INTRODUCTION:

A combination of two drugs – Bromhexine and Cephalexin is used to treat different types of infections (bone/respiratory/skin/genitourinary tract infections). This combination is proven in the treatment of respiratory tract infections, which are accompanied by extreme mucus¹. Bromhexine loosens the mucus and makes it thin. Hence, bromhexine acts as a mucolytic and facilitates comfortable coughing out of mucus².

The interaction of bromhexine and ambroxol (a metabolite of bromhexine) with the receptors of lung cells has shown a beneficial effect in patients with COVID-19, notably in the case of hepatic or lung injury³. The antibacterial nature of cephalexin destroys the bacteria through the prevention of protective cover formation around themselves.

Cephalexin is one of the popular β-lactam antibiotics which is used for the treatment of a variety of bacterial infections. The bacterial cell wall growth is disrupted by cephalexin and is capable of destroying all gram-positive bacteria and a few gram-negative bacteria⁴. Chemically, bromhexine is 2-Amino-3,5-dibromo-N-cyclohexyl-N-methylbenzylamine, and the molecular formula is C₁₄H₂₀Br₂N₂. It is a benzylamine derivative and brominated aniline. Its origin is from a plant-derived ingredient - Vasicin, which is obtained from the Indian lung herb, Adhatoda vasica⁵. Chemically cephalexin is 7-[(aminophenylacetyl)amino]-3- methyl-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene- 2- carboxylic acid. Its chemical formula⁴ is C₁₆H₁₇N₃O₄S (Fig.1).

(a) (b)

Fig.1. Chemical structures of (a) Bromhexine and (b) Cephalexin

Literature study shows that in addition to the methods developed for the quantification of individual drugs –Cephalexin^6-8and Bromhexine^9-10, few methods are also available for simultaneous estimation of these drugs. First-order derivative spectrophotometric and simultaneous equation method was given by Kumar and Krishnaveni¹¹. The RP-HPLC methods proposed for their simultaneous estimation by other researchers comprise acetonitrile as the organic solvent in the mobile phase. 0.1% OPA: acetonitrile (45:55) v/v¹², acetonitrile: TEA buffer pH 4.2 (75:25)¹³, sodium dihydrogen orthophosphate buffer and acetonitrile (45:55 v/v), pH 5.0¹⁴ were the mobile phases in those RP-HPLC methods for simultaneous estimation. Development of green analytical methods is the art of the day^15-17. Though acetonitrile accomplishes the best characteristics of a solvent, it faces major issues – high cost, volatility in supply, and environmental problems, especially in pharmaceutical industries viz, disposal difficulties of acetonitrile wastes, and poor scoring in terms of life cycle management¹⁸. Hence, the aim of the current study is to develop a new green analytical RP-HPLC method for simultaneous estimation of Bromhexine and Cephalexin, followed by the method’s validation as per the current ICH guidelines.

MATERIALS AND METHODS:

Instrumentation:

Performing the chromatographic separation was carried out on a PEAK chromatograph, which was equipped with LC-P7000 isocratic pump. Degassing of the mobile phase and dissolution of samples were carried out by sonicating in an ultrasonicator. Denver electronic analytical balance (SI-234) was used for weighing drug samples and standards. Systronics digital pH meter was used to adjust the pH of the mobile phase.

Chemicals and Solvents:

HPLC grade solvents (methanol, acetonitrile) and AR Grade buffer chemicals were procured from Merck Specialties Private Limited, Mumbai, India. The local market was the source of pharmaceutical formulation.

Preparation of sample solution:

Ten vial formulations of bromohexine and cephalexin (Brocif; Bromohexine – 4 mg and cephalexin- 250 mg) were soaked in 5 ml diluent and were kept for solubility for 1 hour. Then, it was filtered and suitably diluted with the same diluent. The resultant solution was used for the simultaneous estimation of cephalexin and bromohexine in combined dosage forms.

Forced Degradation:

These studies were carried out by comparing the degradants in the chromatogram with the initial values after incubating the Bromhexine and Cephalexin standard in diverse conditions of degradation, like exposing the sample, as mentioned in the results table.

RESULTS AND DISCUSSIONS:

Method Development:

For the simultaneous quantification of the Bromhexine and Cephalexin, the detector’s wavelength was set at 228 nm by considering the absorption maxima of both the drugs from their overlay spectra (Fig.2).

Fig.2. Overlay absorption spectra of Bromhexine and Cephalexin

Till the development of an acceptable peak, optimization of different parameters of the isocratic elution (composition and pH of mobile phase, column, pump pressure) was carried out so that agreeable system suitability conditions could be obtained. Some of the chromatograms pertaining to the method development are shown in Fig. 3. A fixed flow rate (1.0 mL min^–1) and drug concentrations (Bromhexine – 1.2 µg/ml and Cephalexin – 75 µg/ml) were used in the development of isocratic HPLC method for the simultaneous estimation of Bromhexine and Cephalexin. In the initial trial (methanol: acetonitrile (90: 10 v/v); Zodiac C-18 (250 mm x 4.6 mm, 5 μm) column; pH 5.7; Pump Pressure 11.5±4 MPa), only Bromhexine was detected (Fig. 3.i). The change in conditions (methanol: acetonitrile (60: 40 v/v); pH 5.4; Pump Pressure 11.2±4 MPa), didn’t elute out the Cephalexin (Fig. 3.ii). A change in the column to Zodiac C-18 (100 mm x 4.6 mm, 5 μm) (methanol: water (90: 10 v/v); pH5.7; Pump Pressure 13.2±4 MPa), eluted both the drugs –Bromhexine and Cephalexin with an improvement in peak shapes, but USP count (869; 1127) and resolution (1.01) were low (Fig. 3.iii). In spite of an increase in USP count (2153; 2840), resolution (1.59) was low when Hypersil C-18 (250 mm x 4.6 mm, 5 μm) column was used with methanol: water (60:40 v/v);; pH 5.9; Pump Pressure 15.4±4 MPa (Fig. 3.iv). An improvement in resolution (2.91) with high USP plate count (2198; 4504) was observed by a further change in the column to Waters C-18 (250 mm x 4.6 mm, 5 μm at other conditions like Methanol: Acetate Buffer (60: 20 v/v);; pH 5.4; Pump Pressure 13.4±4 MPa, but the peak shape is with less symmetry (USP tailing factors– 2.51; 2.32) (Fig. 3.v). The optimized conditions were obtained by further fine-tuning of the other parameters (methanol: acetate buffer 70:30 (v/v); Waters Symmetry C-18 (250mm x 4.6mm, 5μm) column; pH 5.1; Pump Pressure 10.5±4 MPa), to get a high resolution (7.15), good peak symmetry (USP tailing factors: 0.98; 1.16) and high USP plate count (4492; 7639) (Fig. 3.i). Symmetry C18 columns give high reproducibility in the chromatographic results due to the tightest column specifications. Another advantage of these columns is their delivery of superior peak shape over a wide range of pH (2 to 8) due to two reasons viz., lower residual silanol activity of the packing materials and great bonding density. After fine-tuning the conditions, the system suitability parameters at optimized conditions are listed in Table-1. The current developed method is the green analytical method because the mobile phase used in the present case is methanol, whereas acetonitrile was used by other researchers^12-13.

Table 1. Chromatography conditions of the method

Parameter	Condition
Mode of separation	Isocratic
Column	Waters C-18 column (250 mm × 4.6 mm; 5 µm)
Mobile phase	Methanol: Acetate Buffer (70:30 v/v)
Flow rate	1.0 mL min^–1
Injection volume	2 0 μL
Sample temperature	Ambient
Column temperature	Ambient
Run time	12 min
Detector wavelength	228 nm
Diluents	Mobile phase
Mobile phase pH	5.1

(i)

(ii)

(iii)

(iv)

(v)

Fig.3. Chromatograms of Method Development Trials

Fig.4: Chromatogram of a standard solution of Bromhexine and Cephalexin under the optimized conditions

System Suitability and Specificity:

The suitability of the present proposed method was ensured for the expected application by performing a system suitability test. At the optimized conditions of the present proposed method, the system suitability parameters were detected to be within the agreeable criteria (Table 2). The system’s substantiated performance is clear from the satisfactory values of system suitability parameters, which are tabulated in Table 2. A specificity study was conducted to explore the interference from placebo, blank, and degradation products. None of the placebo/diluents showed interference at the retention times of Bromhexine and Cephalexin. Confirmation of the system's suitability is apparent from the lower tailing factor value (< 2) and high number of theoretical plates (>2000) for the peaks of both Bromhexine and Cephalexin in their chromatograms. Low tailing factors were observed in the present case compared to the reports of Rao et al¹²(1.41 and 1.53) and Sanjeevarani et al¹³ (1.2 and 1.3). A higher number of theoretical plates was observed than the reports of Rao et al. ¹² (2881 and 3914) and comparable values with Sanjeevarani et al. ¹³ (2018) (6280.3 and 6280.3). The performance of the specificity study was meant for the verification of any interference presence due to either potential or degradation impurities at the retention times of Bromhexine and Cephalexin. The current method’s specificity is evident from the lack of peak interference from blank at the retention times of Bromhexine (3.68 minutes) and Cephalexin (6.21 minutes). In the present method, a good separation of peaks with a high resolution (7.15) is obtained in the present case compared to previous researchers with a resolution¹⁴ of 6.0.

Table 2. System suitability parameters at optimized conditions

S. No.	Parameter	Bromhexine	Cephalexin
1	Peak area (µV*Sec)	75092.5	263814.3
2	Retention time (min)	3.68	6.21
3	USP tailing factor	0.98	1.16
4	USP plate count	4492	7639
5	API Concentration (μg/ml)	1.2	75
6	SD of the area	348.9	1698.9
7	%RSD of the area	0.469	0.650
8	Resolution	--	7.15

Stability Indicating Studies:

To understand the stability-indicating nature of the current method, a study of forced degradation was performed, and chromatograms were recorded under various stress conditions. Under each condition of degradation, peaks of the degradants are clearly separated from each other and also from the peaks of both the drugs – Bromhexine and Cephalexin. It indicates the non-interference from the peaks of degradation products. The stability indicating and specific nature of the proposed method was clear from the observed data. The results pertaining to the stress-induced degradation of Bromhexine and Cephalexin are shown in Table –3.a. and 3.b. Towards the oxidation, lower susceptibility was exhibited by Bromhexine. A medium susceptibility was shown by Bromhexine under base, thermal, and photolytic degradation conditions. However, Cephalexin manifested the same under U.V. and photolytic (visible light) degradation conditions. Higher degradation levels were observed for Bromhexine under acid and UV light degradation conditions, whereas Cephalexin exhibited higher degradation under acid, base, thermal, and oxidation conditions. The appropriateness of the current method for routine quality control analysis is evident from these results because it is found to be selective.

Table 3. a. Results of stress study of Bromhexine

Parameter	Stress Conditions	% Assay of the degraded sample (A)	*% Degradation w.r.t. control (B)**
Control sample (No degradation)	No Exposure	100.727	--------
Acid hydrolysis	0.1 ml of 1N HCl and heated at 70 °C for 1 hr	93.87	6.807
Base hydrolysis	1 ml of 1N NaOH and heated at 70 °C for 1 hr	96.83	3.867
Oxidation	0.5 ml of 30% H₂O₂ at 70°C for 1 hr	97.89	2.816
UV light	200-Watt hours/square meter	92.71	7.960
Thermal degradation	Exposed at 80 °C for at least 72 hr	96.46	4.233
Photolytic degradation (light)	Exposed to 1.2 million lux hours of light	97.06	3.641

*B= (100.727- A)/100.727*100

Table 3.b. Results of stress study of Cephlaxine

Parameter	Stress Conditions	% Assay of the degraded sample (A)	*% Degradation w.r.t. control (B)**
Control sample(No degradation)	No Exposure	100.0057	-----
Acid hydrolysis	20 ml of 1N HCl for 24 hrs	89.92	10.037
Base hydrolysis	20 ml of 1N NaOH for 48 hrs	92.69	7.305
Oxidation	20 ml of 30% H₂O₂ for 24 hrs	93.21	6.789
UV light	200-Watt hours/square meter	94.73	5.266
Thermal degradation	Exposed at 80 °C for 24 hr	91.41	8.588
Photolytic degradation (light)	Exposed to 1.2 Million lux hours of light	94.60	5.400

*B= (100.00- A)/100.00*100

Table.4.a. Results of accuracy for Bromhexine

Level of recovery (%)	Amount added (µg mL^–1)	Amount recovered (µg mL^–1)	% Recovered	Statistical evaluation
50	0.6	0.605	100.83	%Mean	101.33
	0.6	0.610	101.70	SD	0.452
	0.6	0.609	101.46	%RSD	0.446
100	0.8	0.797	99.62	%Mean	100.50
	0.8	0.805	100.62	SD	0.821
	0.8	0.810	101.25	%RSD	0.817
150	1	1.013	101.33	%Mean	101.70
	1	1.020	102.01	SD	0.345
	1	1.018	101.75	%RSD	0.339

Table.4.b.Results of accuracy for Cephalexin

Level of recovery (%)	Amount added (µg mL^–1)	Amount recovered (µg mL^–1)	% Recovered	Statistical evaluation
50	37.5	35.79	95.44	% Mean	95.11
	37.5	35.59	94.90	SD	0.288
	37.5	35.62	94.99	%RSD	0.302
100	50	48.40	96.81	%Mean	96.37
	50	47.96	95.91	SD	0.447
	50	48.20	96.40	%RSD	0.464
150	62.5	62.42	99.88	%Mean	99.66
	62.5	62.24	99.59	SD	0.190
	62.5	62.20	99.52	%RSD	0.191

Precision:

The variability in results during the repeated analyses of the Bromhexine and Cephalexin samples with duplicate experimental situations indicates the precision. The study of both precisions (MP–method precision and IP –IP-intermediate precision) was conducted by using six replicates for the validation of the present proposed method. The intermediate precision was performed by maintaining various conditions (analyst/column /instrument). The respective calculated %RSD values for MP of Bromhexine and Cephalexin were 0.480 and 0.596, and similarly for IP were 0.667 and 0.780. It indicates that their values are below the permissible limits specified by ICH (Tables - 5 and 6) as well as a higher degree of precision and ruggedness of the method. A good precision of the method is disclosed from the higher %assay values (above 98%). These %RSD values of MP are comparable to the reports of Band et al¹⁴ (Bromhexine %RSD 0.41 and Cephalexin-%RSD 0.54), Raoet al¹² (2017) (0.5 and 0.4) and Sanjeevarani et al¹³(less than 2%). Similarly, the case with IP.

Robustness:

A thoughtful modification of the main parameters (composition and pH of mobile phase, wavelength) and follow-up evaluation of their impression on the current method in order to realize its robustness by using standard solutions of 1.2 μg/ml of Bromhexine and Cephalexin. An intentional variation in the values of experimental parameters viz., a deviation of ±5% (v/v) in the composition of organic solvent, ±0.2 in pH and ±4 nm in the detection wavelength didn’t significantly affect the characteristics of chromatograms like peak area, retention time, tailing factor and theoretical plates (Table -7). The current method’s robustness is evident from the less than 2.0% change in the peak areas.

Table.5. Results of Precision (Method and Intermediate) Study

S No	% Assay*
	Bromhexine		Bromhexine
	M.P.	I.P.	M.P.	I.P.
1	97.77	98.90	100.26	99.00
2	98.50	98.51	98.79	98.63
3	98.86	98.28	99.20	99.75
4	97.43	97.46	98.24	99.15
5	98.34	97.28	99.00	100.55
6	98.11	97.70	99.81	98.15
Mean (n=6)	98.17	98.02	99.22	99.21
Std.Dev	0.472	0.584	0.662	0.773
% RSD (n=6)	0.480	0.596	0.667	0.780

M.P.: Method Precision I.P.: Intermediate Precision

*at 20 μg mL–1

Table 7. Results of robustness/ruggedness experiment of Bromhexine and cephalexin

Altered Parameter	Actual condition	Altered condition	RT (Min)	Theor plates	Tail factor	Peak area	% Change in peak area
Bromhexine
Control Condition	NA	---	3.68	4492	0.98	75092.5	-------
Mobile phase ratio - Methanol: Acetate Buffer (v/v)*	(70:30)	75:25	3.73	4125	0.91	74963.2	0.172187635
Mobile phase ratio - Methanol: Acetate Buffer (v/v)*	(70:30)	65:35	3.71	4237	0.92	75162.5	-0.093218364
pH	5.1	5.0	3.73	4185	1.02	74778.3	0.418417285
pH	5.1	5.2	3.73	4193	0.94	73692.1	1.864899957
Wave length (nm)	228	234	3.72	4357	0.94	74681.4	0.547458135
Wave length (nm)	228	224	3.70	4193	0.96	75223.6	-0.174584679
Cephalexin
Control Condition	NA	---	6.21	7639	1.16	263814.3	------
Mobile phase ratio – Methanol:Acetate Buffer (v/v)*	(70:30)	75:25	6.23	7629	1.02	262839.4	0.369540241
Mobile phase ratio – Methanol:Acetate Buffer (v/v)*	(70:30)	65:35	6.23	7654	1.14	259342.2	1.69516967
pH	5.1	5.0	6.23	7736	1.19	261947.6	0.707581052
pH	5.1	5.2	6.23	7654	1.31	264278.3	-0.175881292
Wave length (nm)	228	234	6.22	7831	1.12	265361.3	-0.586397326
Wave length (nm)	228	224	6.23	7650	1.17	258757.2	1.916916558

LOD and LOQ:

The respective minimal feasible quantifiable and detectable concentrations in the present method are 0.056583 and 0.018672 µg/ml for Bromhexine whereas 0.1594 and 0.052602 µg/ml for Cephalexin with the tolerable standards of 10 and 3 for S/N ratio. These shorter values of LOD and LOQ imply the applicability of the present method over an extensive concentration range. LOD values are better than those of Band et al¹⁴(0.055 μg/ml and 4.370 µg/ml) and Sanjeevarani et al¹³ (2018) (0.055 μg/ml and 4.370 μg/ml).

Similarly, better LOQ values are observed in the present case than those of Band et al¹⁴(0.167 µg/ml and 13.242 µg/ml) and Sanjeevarani et al¹³ (2018) (0.167 μg/ml and 13.242 μg/ml).

Pharmaceutical formulation analysis:

The combination of these two drugs is available in the market in tablet (brand name Brocif) and capsule (brand names – Mucokef and Resporidex) formulations. In view of the good recovery values of both the drugs – Bromhexine and Cephalexinin the present proposed method, the amounts of these two drugs in the tablet formulation (brand name – Brocif) were determined using this method. Hence, for the determination of Bromhexine and Cephalexin in tablet formulations as per the prevailing guidelines of ICH¹⁹, the present method is employed (Table - 8).In view of the affordability in terms of cost, apart from the spectrophotometric methods^20-21, HPLC methods are used in quality control laboratories of developing countries. Hence, the present method is a significant study.

CONCLUSIONS:

A green analytical RP-HPLC method was developed for simultaneous estimation of Bromhexine and Cephalexinin which a relatively eco-friendly mobile phase (methanol) was used by eliminating the usage of environmentally challenging organic solvents like acetonitrile. A good separation of chromatographic peaks with a high resolution (7.15) is obtained in the present case. The method was validated as per the current ICH guides (Q2R1).

CONFLICT OF INTEREST:

Nil.

REFERENCES:

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Received on 01.12.2023 Revised on 18.06.2024

Accepted on 20.09.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5843-5850.

DOI: 10.52711/0974-360X.2024.00887

System Suitability Parameter	Method Precision		Intermediate Precision
System Suitability Parameter	Bromhexine	Cephalexin	Bromhexine	Cephalexin
USP resolution	------	7.3866	--------	7.51
USP tailing factor	0.955	1.0933	0.9433	1.19
USP plate count	4422	7579	4310	7709
Retention time (min)	3.725	6.227	3.717	6.228
Peak area	74441.6	261486.8	74330.8	261453.5
SD of area	348.91	1698.95	432.50	1983.86
% RSD of area	0.469	0.650	0.582	0.75877981

S No	Drug	Brand Name	Form	Dosage	Amount Prepared	Amount Recovered	% Assay
1	Bromhexine	Brocif	Tablet	4mg	1.2 µg/ml	1.18	98.51
2	Cephalexin	Brocif		250mg	75 µg/ml	74.04	98.72