Development and Validation of Analytical Method for Bromhexine Hydrochloride, Chlorpheniramine Maleate and Guaifenesin by Simultaneous Equation Spectroscopic Method

Ladmeera¹*, Hasumati Raj², Vineet Jain²

¹Research Scholar 2014, Gujarat Technological University, Gujarat

²QualityAssurance Department, Shree Dhanvantary Pharmacy College, Kim, Surat

*Corresponding Author E-mail: _{meeralad235@gmail.com}

ABSTRACT:

A simple, accurate and precise spectroscopic method was developed for simultaneous estimation of Bromhexine hydrochloride, Chlorpheniramine maleate and Guaifenesin in syrup using simultaneous equation Method. In this spectroscopic method, 249.00 nm and 261.00 nm and 274.00 nm wavelengths respectively were selected for measurement of slope and intercept according to its calibration curves. Bromhexine, Chlorpheniramine maleate and Guaifenesin show linearity in a concentration range of 02-10μg/ml, 01-05μg/ml and 25-125μg/ml at their respective λ max. Accuracy, precision and recovery studies were done by QC samples covering lower, medium and high concentrations of the linearity range. The relative standard deviation for accuracy, precision studies were found to be within the acceptance range (<2%). The limit of determination was 0.102μg/ml, 0.169μg/ml and 0.312μg/ml for Bromhexine, chlorpheniramine maleate and guaifenesin respectively. The limit of quantification was 0.311μg/ml, 0.947μg/ml and 0.514μg/ml for Bromhexine, chlorpheniramine maleate and guaifenesin respectively. Recovery of Bromhexine, guaifenesin and chlorpheniramine maleate were found to be 100.02 %, 101.17 and 100.66% respectively confirming the accuracy of the proposed method. The proposed method is recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction.

KEYWORDS: Bromhexine hydrochloride, Guaifenesin, Chlorpheniramine maleate, simultaneous estimation, Simultaneous equation method, analysis method.

INTRODUCTION:

Bromhexine HCl (BRH) is a mucolytic agent used in the treatment of respiratory disorders associated with viscid or excessive mucus. Bromhexine depolymerises muco-polysaccharides directly as well as by liberating lysosomal enzyme network of fibres in tenacious sputum is broken.^[1]

Figure1: Structure of Bromhexine⁽³⁾

Guaifenesin may act as an irritant to gastric vagal receptors, and recruit efferent parasympathetic reflexes that cause glandular exocytosis of a less viscous mucus mixture. Cough may be provoked. Guaifenesin increases fluid and mucus removal from the upper respiratory tract by increasing the volume of secretion and reducing their adhesiveness and surface tension.

Figure2: Structure of Guaifenesin⁽³⁾

Chlorphenamine or chlorpheniramine, commonly marketed in the form of chlorpheniramine maleate, is a first-generation alkylamine antihistamine used in the prevention of the symptoms of allergic conditions such as rhinitis and urticaria. Chlorpheniramine competes with histamine for H₁ receptor sites, there by antagonizing many histamine effects to reduce allergy signs and symptoms. So, it ultimately reduce the cough.

Figure 3: Structure of Chlorpheniramine maleate⁽³⁾

The review of literature regarding quantitative analysis of Bromhexine, Guaifenesin and Chlorpheniramine maleate revealed that no attempt was made to develop analytical methods for these three drugs. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual drugs.^[6-44]The focus of the present study was to develop and validate a rapid, stable, specific, and economic spectroscopic method for the estimation of Bromhexine, Guaifenesin and Chlorpheniramine maleate in Syrup.

MATERIALS AND METHODOLOGY:

· Bromhexine HCl was gifted by Galpha Laboratories Ltd., Ankleshwar.

· Chlorpheniramine Maleate was gifted by Maharshee Laboratories Pvt. Ltd., Bharuch.

· Guaifenesin was gifted by Pan Drugs Limited, Nandesari.

· A double beam UV/Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2 nm, 1 cm quartz cells was used to measure absorbance of all the solutions.

· Spectra were automatically obtained by UV-Probe system software.

· An analytical balance (Sartorius CD2250, Gottingen, Germany) was used for weighing the samples.

· Sonicator (D120/2H, TRANS-O-SONIC)

· Class ‘A’ volumetric glassware were used (Borosillicte)

Standard Solution:

Preparation of the Stock Solution of Bromhexine HCl:

An accurately weighed quantity of Bromhexine HCl (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of Bromhexine (100μg/ml). Sonicate for 10 mins if require.

Preparation of the Stock Solution of Chlorpheniramine Maleate:

An accurately weighed quantity of Chlorpheniramine Maleate (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of Chlorpheniramine Maleate (100μg/ml). Sonicate for 10 mins if require.

Preparation of the Stock Solution of Guaifenesin:

An accurately weighed quantity of Guaifenesin (15 mg) was transferred to a separate 100 ml volumetric flask and dissolved and diluted to the mark with Methanol to obtain standard solution having concentration of Guaifenesin (150μg/ml). Sonicate for 10 mins if require.

Preparation of Test Solution:

From the stock solution of BROM take 0.4ml and from stock solution of CHL take 0.2ml and from the stock solution of GUA take 3.33ml transferred in to 10ml volumetric flask and diluted up to mark with methanol to give a solution having strength of BROM was 4μg/ml, CHL was 2μg/ml and GUA was 50μg/ml.

Calibration curves for Bromhexine HCl:

Pipette out 0.2, 0.4, 0.6, 0.8and 1.0 ml of the stock solution of Bromhexine (100μg/ml), , 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml stock solution of Chlorpheniramine maleate (100μg/ml) and 1.66 ml, 3.33 ml, 5.0 ml, 6.66 ml, 8.33 ml stock solution of Guaifenesin (150μg/ml)into a series of 10ml volumetric flasks and the volume was adjusted to mark with methanol and measured absorbance at 249.00nm, 261nm and 274 nm. Plot the graph of absorbance versus respective concentration of Irbesartan and atorvastatin. Linearityrange of BROM, CHL and GUA was found with correlation co-efficient.

Development and validation of spectroscopic simultaneous equation method:

Selection of wavelength and Method Development For determination of Bromhexine Hydrochloride, Chlorpheniramine Maleate and Guaifenesin:

The standard solution of BROM, CHL and GUA were scanned separately between 200-400nm, and BROM showed absorbance maxima at 249.00 nm and CHL at 261.00 nm and GUA at 274.00 nm (figure 4)

Figure 4: Overlain linear zero order spectra of BRO (Green), CHL (Violet), GUA (Brown) in 2:1:25 ratio, respectively

The concentrations of three drugs are calculated using the following equations:

Where:

S_x, S_y and S_z are the slops of drugs X, Y and Z in their calibration curves:

A₁*,A₂* and A₃* are the modified Absorbance

A₁* = A₁ – (I_x1 + I_y1 + I_z1)

A₂* = A₂ – (I_x2 + I_y2 + I_z2)

A₃* = A₃ – (I_x3 + I_y3 + I_z3)

A₁,A₂ and A₃ are the Absorbance of test at λ₁, λ₂ and λ₃ respectively

I_x, I_y and I_z are the Intercepts of drugs X, Y and Z in their calibration curves

Table 1: Calibration data for BROM, CHL and GUA at249.00 nm and 261.00 nm and 274.00 nm respectively. *(n=6)

Sr no.	Brom	Chlor	Gua	Abs. ± SD (n=6)
				Brom (248 nm)	Chlor (261nm)	Gua (274nm)
1	2	1	25	0.108 ±0.0083	0.016 ±0.0051	0.211 ±0.0009
2	4	2	50	0.196 ±0.0021	0.037 ±0.0020	0.477 ±0.0020
3	6	3	75	0.286 ±0.0021	0.0535 ±0.0005	0.717 ±0.0013
4	8	4	10	0.366 ±0.0016	0.071 ±0.0075	0.932 ±0.0012
5	10	5	125	0.457 ±0.0018	0.0945 ±0.0010	1.190 ±0.0013

_{Table 2: Intraday and Interday precision data for estimation of BROM, CHL and GUA*(n=3)}

Drug	Concentration (ppm)	Precision Mean ± %RSD
		Intraday	Interday
Bromhexine HCl	02	0.181 ± 0.317	0.183 ± 0.546
	04	0.373 ± 0.309	0.376 ± 0.405
	06	0.525 ± 0.190	0.525 ± 0.290
Chlorpheniramine Maleate	01	0.252 ± 0.604	0.183 ± 0.821
	02	0.425 ± 0.489	0.376 ± 0.405
	03	0.622 ± 0.092	0.426 ± 0.590
Guaifenesin	25	0.432 ± 0.266	0.434 ± 0.351
	50	0.653 ± 0.306	0.653 ± 0.233
	75	0.944 ± 0.266	0.945 ± 0.161

Validation parameters:⁽⁴⁵⁾

1. Linearity and Range:

The Zero order (fig.4) showed linear absorbance at 249.00 nm for BROM (02-10 µg/ml), 261.00 nm for CHL (01-05µg/ml) and 274 nm for GUA (25-125µg/ml) with correlation Coefficient (r²) of 0.9997, 0.9959 and 0.9988for BROM, CHL and GUA, respectively. (figure 5, 6 and 7) This method obeyed beer’s law in the concentration range 02-10µg/ml, 01-05µg/ml and 25-125µg/ml for BROM, CHL and GUA respectively. (Table 1)

_{Figure 5: Calibration curve for BROM at 249.00 nm}

_{Figure 6: Calibration curve for CHL at 261.00 nm}

2. Precision:

I. Intraday Precision:

The precision of the developed method was assessed by analysing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1, 2, 3μg/ml for Chlorpheniramine Maleate 2, 4, 6μg/ml for Bromhexine HCl and 25, 50, 75μg/ml for Guaifenesin. Three replicate (n=3) each on same day.It presented in Table 2. These% RSD value was found to be less than ±2. 0 indicated that the method is precise.

_{Figure7: Calibration curve for GUA at 274.00 nm}

II. Interday Precision:

The precision of the developed method was assessed by analysing samples of the same batch in nine determinations with three Standard solutions containing concentrations 1, 2, 3 μg/ml for Chlorpheniramine Maleate, 2, 4, 6μg/ml for Bromhexine HCl and 25, 50, 75 μg/ml for Guaifenesin. Triplicate (n=3) per day for consecutive 3 days for inter-day precision. Interday precision data presented in Table 2. These % RSD value was found to be less than ±2.0indicated that the method is precise.

3. Accuracy:

Accuracy of the method was determined by recovery study from syrup at three level (80%, 100%, 120%)of standard addition. The% recovery values are tabulated in Table 3, 4and 5 Percentage recovery for BROM, CHL and GUA by this method was found in the range of 99.77-100.05%, 99.50-102.22% and99.42-102.27%, respectively, the value of % RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

4. Limit of Detection and Quantitation:

The LOD for BROM, CHL and GUA was conformed to be 0.1026 µg/ml, 0.1696 µg/ml and 0.3126 µg/ml, respectively. The LOQ for BROM, CHL and GUA was conformed to be 0.3110 µg/ml, 0.5141 µg/mland 0.9473 µg/ml, respectively. The obtained LOD and LOQ results are presented in Table 6

5. Robustness and Ruggedness

The obtained Ruggedness and Robustness results are presented in table 7 The % R.S.D was found to be 0.21- 0.83 % for BROM, 0.23- 0.46 % for CHL and 0.11 – 0.35 % for GUA. These %RSD value was found to be less than ± 2.0 indicated that the method is precise. No significant changes in the spectrums were observed, proving that the developed method is rugged and robust.

_{Table 3: Recovery data of BRO*(n=3)}

Conc. Of BRO from formulation (µg/ml)	Amount of Std. BRO added (µg/ml)	Total amount of BRO (µg/ml)	Total amount of BRO found (µg/ml)* Mean± SD	% Recovery (n=3)	% RSD BRO
2	1.6	3.6	3.62±0.0005	100.55	0.18
2	2.0	4.0	3.99±0.0005	99.78	0.17
2	2.4	4.4	4.39±0.0005	99.77	0.16

_{Table 4: Recovery data of CHL*(n=3)}

Conc. of CHL from formulation (µg/ml)	Amount of Std. CHL added (µg/ml)	Total amount of CHL(µg/ml)	Total amount of CHL found (µg/ml)* Mean± SD	% Recovery (n=3)	% RSD CHL
1	0.8	1.8	1.82±0.0005	102.22	0.13
1	1.0	2.0	1.98±0.0005	99.50	0.13
1	1.2	2.2	2.18±0.0005	101.81	0.12

_{Table 5: Recovery data of GUA*(n=3)}

Conc. of GUA from formulation (µg/ml)	Amount of Std. GUA added (µg/ml)	Total amount of GUA (µg/ml)	Total amount of GUA found (µg/ml)* Mean± SD	% Recovery (n=3)	% RSD GUA
25	20	45	44.74±0.001	99.42	0.51
25	25	50	50.14±0.001	100.29	0.29
25	30	55	56.25±0.005	102.27	0.15

_{Table 6 LOD and LOQ data of BROM, CHL and GUA *(n=10)}

Conc. (μg/ml)			BRO(249.0 nm) (n=10)		CHL(261.0 nm) (n=10)		GUA(274.0 nm) (n=10)
DEX	CHL	GUA	Avg. ± SD	%RSD	Avg. ± SD	%RSD	Avg. ± SD	%RSD
2	1	25	0.181 ±0.0013	0.744	0.252 ±0.0009	0.386	0.432 ±0.0009	0.212
LOD(μg/ml)			0.1026		0.1696		0.3126
LOQ(μg/ml)			0.3110		0.5141		0.9473

Table 7: Robustness and Ruggedness data of BROM, CHL and GUA *(n=3)

Drugs	Concentration	Mean ±% RSD
		Instrument I	Instrument II
Bromhexine HCl	02	0.181 ± 0.635	0.182 ± 0.836
	04	0.371 ± 0.410	0.372 ± 0.410
	06	0.525 ± 0.219	0.526 ± 0.290
Chlor-pheniramine Maleate	01	0.250 ± 0.460	0.251 ± 0.229
	02	0.426 ± 0.234	0.427 ± 0.468
	03	0.621 ± 0.245	0.623 ± 0.245
Guaifenesin	25	0.433 ± 0.133	0.434 ± 0.351
	50	0.651 ± 0.177	0.652 ± 0.234
	75	0.942± 0.122	0.944 ± 0.323
Drugs	Concentration	Change in Wavelength (249 ± 0.2 nm, 261 ± 0.2 nm, 274 ± 0.2 nm) Mean ±% RSD
		Wavelength 249.20 nm	Wavelength 248.80 nm
Bromhexine HCl	02	0.182 ± 0.837	0.183 ± 0.833
	04	0.371 ± 0.411	0.372 ± 0.819
	06	0.524 ± 0.397	0.524 ± 0.190
		261.20 nm	260.80 nm
Chlorpheniramine Maleate	01	0.252 ± 0.604	0.254 ± 0.988
	02	0.424 ± 0.271	0.425 ± 0.359
	03	0.621 ± 0.161	0.623 ± 0.424
		274.20 nm	273.80 nm
Guaifenesin	25	0.433 ± 0.230	0.434 ± 0.479
	50	0.652 ± 0.234	0.653 ± 0.306
	75	0.942 ± 0.162	0.944 ± 0.105

Application of the proposed method for analysis of BROM, CHL and GUAIN combined capsule dosage form:

· Pipette out 5 ml of liquid sample, Add 10 ml of methanol, basify with 10 ml 1 M NaOH solution.

· Extract with 10 ml chloroform 4 times. Combine chloroform and evaporate.

· Take all residue in 20 ml methanol and make the volume upto 100 ml.

· Finally, measure absorbance at 249.0 nm, 261.0 nm and 274.0 nm.

· Determine the concentration of drugs using equation

Drugs	% Assay ± % RSD(n=3)	IP Limit
Bromhexine HCl	100.83 ± 0.0010	98.5-101.5%
Chlorpheniramine Maleate	100.50 ± 0.0005	98.0-101.0%
Guaifenesin	100.46 ± 0.0058	98.0-101.5%

Summary of validation parameter:

Table 8: Summary of validation parameters

Parameter	Bromhexine HCl	Chlorpheniramine Maleate	Guaifenesin
λmax	249.0 nm	261.0 nm	274.0 nm
Linearity	2 – 10 ppm	1 – 5 ppm	25 – 125 ppm
Regression equation	y = 0.043x + 0.0223	y = 0.0189x – 0.0022	y = 0.0097x - 0.0183
Correlation coefficient (r^2)	0.9997	0.9959	0.9988
Accuracy	100.02 ± 0.628	101.17 ± 1.466	100.66 ± 1.460
LOD (n=10)	0.1026 ppm	0.1696 ppm	0.3126 ppm
LOQ(n=10)	0.3110 ppm	0.5141 ppm	0.9473 ppm
Precision Intra-day(n=3) Inter-day (n=3)	%RSD 0.190 – 0.317 0.290 – 0.546	%RSD 0.092 – 0.604 0.321 – 0.821	%RSD 0.266 – 0.306 0.161 – 0.351
Assay	100.833 ± 0.001%	100.50 ± 0.0005%	100.46 ± 0.005%
Robustness(%RSD)	0.219 – 0.836%	0.234 – 0.468 %	0.122– 0.351 %

CONCLUSION:

A new, Simultaneous Equation method has been developed for estimation of Bromhexine hydrochloride, Chlorpheniramine maleate and Guaifenesin in syrup. The method was validated by employment of ICH(18) guidelines. The validation data is indicative of good precision and accuracy, and prove the reliability of the method.

REFERANCE:

1. Tripathi KD. Essentials of Medical Pharmacology; 6^thEdn; Jaypee Brothers Medical Publishers (P) Ltd, 2008, pp 213-215.

2. Lad MV, Jain V, Raj HA, “A review of analytical methods for determination bromhexine hydrochloride in pharmaceutical and biological samples.” Pharmatutor, 2014, 2, 35-41.

3. DrugBank, “Open Data Drug and Drug Target Database”, DB09019 Bromhexine Hydrochloride, DB01114 Chlorpheniramine, DB00874 Guaifenesin”, Oct 2013, www.drugbank.ca/drugs.html.

4. ChemicalBook,www.chemicalbook.com/ProductChemicalPropertiesCB3249453_ EN.htm#MSDS.

5. Jones and Bartlett Learning. Nurse’s Drug Handbook; 10^thEdn; Jones and Bartlett Learning International, London, 2011,pp 301, 493.

6. Susmitha K, Thirumalachary M and Venkateshwarlu G, “Spectrophotometric determination of Bromhexine hcl in pure and pharmaceutical forms.” Int. Scholarly Res. Notices Ana. Chem.2013, 5, 1-7.

7. Liu Suying FS and Wong Kwok TX, “Bromhexine hydrochloride film content on high performance liquid chromatography.” Res. Papers Center. 2012, 12, 56-57.

8. Mehran J, Mohammad HN and Behrouz A, “Molecularly imprinted solid-phase extraction for the selective determination of Bromhexine in human serum and urine with high performance liquid chromatography.” Talanta. 2009, 80, 133-138.

9. EEndang S and Gunawan I, “TLC densitometric determination of Bromhexine hydrochloride in pharmaceuticals and its validation.” J. Liq. Chrom.and Related Tech.2005, 27, 2047-2056.

10. Rao Monica RP, Kumar M, Aghav S and Sukre Girish, “Development and validation of HPTLC method for determination of Bromhexine hydrochloride in human plasma.” Res. J. Pharm. and Tech.2012, 5, 1054-1057.

11. Wen Z, Fang F and Ya Wang, “Bioequivalence study of Bromhexine by liquid chromatography–electrospray ionization-mass spectrometry after oral administration of Bromhexine hydrochloride tablet.” J. P’ceuticaland Biomed. Ana.2008, 48, 1206-1210.

12. Dhoka MV, Gawande VT and Joshi PP, “Simultaneous estimation of Amoxicillin trihydrate and Bromhexine hydrochloride in oral dosage forms by spectrophotometric method.” Int. Res. J. Pharm. 2011, 2, 197-201.

13. Tantishaiyakul V, Poeaknapo C, Sribun P and Sirisuppanon K, “Simultaneous determination of Dextromethorphan HBr and Bromhexine hcl in tablets by first-derivative spectrophotometry.” J. P’ceuticaland Biomed. Ana.1997, 17, 237-243.

14. Mevada D, Bhalodiya K, and Faldu S, “Development and validation of first order derivative spectrophotometric method for simultaneous estimation of Bromhexine hydrochloride and Phenylephrine hydrochloride in their combined pharmaceutical dosage form.” Pharmatutor. 2014, 2, 132-138.

15. Amit K and Sanju N, “A validated high performance liquid chromatographic method for estimation of Bromhexine and Terbutaline in bulk and tablet dosage forms.” Int. J. ChemTech Res. 2011, 2, 218-222.

16. Lalit VS and Sanjaykumar BB, “Development and validation of RP-HPLC method for the simultaneous estimation of Amoxicillin trihydrate and Bromhexine hydrochloride from oily suspension.” P’ceutical Ana. Acta. 2010,1, 107.

17. Satyanarayana PV, Murali M and Venkateswara RP, “Simultaneous determination of Terbutaline and Bromhexine in combined pharmaceutical dosage form by RP-HPLC method.” Int. J. ChemTech Res.2012, 4, 240-246.

18. Shujuan L, Liang Z, Xia L, Feng Z and Shengxiang J, “Simultaneous determination of Bromhexine HCl and Baicalin in chinese compound medicine by a reversed-phase ion-pair HPLC.”J. Food and Drug Ana. 2004, 12, 306-310.

19. Pai PN, Rao GK, Murthy MS, Agarwal A and Puranik S, “Simultaneous determination of Salbutamol sulphate and Bromhexine hydrochloride in tablets by reverse phase liquid chromatography.” Ind. J. P’ceutical Sci.2009, 71, 53–55.

20. Venkata R, Ramana R, Raghuveer S and Khadgapathi P, “Gas-liquid chromatographic and ion-pair high-performance liquid chromatographic determination of Pseudoephedrine hydrochloride and Bromhexine hydrochloride in P’ceuticals.” Ana.1987, 112, 871-874.

21. Denise CO, Karina TV, Alma L and Revilla V, “Simultaneous determination of Bromhexine and Amoxicillin in pharmaceutical formulations by capillary electrophoresis.” J. Mex. Chem. Soc. 2011, 55, 79-83.

22. Bhattacharyya E, “Estimation and validation of stability indicating UV spectrophotometric method for the determination of Guaifenesin in presence of its degradant products.” Int. J. Pharm. and P’ceutical Sci.2013, 5, 262-268.

23. Sharaf MH and Stiff DD, “Determination of Guaifenesin in human serum by capillary gas chromatography and electron capture detection.” J Pharm. Biomed Ana.2004, 35, 801-806.

24. Sridharan D, “Bio analytical method development and validation of Guaifenesin in human plasma by liquid chromatography coupled with tandem mass spectroscopy.”Pharm. Sci. Monitor. 2011, 2, 73-82.

25. Ola MA, “Sensitive spectrophotometric method for quantisation of Guaifenesin and Dropropizine in their dosage forms.” Int. J. Ana. Chem.2010, 2010, 5-10.

26. Bankar AA, “Spectrophotometric estimation of Guaifenesin and Salbutamol in pure and tablet dosage form by using different methods.” Der Pharm. Chem.2013, 5, 92-97.

27. Dubey N, Sahu S and Shing GN, “Development of HPLC method for simultaneous estimation of Ambroxol, Salbutamol and Guaifenesin in single dosage form.” Ind. J. Chem.-B. 2012, 51, 1633-1636.

28. Walode SG, Deshpande SD and Deshpande AV, “Stability indicating RP-HPLC method for simultaneous estimation of Salbutamol sulphate and Guaifenesin.” Der Pharm. Sinica.2013, 4, 61-67.

29. Senthilraja M and Giriraj P, “RP-HPLC method for the simultaneous estimation of Terbutaline sulphate, Bromhexine hcl and Guaifenesin in cough syrup.” Asian J. P’ceutical and Clinical Res. 2011, 4, 13-15.

30. Nalluri BN, “Simultaneous estimation of Phenylephrine hcl and Guaifenesin in bulk drug and pharmaceutical dosage forms by RP-HPLC method.” J. Chem. andP’ceutical Res.2013, 5, 188-194.

31. Mallu UR, Bobbarala V and Penumajji S, “Analysis of cough and analgesic range of pharmaceutical active ingredients using RP-HPLC method.”Int. J. Pharm. and Bio Sci.2011, 2, 439-452.

32. Harsono T, Yuwono M and Indrayanto G, “Simultaneous determination of some active ingredients in cough and cold preparations by gas chromatography, and method validation.” J. AOAC Int.2005, 88, 1093-1098.

33. Athanikar NK, “Rapid quantitative analysis of Chlorpheniramine in plasma, saliva and urine by high-performance liquid chromatography.” J. Chro. B: Biomed. Sci. andAppli.1979, 162, 367–376.

34. Subramaniyan SP and Das SK, “Rapid identification and quantification of Chlorpheniramine maleate or Pheniramine maleate in pharmaceutical preparations by thin-layer chromatography-densitometry.” J. AOAC Int.2004, 87, 1319-1322.

35. Midha KK, “Subnanogram quantitation of Chlorpheniramine in plasma by a new radioimmunoassay and comparison with a liquid chromatographic method.” J. P’ceutical Sci.1984, 73, 1144–1147.

36. Sawant R, “Simultaneous estimation and validation of Paracetamol, Phenylephrine hcl and Chlorpheniramine maleate in tablets by spectrophotometric method.” J. P’ceutical Res. and Health Care, 2011, 3, 23.

37. Kaura A, ”Spectrophotometric determination of Chlorpheniramine maleate and Phenylpropanolamine hydrochloride in dosage forms.” Int. Current P’ceutical J.2013, 2, 97-100.

38. Qi ML. “Simple HPLC method for simultaneous determination of Acetaminophen, Caffeine and Chlorpheniramine maleate in tablet formulations.” Chro.2002; 56, 295-298.

39. Şenyuva H. and Ozden T, “Simultaneous high-performance liquid chromatographic determination of Paracetamol, Phenylephrine hcl and Chlorpheniramine maleate in pharmaceutical dosage forms.” J. Chro. Sci.2002; 40, 97-100.

40. Maithani M, “Development and validation of a RP-HPLC method for the determination of Chlorpheniramine maleate and Phenylephrine in pharmaceutical dosage form.” Pharm. Globale (IJCP). 2010, 5, 1-4.

41. Kommana R and Basappa P, “Validated stability indicating RP-HPLC method for simultaneous estimation of Codeine phosphate and Chlorpheniramine maleate from their combined liquid dosage form.” Chro. Res. Int.2013, 2, 7-14.

42. Rajurkar S, “Simultaneous determination of Chlorpheniramine maleate, Paracetamol and Pseudoephedrine hcl in pharmaceutical preparations by HPLC.” Int. J. Life science and Pharm. Res.2011, 1, 94-100.

43. Al-Shaalan NH, “Simultaneous estimation of Chlorpheniramine maleate, Dextromethorphan HBr and Phenylephrin HCl in codilar syrup using HPLC.” J. of Ph’ceuticaland Sci. Inno. 2012, 1, 29-32.

44. RedasanI VK, “Simultaneous determination of Chlorpheniramine maleate, Phenylephrine hydrochloride, Paracetamol and Caffeine in pharmaceutical preparation by RP-HPLC.” Chem. Industry and Chem. Eng. Qua. 2013, 19, 57-65.

45. Virani P, Raj H, Jain V and Jain P. Updated review: validation and method validation parameters. Pharma tutor, 2(10); 2014: 27-37.

Received on 04.03.2015 Accepted on 21.03.2015

© Asian Pharma Press All Right Reserved

Asian J. Res. Pharm. Sci. 5(2): April-June 2015; Page 76-82

DOI: 10.5958/2231-5659.2015.00013.2