The Development and Validation of a new HPLC Method for the Simultaneous Determination of Impurities in Guaifenesin, Terbutaline Sulfate and Ambroxol Hydrochloride

Vikram S. Gharge*, Anant V. Patil, Balasaheb S. Jadhav, Bhagyashree G. Tayade,

Chetan T. Parde, Sushma N. Kakade, Vitthal A. Dighe

Research and Development, Zuventus Healthcare Limited, Pune 411057, Maharashtra, India.

*Corresponding Author E-mail: Vikram.gharge@zuventus.com

ABSTRACT:

A new liquid chromatography method was developed and applied for the simultaneous determination of Ambroxol Hydrochloride (AH), Guaifenesin (GN) and Terbutaline Sulfate (TS), which are present in raw materials with potential impurities in drugs and pharmaceutical products. This method uses YMC Triart C18 chromatography column. A gradient mixture of solvents A and B is used as the mobile phase, and a UV (ultraviolet) detector is used to detect at 210 nm. The column temperature is 50°C chromatographic separation resolution. TS, GN, AH and their impurities are more than 1.5. Regression analysis showed a high correlation coefficient (r> 0.998), indicating the reliability of the method. The developed method is capable of identifying 0.625% of impurities in test solution, GN and AH & TS based on the standard measurement of 1.0 mg/mL, 0.4 mg/mL and 0.025 mg/mL respectively. The model showed excellent inter-day and intra-day precision with percentage RSD values less than 3.0 for all impurities in TS, GN and AH. The process also shows that there is a recovery. Moreover, these chemicals are exposed to stresses such as acid, alkali, aqueous hydrolysis, oxidation, photolysis and thermal degradation according to the guidelines developed by the International Conference on Harmonization (ICH). Liquid Chromatography method provides a robust, sensitive and reliable method for the determination of TS, GN and AH and their impurities in pharmaceutical preparations.

KEYWORDS: Ambroxol Hydrochloride, Terbutaline Sulphate, Guaiphenesin, Related substances, Method development, Method Validation.

INTRODUCTION:

Guaifenesin (GN), Terbutaline Sulfate (TS), and Ambroxol Hydrochloride (AH) are the main ingredients of oral syrup formulations used to treat respiratory diseases. GN, also known as 3-(2-methoxyphenoxy)-1,2 propylene glycol, acts as an expectorant by reducing sputum viscosity and helps in the elimination of sputum¹. TS, defined as 1,3-benzenediol, 5- [2- [(1,1 dimethylethyl) amino]-1-hydroxyethyl]-, sulfate (2:1) (salt), is a bronchodilator targeting β2-adrenergic receptors, especially in the treatment of bronchial asthma². Chemically identified as [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexanol hydrochloride], AH acts as a mucolytic agent and helps reduce hyperreactive bronchi, stimulates the production of cellular surfactants, and has anti-inflammatory properties^3-7.

To create effective oral syrup forms, these active ingredients are often combined with antibiotics, antiseptics, sweeteners, acidifiers, antioxidants and flavouring agents. These compounds are an important part of the cough syrup formulation. To make this preparation safe and effective, a clear identification of impurities associated with the active ingredients is essential. Although different analytical methods have been reported for the quantification of GN, TS and AH in various drug formulations, no method exists for the simultaneous determination of these three substances and their impurities in one test⁸.

Previous studies have described and identified methods such as capillary gas chromatography and liquid chromatography-tandem mass spectrometry for the determination of GN in human urine and blood, and liquid chromatography for the definitive determination of TS in human urine and blood drugs. However, there are significant differences in the data regarding stability.

LC methods can distinguish and identify impurities associated with TS, GN, and AH, specifically for oral drugs⁹. The development of this method will highlight the urgent need for reliable and high-quality testing to ensure the quality and safety of cough medicines containing active ingredients.

METHOD AND MATERIAL:

Chemicals and reagents:

The purity test of Ambroxol Hydrochloride IP standard seven was determined as 100.08%, the purity of Terbutaline Sulfate was 99.69%, the purity of Guaifenesin was 99.71% and the purity of Isoguaifenesin was 100.00%. The purity levels of Ambroxol impurities are as follows: Ambroxol impurities A (99.40%), B (98.72%), C (95.80%), D (99.14%) and E (99.09%), respectively. Impurity samples are shown in Table 1 and control samples were purchased from Zuventus Healthcare Ltd. The analytical reagents utilized for the forced degradation studies included milli-Q water, HPLC grade Methanol and Acetonitrile, Phosphoric acid (OPA), Triethylamine, Hydrochloric acid (HCl), Sodium Hydroxide (NaOH), Hydrogen peroxide (H₂O₂) were purchased from Zuventus Healthcare Ltd.

Equipment:

Chromatographic analysis was performed using a Waters Alliance HPLC system from Milford, United States (USA), consisting of a square pump integrated in the separation unit with an auto-injector and a photo array analyser result from the analysis monitored using the Empower-3.0 software. A pH meter from Lab-India was used to measure the pH of the solution. Prior to analysis, all solutions were decanted using ultrasonic equipment (PreciSonic) and filtered through a 0.45 μm nylon filter from PALL Life Sciences, USA.

Chromatographic conditions:

The analysis method was developed using a YMC Triart C18 section (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of a gradient mixing scheme of solvents A and B. Solvent A was 0.05 M Potassium dihydrogen orthophosphate and 2.0 ml Triethylamine, pH adjusted to 5.0 using OPA acid diluted as a buffer. Solvent B is a mixture of ACN and MeOH in a ratio of 70:30 (v/v)¹⁰.

The Liquid Chromatography (LC) gradient program (time / %B) is programmed as follows: 0.01 min / 18% B, 35 min / 80% B, 50 min / 50% B, 75 min / 75% B, 76 min / 18% B, 80 min / 18% B. The oven temperature was set to 50°C and the sample temperature was kept at 5°C. Detection was performed at a wavelength of 210 nm. The volume is 20μL and the diluent is Acetonitrile: Water (50:50).

Preparations of Analytical Solution:

Preparation of solution process

AH (0.004mg/mL) and GN (0.01mg/mL) solutions were prepared by separating the amount of each drug in the diluent. Then, the mixed process was prepared to obtain the content of AH (4µg/mL) and GN (10 µg/mL).).

Preparation of Sample Solution

Weigh carefully a syrup sample containing 20mg of AH and transfer it to a 50mL volumetric flask. Then add 30 mL of diluent and sonicate the mixture for 15 min with occasional shaking. Adjust the volume to 50mL with diluent and mix well. The sample solution contains AH at a concentration of 0.4mg/mL, GN at a concentration of 1.0mg/mL, and TS at a concentration of 0.025 mg/mL. The solution was filtered through a 0.45µm nylon filter.

Preparation of Placebo Solution:

Carefully measure the placebo syrup equivalent to 20mg AH from the standard preparation and transfer it to a 50 mL volumetric flask. Then, 30mL of diluent was added to the flask and the mixture was sonicated for approximately 15 minutes with occasional shaking. Then, the volume then adjusted to the mark with diluent and thoroughly mixed. Finally, the solution was filtered through a 0.45μm nylon membrane filter.

RESULTS AND DISCUSSION:

Method Development:

Selection of Chromatographic method

The choice of chromatographic method (HPLC in this case) depends on the nature of the molecules analysed, such as AH, TNF and GN. The HPLC method was chosen for its simplicity, suitability, and ruggedness in the separation and quantification of active pharmaceutical ingredients and related products¹¹.

In the initial test, different phases of the mobile phase were tested to ensure separation of compounds. Test-1, using a mobile phase containing 0.05M Potassium dihydrogen orthophosphate and Trimethylamine, failed to achieve the desired separation and the placebo peak merged with the Guaifenesin peak. Test-2, using a modified ACN: MeOH (80:20) mobile phase composition, showed a hump in the Ambroxol peak retention time, representing incomplete separation¹².

To address these issues, Trial-3 was conducted with a further modified mobile phase composition of Acetonitrile: Methanol (70:30). In this trail, solvent B is optimized by preparing a mixture of Methanol and Acetonitrile in a specific ratio (700:300, v/v) for rapid elution of the compound, good solubility and highest quality of the product of interest: Ambroxol Hydrochloride, Terbutaline Sulphate and Guaifenesin and their respective impurities.

By optimizing the mobile phase composition and gradient elution, Trial-3 successfully achieved the desired separation and resolution of target compounds and related substances, demonstrating the importance of method development and optimization in HPLC analysis for pharmaceutical applications resulting chromatogram show in Figure.1

(1A) Blank chromatogram

(1B) Placebo chromatogram

(1D) Sample spiked chromatogram

Figure 1-Typical chromatograms, (1A) Blank chromatogram, (1B) Placebo chromatogram, (1C) Standard chromatogram, (1D) Sample spiked chromatogram

VALIDATION OF THE METHOD:

The estimation procedure was validated according to ICH guidelines^13-16.

The accuracy of the test method was evaluated through repeatability by injecting six cough syrup test preparations. The %RSD was calculated for each impurity concentration and in the precision and intermediate precision study the results were found to be less than 5.0% for all known impurities¹⁷. Results shown in Table 2, Table 3.

Table 2.-Results of Method Precision

Sample No.	% Isoguaifenesin imp.	% Ambroxol Imp.D	% Ambroxol Imp.A	% Ambroxol Imp.B	% Ambroxol Imp.C+E	% Any individual impurity	% Total impurities
Mean	0.332	BDL	BDL	0.096	BDL	BDL	0.475
SD	0.001	BDL	BDL	0.004	BDL	BDL	0.018
%RSD	0.44	BDL	BDL	4.01	BDL	BDL	3.70

Table 4-Peak purity of sample solution and impurities.

Sample details	Retention time	Peak purity Threshold	Peak purity Angle	Peak purity
Impurity Spike test solution
Isoguaifenesin	13.72	0.581	0.416	Pass
Ambroxol impurity D	45.92	1.145	1.026	Pass
Ambroxol impurity A	54.97	0.574	0.347	Pass
Ambroxol impurity B	58.11	1.157	0.888	Pass
Ambroxol impurity C	63.78	1.502	1.197	Pass
Ambroxol impurity E	63.74	1.100	0.669	Pass
Test solution
Guaifenesin	17.70	2.067	1.536	Pass
Ambroxol HCl	39.44	0.263	0.055	Pass

Table 5-Peak purity of Identification solution.

Sample details	Retention time	Peak purity Threshold	Peak purity Angle	Peak purity
Terbutaline	3.40	1.048	0.601	Pass
Isoguaifenesin	13.66	0.786	0.519	Pass
Guaifenesin	17.75	0.793	0.610	Pass
Ambroxol HCl	39.63	2.241	1.590	Pass
Ambroxol impurity D	45.89	1.083	0.914	Pass
Ambroxol impurity A	54.91	0.524	0.357	Pass
Ambroxol impurity B	58.05	1.411	1.039	Pass
Ambroxol impurity C	63.76	1.431	1.077	Pass
Ambroxol impurity E	63.73	0.909	0.689	Pass

Table 6-Forced degradation

Sr.

No.

Stress condition

%Isoguaifen esin imp.

%Ambroxol

Imp. D

%Ambroxol

Imp. A

%Ambroxol

Imp. B

%Ambroxol

Imp. C+E

% Any individual impurity

% Total impurities

As such sample

0.328

BDL

0.328

Acid degradation (10 mL 0.1 M HCl, kept sample solution in water bath at 70°C for 60 minutes

0.384

BDL

1.387

3.939

Alkali degradation (10 mL 0.1 M NaOH, kept sample solution in water bath at 70°C for 60 minutes.)

0.359

BDL

0.359

Oxidation degradation (10mL, 1.0% H2O2, kept sample solution in water bath at 70°C for 60 minutes.)

0.287

0.04

BDL

2.215

BDL

3.042

7.616

Heat degradation (solid State) (Exposed the sample at 70°C for 24 hours in oven.)

0.639

BDL

0.133

1.144

Humidity degradation (Exposed the sample at 40°C/75% RH for 24 hours.)

0.318

BDL

0.875

1.414

(2A) Sample

(2B) Sample in 0.1 N HCl condition

(2C) Sample in 0.1 N NaOH condition

(2D) Sample in1% Peroxide condition

(2E) Exposed the sample at 70°C for 24 hours in oven

(2F) Humidity degradation

Figure 2-Forced degradation chromatograms, (2A) Sample (2B) Sample in 0.1 N HCl condition, (2C) Sample in 0.1 N NaOH condition, (2D) Sample in1% Peroxide condition, (2E) Exposed the sample at 70°C for 24 hours in oven, (2F) Humidity degradation (Exposed the sample at 40°C/75% RH for 24 hours.)

Linearity:

The studies included the preparation of Ambroxol hydrochloride, Ambroxol Imp-A, Ambroxol Imp-B, Ambroxol Imp-C, Ambroxol Imp-D, Ambroxol Imp-E, Isoguaifenesin-I and a series of linear standard solution problems of Isoguaifenesin across a concentration range from 0.3125% to 150% of the specification limit. A graph was constructed for each compound depicting the relationship between the concentration and the response from the limit of quantification (LOQ) 0.625% to 150% of the standard concentration²⁰.

The results show that the method leaves between LOD % and 150% of Ambroxol Hydrochloride and its impurities (A, B, C, D, E), Isoguaifenesin impurity, and Guaifenesin. This suggests that the analytical method used for quantification was capable of providing accurate and reliable measurements across a wide concentration range for the specified compounds. Coefficient linear regression (R²) of impurities shown in Table 7.

Table 7-Coefficient linear regression (R²)

Compound

Isoguaifenesin Imp (% RSD)

Ambroxol Imp D

(% RSD)

Ambroxol Imp A

(% RSD)

Ambroxol Imp B

(% RSD)

Ambroxol Imp C

(% RSD)

Ambroxol Imp E

(% RSD)

R²

0.9991

0.9996

0.9995

0.9997

0.9992

0.9998

Table 8-The percentage recovery of impurities

% Level	Isoguaifenesin impurity (% RSD)	Ambroxol impurity D (% RSD)	Ambroxol impurity A (% RSD)	Ambroxol impurity B (% RSD)	Ambroxol impurity C (% RSD)	Ambroxol impurity E (% RSD)
LOD	105.23 (1.86)	82.71 (2.14)	109.80 (3.09))	96.70 (0.33)	93.33 (0.62)	83.91 (2.18)
50	100.37 (0.18)	107.32 (0.41)	102.96 (0.08)	92.20 (0.47)	108.01 (0.84)	106.83 (0.27)
100	92.00 (0.95)	107.94 (0.38)	101.10 (0.36)	92.38 (1.55)	106.25 (0.96)	105.84 (0.48)
150	96.82 (0.07)	95.53 (0.14)	100.33 (0.01)	96.70 (0.55)	104.92 (0.11)	107.48 (0.74)

Table 9-Solution Stability

Sample No.	% Isoguaifenesin Imp.	%Ambroxol Imp. D	%Ambroxol Imp. A	%Ambroxol Imp. B	%Ambroxol Imp. C+E	% Any individual impurity	% Total impurities
Initial	0.335	BDL	0.047	0.097	BDL	0.067	0.547
8 Hr.	0.335	BDL	0.056	0.103	BDL	0.071	0.565
13 Hr.	0.335	BDL	0.051	0.103	BDL	0.072	0.562
20 Hr.	0.337	BDL	0.050	0.105	BDL	0.073	0.597
24 Hr.	0.338	BDL	0.053	0.104	BDL	0.072	0.598
30 Hr.	0.338	BDL	0.050	0.104	BDL	0.074	0.599
36 Hr.	0.336	BDL	0.051	0.102	BDL	0.075	0.604
48 Hr.	0.333	BDL	0.031	0.128	BDL	0.072	0.611
Mean	0.336	BDL	0.049	0.106	BDL	0.072	0.585
SD	0.002	BDL	0.008	0.009	BDL	0.002	0.024
% RSD	0.51	BDL	15.58	8.81	BDL	3.32	4.04

Accuracy:

All impurities were spiked at 50%, 100% and 150% LOQ of the desired range with impurity stock solutions and these spikes were used in triplicate in mixed placebo powders¹⁹. The percentage recovery for impurities, Isoguaifenesin IGN impurity, AH impurities (A, B, C, D, E) and reference materials for each recovery solution is shown in Table 8.

Stability of Solution

The reference and test solutions, spiked with impurities where prepared according to this procedure and stored at 5°C. Samples and sample solutions are tested at the initial, 8 hr, 13 hr, 20 hr, 24 hr, 30 hr, 36 hr, and 48 hr intervals ²⁰ and result shown in Table 9.

Stability studies show that the test solution is stable for up to 48 hours at 5°C and the standard solution is stable for up to 72 hours at 5°C.

Robustness:

The robustness studies have evaluated the performance of the method under various conditions including changes in flow rate, column, mobile phase buffer pH, and column oven temperature. The result show that the method is robust to changes in column throughput, flow rate, mobile phase buffer solution pH, and column oven temperature within the specified range. However, the method has been shown to be sensitive to changes in column oven temperature (+5°C) and not the pH of the mobile phase buffer solution^21-26.

CONCLUSION:

The focus of the review is on the development and validation of relevant methods for the analysis of Ambroxol Hydrochloride, Terbutaline Sulfate and Guaifenesin in a syrup formulation is a critical aspect of pharmaceutical analysis by High-Performance Liquid Chromatography (HPLC). This method is important for determining the purity, quality and stability of the drug in dosage form. Firstly, the development method has optimization for the chromatographic system, such as selection of stationary phase, mobile phase, flow rate and detection wavelength, separation and identification of relevant components in syrup. This step is important to ensure the analysis of target compounds.

Validation of the design is necessary to demonstrate its reliability, accuracy, and reproducibility. Validation parameters typically include specificity, linearity, precision, accuracy, robustness, and system compatibility. Specificity ensures that the method can discriminate between the target analyte and impurities or degradation products. Linearity establishes a relationship between the concentration of the analyte and the response of the analyzer, while precision measures the repeatability and intermediate precision of the method. Accuracy is determined by comparing measured values with actual values, usually through regression analysis. Robustness measures the ability of a method to remain unaffected by small changes in the experiment. Appropriate tests were performed to ensure the performance of the chromatographic system prior to sample analysis.

The Development and validation of a new HPLC method for the simultaneous determination of impurities in Guaifenesin, Terbutaline Sulfate and Ambroxol Hydrochloride in syrup involves careful planning and thorough testing. This method will help pharmaceutical labs ensure the quality and safety of their products, making it a valuable tool for patient care.

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Received on 26.08.2024 Revised on 16.11.2024

Accepted on 20.01.2025 Published on 27.02.2025

Available online from March 05, 2025

Asian J. Pharm. Tech. 2025; 15(1):17-24.

DOI: 10.52711/2231-5713.2025.00004

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Sr. No.	Related substance	Structure
1.	Isoguaifenesin (2-(2-Methoxyphenoxy) propane-1,3-diol)
2.	Ambroxol impurity D (1s,4s)-4-((2-Amino-3,5-dibromobenzyl) amino) cyclohexanol Hydrochloride; cis-4-[[(2-Amino-3,5-dibromophenyl) methyl] amino] cyclohexanol Hydrochloride;
3.	Ambroxol impurity [A 2-Amino-3,5-dibromophenyl) methanol]
4	Ambroxol impurity B [4-(6,8-dibromo-2,4-dihydro-1H-quinazolin-3-yl) cyclohexan-1-ol]
5	Ambroxol impurity C (trans-4-[[(E)-2-Amino-3,5-dibromobenzyliden] amino] cyclohexanol)
6	Ambroxol impurity E (Amino-3,5-dibromobenzaldehyde; 3,5-Dibromo-2- amino benzaldehyde)

Sample No.	% Isoguaifenesin imp.	% Ambroxol Imp.D	% Ambroxol Imp.A	% Ambroxol Imp.B	% Ambroxol Imp.C+E	% Any individual impurity	% Total impurities
Mean	0.311	BDL	BDL	0.083	BDL	BDL	0.394
SD	0.003	BDL	BDL	0.003	BDL	BDL	0.005
%RSD	0.95	BDL	BDL	3.51	BDL	BDL	1.24