Analytical Methods for Quantitative Estimation of Ambroxol HCl in Pharmaceutical Preparation: A Review

Veena Devi Singh¹*, Sanjay J. Daharwal²

¹Research Scholar, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur CG 492010 

²Assistant Professor, University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur CG 492010 

ABSTRACT:

Context: Ambroxol Hcl is an active metabolite of bromohexine. It is an expectoration improver and a mucolytic agent used in the treatment of bronchial asthma and chronic bronchitis.The available formulation of Ambroxol was authorized first in market since 1978.

Objective:This article highlights on published analytical methods reported in the literature for the determination of Ambroxol Hcl in biological samples and pharmaceutical formulations.

Material and Methods: Various techniques like electrochemical, spectrophotometry, high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC–MS), Gas chromatography, Ultra performance liquid chromatography (UPLC)  and high-performance thin layer chromatography (HPTLC) were used for the qualitative and quantitative estimation of Ambroxol Hcl.

Result and Discussion: Literature reveals that most widely used diluents are methanol and distilled water in HPLC methods, which prolonged the run times with greater tailing factor. For spectrometric determination, the presence of multiple entities and excipients includes complexity with multi- component dosage forms, which could produce significant challenge to the analytical chemist during the development of assay procedure. For such instances, chemo-metric methods can be preferred to routine spectrophotometric methods.

Conclusion: Amongst various analytical techniques available for the quantification of single and multicomponent dosage form. HPLC methods are most extensively used for analysis of Ambroxol Hcl.

INTRODUCTION:

Ambroxol hydrochloride is chemically 4-{((2-amino-3,5-dibromophenyl)-methyl)-amino)}-cyclohexanol or N-{((trans-phydroxycyclohexyl),(2-amino-3,5-dibromo benzyl))}-amine,(Figure 1.) which is a semi synthetic derivative of vasicine obtained from the Indian herbal shrub “Adhatoda vasica”. Ambroxol HCl is an active metabolite of bromohexin, and it is a mucolytic agent, used as an expectoration improver as well as in the treatment of chronic bronchitis and bronchial asthma (Pai et al.; 2005). It was authorized first in market since 1978. Here, many formulations are available such as tablet, syrup, pastilles, sachet and dry powder, drops, ampoules and inhalation solution, as well as effervescent tablet in market in individual component and in combination with other drugs.

The drug is official in Indian Pharmacopoeia (Indian Pharmacopoeia 2007), British Pharmacopoeia (British Pharmacopoeia 2005), and European Pharmacopoeia (European Pharmacopoeia 2005).It is sparingly soluble in water and ethanol, soluble in dimethyl formamide (DMF), methanol and insoluble in chloroform and benzene. It is a white yellowish crystalline powder; melting point 240°C; administered orally. The dose of Ambroxol hydrochloride is 30-60 mg per day. The molecular weight of Ambroxol Hydrochloride is 414.6, and their Pharmacological properties namely surfactant stimulatory, anti-inflammatory, and anti-oxidant and local anesthetic effects in addition to the muco-kinetic and mucociliary effects of the parent compound. Recognition of the surfactant stimulatory and anti-inflammatory properties of the drug has led to the resurgence of interest in the molecule in the management of difficult to treat obstructive airway disorders. (Gupta 2006)

Figure-1. Structure of Ambroxol HCl.

The present review of literature stated that till date various methods have been reported for the determination of Ambroxol in biological samples and pharmaceutical formulations. Techniques such as Electrochemical, Spectrophotometry, High performance liquid chromatography (HPLC), Ultrathin layer chromatography, Gas chromatography, High-performance thin layer chromatography (HPTLC) and Liquid chromatography–mass spectrometry (LC–MS), from which HPLC methods are used most extensively. Overview of the methods for determination of Ambroxol shown in (Figure. 2).

2. SAMPLE PREPARATION:

2.1. Solubility

With reference to Biopharmaceutics Classification System (BCS), classification of Ambroxol comes  under BCS class-I, it means Ambroxol  has high  solubility and high permeability (Yazdanian et al. 2005; Polli et al. 2004). To control the release of drug, various strategies like hydrophilic matrix system (Shaikh et al. 2011) and Microsphere (Kumar 2014) have been proposed . The solubility of the drug was tested in solvents routinely used for analytical methodology.

2.2. Sample preparation strategies

Sample preparation is an important part of analytical methodology, and about approximately 30% error generated in sample analysis was reported due to sample preparation (Hendriks  et al. 1996).(Figure. 3) shows various diluents used for the analysis of Ambroxol. In major cases methanol was used as a diluent. The sample preparation techniques for the extraction of Ambroxol from biological matrices (plasma, serum and urine) (Hu et al. 2008) include protein precipitation, Liquid-Liquid Extraction had been chosen among various techniques. The solvents like acetonitrile, ethanol and n-Hexane etc. were tried alone as well as in combination (Tompe et al.  2013).

3. ANALYTICAL METHODS

3.1. Electrochemical methods

Yuanzhe et al. constructed a new electrochemical method for determination of Ambroxol HCl, the method was based on MWCNT/ Nafion modified glassy carbon electrodes. In cyclic voltammetry, the compound showed an irreversible oxidation peak. It was compared with the results on bare glassy carbon electrode and Nafion modified glassy carbon electrode, and obtained significant improvements in the sensitivity on MWCNT/Nafion modified glassy carbon electrode. The differential pulse voltametric detection limits were determined to be 3×10^-8 M, 4×10^-9 M and 1×10^-9 M on bare glassy carbon electrode, Nafion modified glassy carbon electrode and MWCNT/Nafion modified glassy carbon electrode, respectively. At the optimized condition, a sensitive linear differential pulse voltametric response range for the Ambroxol was between 1×10^-8 and 1.8×10^-6 M on MWCNT/Nafion modified glassy carbon electrode.

(Piao 2012)

Habib et al. constructed an electrochemical procedure for the determination of Ambroxol in mucolytic. The method was based on adsorptive accumulation of the species at the hanging mercury drop electrode (HMDE), The behaviour of adsorptive stripping response was studied by using various experimental conditions, such as  type of supporting electrolyte, pH, accumulation time, pulse amplitude, scan rate and mode of sweep. In Britton-Robinson buffer solution, an irreversible reduction process involving transfer of one electron and one proton was took place. The response was linear over the 0.2-6 µ/ml concentration range. The average of determinations obtained by the square wave adsorptive voltametric method with its relative standard deviation was 99.8 +/- 2.40%. (Habib et al. 2005)

Figure-2. Overview of analytical methods for estimation of Ambroxol HCl in biological and pharmaceutical samples.

Figure-3. Various diluents used for the analysis of Ambroxol HCl.

3.2. Spectrophotometry

In the literature about 17 methods were reported for the estimation of Ambroxol using Spectrophotometry (Akhtar et al. 2013; Ponnilavarasan et al. 2011; Prasanthi et al. 2010 ) of which 1method are for determining Ambroxol alone, while the others are for quantifying Ambroxol in combination with other drug substances.  Table 1. shows the summary of the reported spectrophotometric methods indicating the basic principle, λ max, solvent and limit of detection (LOD).

3.3. Capillary Electrophoresis (CE)

CE methods have excellent performance for separation of pharmaceuticals, which makes it the first-choice technique for separation of stereoisomer’s. For Ambroxol HCl analysis few authors have used CE as a separation and determination technique.

Tomas et al. constructed a sensitive capillary electrophoretic method combined with laser-induced fluorescence detection for the determination of Ambroxol HCl. Samples were derivatized with 5·10⁻⁴Mfluorescein isothiocyanate. A linear relationship between concentration and peak area was in the concentration range 0.008–42 μg ml−1 with a correlation coefficient of 0.9999. The method was demonstrated their applicability to serum and urine samples. (Tomas et al. 2000)

3.4. Chromatography

3.4.1. HPLC

3.4.1.1. Biological samples.

Various methods for the determination of Ambroxol in biological samples like plasma, serum and urine(Suresh et al. 2012; Dharuman et al. 2011 ) are listed in Table 2.

3.4.1.2. Pharmaceutical samples.

Analytical methods for the determination of Ambroxol HCl in pharmaceutical dosage forms using HPLC (Mallapur et al.  2011; Kotkar et al. 2012 )are shown in Table 3.

3.5. HPTLC

In the literature about 6 methods were reported for the determining Ambroxol HCl alone, and in combination with other drug substancesin pharmaceutical formulation and in biological samples by using high performance thin layer chromatography (Kasture et al. 2010; Bagada et 2013).The HPTLC methods are shown in Table 4.

Table 1. Representative  spectrophotometric  method for the analysis of Ambroxol Hydrochloride.

Table 2. Summary of HPLC methods to determine Ambroxol hydrochloride in biological sample.

.

Table 3. Reported analytical HPLC methods for determination of Ambroxol HCl either alone or in combination with other drugs in pharmaceutical dosage form.

Table 4. Representative HPTLC methodsfortheanalysisof Ambroxol Hydrochloride.

Simultaneous determination of Roxythromycin (ROX) and Ambroxol Hydrochloride (AMB) in tablets

Benzene: Diethyl ether:Triethylamine

( 4 :5: 1 v/v/v)

365 (ROX)

255

(AMB)

0.95(ROX) 0.36(AMB)

Cold Formulations

Chloroform: Methanol: Ethyl acetate: acetic acid

(70: 8: 2:10v/v/v/v)

270

0.14(AMB)

0.66(GUA)

0.79(AD)

0.88  (GD)

3.6. Ultra Performance Liquid Chromatography (UPLC) Method

Trivedi et al. (2011) constructed stability indicating reversed phase ultra-performance liquid chromatography (RP-UPLC) method for simultaneous determination of Ambroxol hydrochloride (AMB), Cetirizine hydrochloride (CTZ), Methylparaben (MP) and Propylparaben (PP) in liquid pharmaceutical formulation. Chromatographic separation was carried outby using Agilent Eclipse plus C18, 1.8 μm (50 x 2.1 mm) column, at 237 nm detector wavelength. The optimized mobile phase containing  mixture of 0.01 M phosphate buffer and 0.1 % triethylamine as a solvent-A and acetonitrile as a solvent-B. The developed method separates AMB, CTZ, MP and PP in presence of twelve known impurities/degradation products and one unknown degradation product within 3.5 min.  At a flow rate of 0.5 mL/min at 50°C (column oven) temperature. Under the backpressure in the system was about 6,000 psi. The LLOQ (µg/ml) were found to be 0.12, 0.18, 0.13 and 0.16 of MP, CTZ, PP, and AMB respectively, and %RSD were found to be 3.5, 4.3, 5.7 and 4.8 of methyl paraben (MP), Cetirizine hydrochloride (CTZ), Propylparaben (PP),and Ambroxol hydrochloride (AMB) respectively.( Trivedi et al. 2011)

3.7. Gas Liquid Chromatography

Marucci et al. studied Ambroxol HCl in biological material by using gas chromatography with electron-capture detection. Pinazepam, was used as internal standard, at a concentration of 5 ng/ml in methanol. The % Recovery was found to be 100 ±2.3 % R .S.D. The limit of detection was 4 ng/ml of Ambroxol HCl for all the biological samples. (Colombo et al. 1990)

3.8. LC–MS

Xin et al. developed a novel, rapid and sensitive LC-MS/MS method operated in segmental and multiple reactions monitoring for the simultaneous determination of amoxicillin and Ambroxol HCl in human plasma. Amoxicillin degrades in plasma at room temperature and readily undergoes hydrolysis by the plasma amidase. The degradation of amoxicillin in plasma was well prevented by immediate addition of 20 μL glacial acetic acid to 200 μL aliquot of freshly collected plasma samples before storage at −80°C. The lower limits of quantitation of Ambroxol and Amoxicillin were 0.5ng/mL and 5 ng/mL respectively. Furthermore, the mass response saturation problem with amoxicillin was avoided by diluting the deproteinized plasma samples with water before injection into the LC-MS/MS system. (Dong et al. 2013)

Kim et al. constructed a sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry (LC-MS/MS) for the quantification of Ambroxol HCl in human plasma. Domperidone was used as internal standard, with plasma samples extracted using diethyl ether under basic condition. A centrifuged upper layer was then evaporated and reconstituted with 200 µl methanol. The reconstituted samples were injected into a C-18 XTerra MS column (2.1 x 30 mm) with 3.5 µm particle size. The analytical column lasted for at least 600 injections. The mobile phase was composed of 20 mM ammonium acetate in 90% acetonitrile (pH 8.8), with flow rate at 250 µl/min. The mass spectrometer was operated in positive ion mode using turbo electron spray ionization. Nitrogen was used as the nebulizer, curtain, collision, and auxiliary gases. Using MS/MS with multiple reactions monitoring (MRM) mode, Ambroxol HCl was detected without severe interferences from plasma matrix. Ambroxol HCl produced a protonated precursor ion ((M+H)(+)) at m/z 379 and a corresponding product ion at m/z 264. And internal standard (domperidone) produced a protonated precursor ion ((M+H)at m/z 426 and a corresponding product ion at m/z 174. Detection of Ambroxol HCl in human plasma was accurate and precise, with quantification limit at0.2 ng/ml. (Kim et al. 2003)

Wen et al. developed LC-MS/MS method for simultaneous determination of amoxicillin and Ambroxol in human plasma using Clenbuterol as internal standard (IS). The plasma samples were subjected to a simple protein precipitation with methanol. Separation was achieved on a Lichrospher C(18) column (150 mm x 4.6mm ID, dp 5 microm) using methanol (containing 0.2% of formic acid) and water (containing 0.2% of formic acid) as a mobile phase by gradient elution at a flow rate of 1.0 mL/min. Detection was performed using electron spray ionization in positive ion multiple reaction monitoring (MRM) mode by monitoring the ion transitions from m/z 365.9-->348.9 (amoxicillin), m/z 378.9-->263.6 (Ambroxol HCl) and m/z 277.0-->203.0 (IS). Calibration curves were linear in the concentration range of 5-20,000 ng/mL for amoxicillin, and 1-200 ng/mL for Ambroxol HCl, with the intra- and inter-run precisions of <9% and the accuracies of 100+/-7%.(Wen et al. 2008)

Thummala.et al. studied the isolation, identification, and characterization of Ambroxol HCl’s unknown impurity. One unknown impurity of Ambroxol was formed in the formulated drug under stress conditions (40°C /75% relative humidity (RH) for 6 months) with the relative retention time (RRT) 0.68 in RP-HPLC. The impurity was enriched by exposing it to heat and it was isolated by using preparative HPLC. The enriched impurity was purified and characterized using the following sophisticated techniques: 2D NMR (gDQ-COSY, gHSQC, and gHMBC), FTIR, and LC-MS/MS. On the basis of the spectral data, the impurity was characterized as trans-4-(6,8-dibromoquinazolin-3(4H)-yl)cyclohexanol.( Thummala.et al. 2014). The comparative sensitive methods of various techniques are shown in Figure. 4.

Figure-4. Comparative sensitive methods of various techniques.

4. CHALLENGES:

Ambroxol HCl is used in preventing bronchial hyper-reactivity, as well as decreased airway hyper-reactivity by either increasing the lyso-phosphatidyl-choline turnover, or modifying epithelial secretion. As discussed earlier, Ambroxol HCl belongs to BCS class-I, it means Ambroxol has high solubility and high permeability. However, for successful treatment a constant and uniform supply of drug is needed. Hence, it represents significant formulation challenges. To overcome these problems and challenges an important strategy is considered, hydrophilic matrices are commonly used which achieves slow release of drug over an extended period of time. The onset of its pharmacologic action is often delayed and the duration of its therapeutic effect is sustained. Hence, in development of hydrophilic matrix, there is having chances to interference of excipients to drug. Furthermore, it becomes necessary to analysis of drugs, and selection of solvent is greater challenges for the analysis. The review of literature reveals that most widely used diluents are methanol and distilled water in HPLC methods, which prolonged the run times with greater tailing factor. It was observed that the drug gets slowly degraded in acidic conditions over a period of time. For spectrometric determination, the presence of multiple entities and excipients includes complexity with multi- component dosage forms, which could produce significant challenge to the analytical chemist during the development of assay procedure. Estimation of the individual drugs in these multicomponent dosage forms becomes difficult. For such instances like multicomponent dosage forms, chemo-metric methods can be preferred to routine spectrophotometric methods.

5. CONCLUSION:

The review   is concluded that    various methods such as spectroscopy, chromatography, electrophoresis and hyphenated technique are available for the quantification of single and multicomponent dosage form of Ambroxol HCl in biological fluids and pharmaceutical formulations. HPLC was extensively used for the determination of Ambroxol HCl in various matrices like plasma, serum and urine. For determination of Ambroxol HCl in biological samples, were commend the LC–MS/MS method, since this method combines the LC separation ability with MS sensitivity and selectivity, allowing the unambiguous identification of Ambroxol HCl and its metabolites. For analysis of Ambroxol HCl in pharmaceuticals, HPLC with UV detection is applicable because this method provides accurate results and low cost compared to more advanced detection techniques. This review revealed an overview of the recent state-of-art analytical methods for the determination of Ambroxol HCl.

6. DECLARATION OF INTEREST:

There is no conflict of interest with any financial organization regarding the material discussed in the manuscript. The authors alone are responsible for the content and writing of the manuscript.

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Received on 20.07.2014          Modified on 10.08.2014

Accepted on 18.08.2014          © RJPT All right reserved