A REVIEW OF ANALYTICAL METHODS FOR DETERMINATION BROMHEXINE HYDROCHLORIDE IN PHARMACEUTICAL AND BIOLOGICAL SAMPLES

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ABOUT AUTHORS:
Meera V. Lad¹*, Vineet Jain², Hasumati Raj¹
¹Department of Quality Assurance,
²Department of Pharmacognosy,
Shree Dhanvantary Pharmacy College, Kim, Gujarat
meeralad235@gmail.com

ABSTRACT:
Bromhexine HCl (BRH)is a mucolytic agent used in the treatment of respiratory disorders associated with viscid or excessive mucus, chemically named 2-amino-3,5-dibromo-N-cyclohexyl-N-methyl benzenemethanamine hydrochloride. According to IUPAC it is 2,4-dibromo-6-[[cyclohexyl(methyl)amino]methyl] aniline hydrochloride. Because of its physiological importance, the drug has been quantified by exploiting its chemical and physical properties. Bromhexine is a weak base and its precipitate out at pH value above 6. Bromhexine is a synthetic benzyl amine derivative ofvasicine. The different analytical methods used to quantify the drug as a single active pharmaceutical ingredient include flow injection analysis with ionselectiveelectrodes, inductively coupled plasma mass spectrometry, electrokinetic chromatography, electrochemical oxidation at the glassy carbon electrode, liquid chromatography, liquid gas chromatography, GC with mass detection, and voltammetry. The drug has also been quantified in its combined formulations using HPLC, direct and derivative UV spectrophotometry.

REFERENCE ID: PHARMATUTOR-ART-2276

PharmaTutor (ISSN: 2347 - 7881)

Volume 2, Issue 11

Received On: 10/09/2014; Accepted On: 19/10/2014; Published On: 01/11/2014

How to cite this article: MV Lad, V Jain, R Hasumati; A Review of Analytical Methods for Determination Bromhexine Hydrochloride in Pharmaceutical and Biological Samples; PharmaTutor; 2014; 2(11); 35-41

INTRODUCTION:^[1-9]
Bromhexine hydrochloride is a mucolytic agent rendering the sputum less viscous thereby facilitating easy expulsion of it from the respiratory tract.

The drug is official in IP and BP.

CHEMICAL AND PHYSICAL INFORMATION OF BROMHEXINE HYDROCHLORIDE:

STRUCTURAL FORMULA:

Figure 1: Chemicalstructures of Bromhexine Hydrochloride

MOLECULAR FORMULA:C₁₄H₂₀Br₂N₂.HCl

MOLECULAR WEIGHT:412.59 g/mol

CHEMICAL NAME:
2-Amino-N-cyclohexyl-3,5-dibromo-N-methylbenzylamine hydrochloride

CATEGORY: Mucolytics

DOSE: 8 mg

DESCRIPTION: white crystalline powder

SOLUBILITY:
Slightly soluble in alcohol and methylene chloride,
Sparingly soluble in water.

PHARMACOLOGICAL ACTION:^[10]
Bromhexine hydrochloride (Figure 1) is a mucolytic expectorant whichexhibits its action by increasing bronchial secretions and reducing their viscosity. In addition, it produces in an increase in immunoglobulin levels inairway secretions. This agent was recently recommended as a new therapyfor pathological states, such as alcoholic chronic pancreatitis where there isan increased viscosity of the pancreatic juice. Bromhexine hydrochloride may be administered in combination with antimicrobial agents in the treatment ofrespiratory infections, due to its capacity to disrupt the mucopolysaccharidesof bronchial secretion and as results in enhancing the bronchial penetrationof antimicrobial drugs.

PHARMACOKINETICS:^[11]
Onoraladministration,Bromhexine hydrochloride is rapidly absorbed from the gastrointestinal tract and undergoes extensive first-pass metabolism in the liver. Its oral bioavailability is stated to be only about 20%. It is widely distributed to body tissues and is highly bound to plasma proteins. About 85 to 90% of a dose is excreted in the urine mainly as metabolites. It has a terminal elimination half-life of up to about 12 hours. Bromhexine crosses the blood brain barrier and small amounts cross the placenta.

SIDE EFFECTS:^[11]
Gastrointestinal side effects may occur occasionally with bromhexine and a transient rise in serum aminotransferase values has been reported.

Other reported side effects include headache, vertigo (dizziness), sweating and allergic reactions.

ANALYTICAL METHODS:
This all are the methods which are used for the determination of Bromhexine Hydrochloride in marketed formulation and in biological fluids. This all analytical methods are reported which are seen during the literature survey. This article describes the review on the all reported analytical methods with specific conditions.

I. COMPENDIAL METHODS:

Table No.1: Summary of Compendial Methods of Bromhexine hydrochloride

PHARMACOPOEIA	METHOD
IP^[3]	Potentiometry: Weigh accrately about 0.3.g, dissolve in 70 ml of ethanol(95%), add 1 ml of 0.1. M hydrochloric acid and titrate with 0.1 M sodium hydroxide, determining the end point potentiometrically. Record the volume added between the two inflections. 1 ml of 0.1 M sodium hydroxide is equivalent to 0.04126 g of C₁₄H₂₀Br₂N₂,HCL.
BP^[4]	Potentiometric Titration: Dissolve 0.300 gm in 70 ml of alcohol R and add 1 ml of 0.1 M hydrochloric acid. Carry out a potentiometric titration, using 0.1 M sodium hydroxide. Read the volume added between the two points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 41.26 mg of C₁₄H₂₁Br₂ClN₂.
EP^[12]	Potentiometric Titration: Dissolve 0.300 g in 70 ml of alcohol R and add 1 ml of 0.1 M hydrochloric acid. Carry out a potentiometric titration, using 0.1 M sodium hydroxide. Read the volume between the 2 points of inflexion. 1 ml of 0.1 M sodium hydroxide is equivalent to 41.26 mg of C₁₄H₂₁Br₂ClN₂.

II. CHROMATOGRAPHIC METHODS:^[13-21]
Various chromatographic methods are used for the determination of the Bromhexine Hydrochloride alone or combination with other drugs in various marketed formulation and in biological fluids like human plasma and urine. Chromatographic methods like High performance liquid chromatography (HPLC/RP-HPLC), High performance thin layer chromatography (HPTLC), High performance liquid chromatography (HPLC) with Solid Extraction method, Thin Layer Chromatography(TLC) Densitometric method are used. In which the stationary phase commonly used is C₁₈ column and mobile phase is commonly used is acetonitrile & phosphate buffer &methanol, its proportion is varies with condition of methodand range of pH is 3 t0 4. commonly used wavelength for detection is in the range of 250-270 nm. Below in table describes the summary of the various chromatographic methods are used with the method description.

Table No.2: Summary of Chromatographic Methods of Bromhexinehydrochloride

Title	Method	Mobile Phase	Stationary Phase	Wavelength (nm)
Bromhexine hydrochloride film content on High Performance Liquid Chromatography^[13]	HPLC	Acetonitrile 0.05 mol·L^-1:Phosphate buffer (containing 0.2% triethylamine,)(30:70)	an Altima C₁₈ column (4.6 mm × 150 mm, 5 μm),	249 nm
Molecularly imprinted solid-phase extraction for the selective determination of bromhexine in human serum and urine^[14]	Solid phase Extraction with HPLC	3× 1 mL Methanol/Acetic Acid (10/1, v/v)	Octadecylsilica column (55 mm × 4 mm, 3 μm particles)	270 nm
Estimation Of Bromhexine And Terbutaline In Bulk And Tablet Dosage Forms^[15]	HPLC	Phosphate buffer (0.05 M, pH 3): Acetonitrile (70:30 v/v)	ODS C₈ column (length 250 mm and internal diameter 4.6 mm)	270 nm
Simultaneous Estimation of Amoxicillin Trihydrate and Bromhexine Hydrochloride from Oily Suspension^[16]	RP-HPLC	Methanol and Glacial Acetic Acid (50:50 v/v)	ODS C18 (250 X 4.5mm ID),	254 nm
Simultaneous determination of Terbutaline and Bromhexine in Combined Pharmaceutical Dosage Form^[17]	RP-HPLC	Methanol: Acetonitrile: Ortho-Phosphoric acid in the ratio of 80:10:10 (v/v/v)	Inertsil ODS C-18 column 5μm column having 250 x 4.6mm internal diameter	270 nm
Simultaneous Determination of Bromhexine HCl and Baicalin in Chinese Compound Medicine^[18]	RP-ion pair HPLC	Water/Acetonitrile/Phosphoric-acid/Triethylamine (78/22/0.1/0.1 v/v/v/v)	C₁₈Column (250 x 4.6mm internal diameter, 5 μm)	225 nm
Simultaneous Determination of Salbutamol Sulphate and Bromhexine Hydrochloride in Tablets^[19]	RP-HPLC	Acetonitrile, Methanol and Phosphate buffer, pH 4 in the ratio 60:20:20 v/v.	SS Wakosil-II C-18 column	224 nm
TLC Densitometric Determination of Bromhexine Hydrochloride in Pharmaceuticals^[20]	TLC Densitometric Method	n?Butanol–Glacial Acetic Acid–Water (26:7.5:7.5)	Pre-coated silica gel plates	325 nm
Determination of Bromhexine hydrochloride in Human Plasma^[21]	HPTLC	a mixture of n-Butyl acetate: Methanol : GAA: Water(HPL grade) in the ratio of 5:2.5:2.5:1v/v/v/v.	TLC plates precoated with silica gel 60 F254	246 nm

II.UV SPECTROSCOPIC METHOD:^[22-25]
A simple, precise and economical spectrophotometric method for the estimation of Bromhexine Hydrochloride in pharmaceutical bulk and tablet dosage form was developed and validated. Identification was carried out usinga UV- visible double beam spectrophotometer detector with working wavelength in the range of 250-270 nm in methanol medium. The method was validated with respect to its specificity, linearity range, accuracy, and precisionin analytical media. Bromhexine Hydrochloride shows the maximum absorbance(λ_max) at248nm. Simple UV spectroscopy, first derivative spectroscopy, AUC method and simultaneous equation methods are reported for determination of the Bromhexine Hydrochloride in marketed formulation. Below in table describes the various spectroscopic methods with the method description and condition which are reported on review literature.

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Table No.3: Summary of UV spectroscopic methods of Bromhexine Hydrochloride

Title	Method	Medium	λ_max (nm)	R²
Spectrophotometric Determination of Bromhexine HCl^[22]	Spectrophotometric Method	Doubly Dis. Water	415 nm	1.00
		1,2-dichloroethane	366 nm
		Alkaline KMnO₄	610 nm
Simultaneous Estimation of Amoxicillin Trihydrate and Bromhexine Hydrochloride in Oral Solid Dosage Forms^[23]	1^st Order Derivative Method	Methanol	283 & 218.6 nm	0.9998
	Area Under Curve Method	Methanol	271-274 & 244-248 nm	0.9998
Simultaneous Determination of Dextromethorphan HBr and Bromhexine HCl in tablets by First- Derivative Spectrophotometry^[24]	Zero-Crossing Mesurement	50 % Methanol in Phosphate Buffer (pH-6)	234 nm	0.9999
	Peak to Base Mesurement	50 % Methanol in Phosphate Buffer (pH-6)	324 nm	0.9999
Simultaneous Estimation Of Bromhexine Hydrochloride And Phenylephrine Hydrochloride^[25]	Simultaneous Equation Method	Methanol	250 & 275 nm	0.998 & 0.999

III.MISCELLANOUS METHODS:^[26-28]
Most widely used methods are mainly HPLC, UV and HPTLC for determination of Bromhexine Hydrochloride in various formulation or in biological fluids but along with that other methods are also used which are seen during the literature survey. The summary of that methods are described below in table.

Table No.3: Summary of Miscellaneous methods of BromhexineHydrochloride

Sr.No.	Title	Method
1	Bioequivalence study of bromhexine by liquid chromatography–electrospray ionization-mass spectrometry after oral administration of bromhexine hydrochloride tablets^[26]	LC-MS
2	Determination of Pseudoephedrine Hydrochloride and Bromhexine Hydrochloride in Pharmaceuticals ^[27]	Gas Liquid Chromatography & Ion Pair HPLC
3	Simultaneous Determination of Bromhexine and Amoxicillin inPharmaceutical Formulations	Capillary Electrophoresis
4	bromhexine hydrochloride with morin as chemiluminescent reagent^[28]	Chemiluminescence

SR NO.	BRAND NAME	COMPANY NAME	FORMULATION	DOSE(mg)
1.	Bromhexine (8 mg)	Intima(IPCA Laboratories Ltd)	TABLET	8 mg
2	Bisolvon(8 mg)	German Remedies (ZydusCadila Healthcare Ltd)	TABLET	8 mg
3.	Bromex (8 mg)	Cipla Limited	TABLET	8 mg
4.	RTMox Kid (4+125)	Fourrts Laboratories Pvt Ltd	TABLET	Brom-4 mg Amoxi-125 mg
5.	Etoxin B	Ind-Swift Limited	TABLET	Brom-8 mg Ter-2.5 mg

(Brom-Bromhexine, Amoxi- Amoxicillin, Ter- Terbutaline)^[29]

CONCLUSION
The presented review highlights on various analytical methods reported on Bromhexine Hydrochlorideand in combination with other drug. HPLC-HPTLC-UV methods were found to be most widely used. Various chromatographic conditions are presented in under Table. The faster time, high sensitivity; specificity and better separation efficiency enable HPLC to be used frequently for the determination of Bromhexine Hydrochloride in the comparison with the other methods. There is no doubt on the fact that these chromatographic methods are rapid and far more economical. Other methods are also useful. In this way various analytical methods for the estimation of Bromhexine Hydrochloride in bulk or in various matrixes like plasma, alone or in combination with other drugs is discussed. The presented information is useful for the researchers especially those involved in the formulation development of Bromhexine Hydrochloride in individually and combination with other drug because there are various marketed formulation of Bromhexine Hydrochloride and with other combination. No method reported for degraded product of Bromhexine hydrochloride.

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