	99.50± 0.0021

 

Precision:

System precision was established by measuring the absorbance’s at 260, 290 and 268nm for six (06) replicates of standard solutions (10μg/mL) of DOX succinate and PYD HCl at optimized spectroscopic conditions (Table 5). Percentage (%) RSD was lies within the acceptance criterion (<2%).

 

Table 5: System precision data of drugs (n = 3)

	Absorbance of DOX succinate			Absorbance of PYD HCl
	260 nm	290 nm	268 nm	260 nm	290 nm	268 nm
1	0.522	0.034	0.495	0.135	0.949	0.276
2	0.522	0.034	0.495	0.135	0.949	0.276
3	0.521	0.034	0.494	0.135	0.948	0.275
4	0.522	0.036	0.495	0.135	0.949	0.275
5	0.522	0.034	0.495	0.135	0.948	0.276
6	0.522	0.035	0.495	0.134	0.948	0.276
Mean±SD	0.521±0.0004	0.034±0.0008	0.495±0.0004	0.135±0.0004	0.9485±0.00054	0.275±0.0005
%RSD	0.076	1.856	0.080	0.296	0.055	0.181

 

The method precision was established by measuring the absorbance’s at 260, 290 and 268nm for six replicates of sample solutions (10μg/mL) of DOX succinate and PYD HCl at optimized spectroscopic conditions (Table 6). Percentage (%) assays and % RSD of the assay were calculated with proposed methods which showed the results were within the limits with repeatability.

 

Table 6: Method precision data of drugs (n = 3)

S. No.	Absorbance			Assay (%)
	260 nm	290 nm	268 nm	SEM		Q-ARM
				DOX succinate	PYD HCl	DOX succinate	PYD HCl
1	0.453	0.682	0.528	98.2	98.9	100.4	99.5
2	0.453	0.682	0.528	98.2	99.0	100.4	99.5
3	0.453	0.683	0.528	98.2	98.9	100.4	99.4
4	0.453	0.682	0.527	98.2	98.9	100.5	99.5
5	0.453	0.682	0.526	98.2	98.8	100.4	99.6
6	0.452	0.682	0.528	98.1	98.9	100.3	99.4
Mean ±SD	0.453±0.000408	0.682±0.000408	0.528±0.000837	98.2±0.040	98.9±0.063	100.3±0.062	99.5±0.083
% RSD	0.008	0.058	0.151	0.041	0.063	0.061	0.083

 

Robustness:

When the detection wavelength and pH of the buffer was varied the % RSD found was to be <2% for both the drugs in three determinations (Table 7 and 8). The results indicated that the method was robust by marginal changes in wavelength and pH of the medium.

 

Table 7: Robustness data of change in wavelength of DOX and PYD (n = 3)

S. No.	Change in Wavelength	Absorbance of DOX succinate (Mean ± SD)	% RSD	Change in wavelength	Absorbance of PYD HCl (Mean ± SD)	% RSD
1	Low (258 nm)	0.632±0.0088	1.392	Low (288nm)	0.978±0.001	0.102
2	Original (260nm)	0.640±0.0015	0.234	Original (290nm)	0.986±0.0011	0.111
3	High (262 nm)	0.651±0.0005	0.076	High (292nm)	0.996±0.0021	0.21

 

Table 8: Robustness data of change in buffer pH of DOX succinate and PYD HCl (n = 3)

S. No	Change in pH	Absorbance of DOX succinate at 260 nm (Mean ± SD)	% RSD	Absorbance of PYD HCl at 290 nm (Mean ± SD)	% RSD
1	Low (4.8)	0.458±0.001	0.218	0.114±0.0021	1.84
2	Original (5.0)	0.639±0.0017	0.266	0.140±0.0012	0.857
3	High (5.2)	0.440±0.0024	0.545	0.138±0.0021	1.51

 

Ruggedness:

The sample solution (10μg/mL) was analyzed by measuring their relative absorbances at variable conditions; all the experiments were conducted to get the triplicate values. At these variable conditions the % RSD of the results analogous to the absorbance’s were within acceptance criteria i.e., 1.306-1.90 for DOX succinate whereas 0.618-0.765 for PYD HCl.

 

LOD and LOQ:

The calculated LOD and LOQ values of DOX succinate were 0.337 and 1.024μg/mL and for PYR HCl were 0.134 and 0.405μg/mL, respectively. These results indicated a good sensitivity to the methods.

In the method validation, linearity was achieved in the range of 10-35µg/mL. As per the calibration curve plots it seems to be there is linearity in absorbance to that of the tested concentration of DOX succinate and PYD HCl. In accuracy determination, the percentage of drug recovered was achieved within the potency range. LOD and LOQ values showed good sensitivity of the methods. The developed methods showed good resolution between both the analyte drugs (DOX succinate and PYD HCl) with a quick and accurate analysis time. The assay and validation results data was postulated in the following Table 9.

 

Table 9: Results data of assay and validation.

Parameters	Results		Acceptance limits
	DOX succinate	PYD HCl
Specificity	-	-	No interference
Linearity	r² = 0.9949	r² = 0.9935	r² = 0.999
Accuracy	98.1-100.5 %	99.5-99.85 %	98-102%
Precision System precision Method Precision	0.076-1.856 0.041-0.063	0.055-0.296 0.061-0.083	RSD < 2%
Robustness Change in wavelength Change in buffer pH	1.406 1.102	0.912 1.102	RSD < 2%
LOD ( µg/mL)	0.337	0.134	Signal noise ratio should be more than 3:1
LOQ ( µg/mL)	1.024	0.405	Signal noise ratio should be more than 10:1
Assay SEM Q-ARM	98.10 ± 0.00184% 100.5 ± 0.0215%	98.8 ±0.00124% 99.05± 0.0134 %	98-102%

 

CONCLUSIONS:

Simple, accurate and precise analytical methods (SEM and Q-ARM) were developed for the determination of DOX succinate and PYD HCl in their tablet dosage form using UV-spectrophotometry. Phosphate buffer (pH.5.0, 0.05M) used as a solvent system, in which the absorbance maxima were found at 260 and 290nm, respectively. Specificity, precision, linearity, accuracy, robustness, LOD and LOQ parameters were validated for proposed methods according to standard guidelines (ICH Q₂(R₁)) and the methods were satisfied the acceptance criterion. Both the methods imply simple, specific, and also requires a shorter analysis time. Hence, it was concluded that the present methods were well suitable and can be applied for routine analysis of DOX succinate and PYD HCl in their pharmaceutical dosage formulations.

 

ACKNOWLEDGMENTS:

We thank the Principal, KL College of Pharmacy, KL Deemed to be University, Vaddeswaram and Vijaya Institute of Pharmaceutical Sciences for Women (VIPW), Vijayawada for their co-operation and also providing facilities to conduct the experiments.

 

CONFLICT OF INTEREST:

Authors declare no conflict of interest.

 

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Received on 18.12.2019          Modified on 03.02.2020

Accepted on 19.03.2020         © RJPT All right reserved