INTRODUCTION:

Analytical chemistry plays a vital role in maintaining the quality of drugs. It consists of Qualitative and Quantitative estimations¹. To develop a new HPLC method for any drug, knowledge of its molecular weight, polarity, ionic character, pK_a values, wavelength of absorption, purity of compound and the solubility should be known. Method development involves considerable effort and time.²

Acute respiratory infections (ARI) may cause inflammation of the respiratory tract anywhere from nose to alveoli, with a wide range of combination of symptoms and signs. ARI is often classified by clinical syndromes depending on the site of infection and is referred to as ARI of upper (AURI) or lower (ALRI) respiratory tract.³ Upper respiratory tract comprises of the airways from nostrils to the vocal cords in the larynx, plus the paranasal sinuses and the middle ear. Upper respiratory tract infection (URTI) includes the common cold, laryngitis, pharyngitis/tonsillitis, acute rhinitis and acute otitis media. The lower respiratory tract includes the furtherance of the airways from the trachea and bronchi to the bronchioles and the alveoli. Lower respiratory tract infection (LRTI) includes acute bronchitis, bronchiolitis and pneumonia⁴.

WHO reported more than four million deaths a year from acute respiratory infections in the developing world quarters. Mortality may be greater in developing countries because of low resistance of children due to malnutrition, overcrowding and poor environmental circumstances such as indoor air pollution⁵. Respiratory problems are responsible for a large proportion of pediatric admissions and outpatient attendance. Children are an embodiment of our dreams and hopes of the future.⁶

Roxithromycin macrolide category wide spectrum antibacterial drug that inhibits bacterial protein biosynthesis by binding reversibly to the subunit 50S of the bacterial ribosome, thereby inhibiting translocation of peptidyl-Trna⁷. It acts on gram-positive and gram-negative bacteria. It is used to treat respiratory tract, urinary and soft tissue infections. ROX is derived from erythromycin, containing the same 14-membered lactone ring⁸.

ROX is erythromycin 9-[O-[(2)-methoxyethoxy) methyl] oxime, a semi synthetic macrolide antibiotic drug, very slightly soluble in water and aqueous fluids and its absorption is dissolution rate limited. ROX is used in the treatment of UTI, RTI, ENT, genital tract, skin and soft tissue infections⁹.

Fig 1: Chemical Structure of Roxithromycin¹⁰

Ambroxol is an expectorant useful in the treatment of bronchial asthma and chronic bronchitis. Moreover, it exhibits antioxidant and anti-inflammatory properties.¹¹

Ambroxol hydrochloride is metabolite of bromhexine and it is administered as hydrochloride salt. It is used in a variety of disorders including chronic bronchitis, cystic fibrosis and infant’s respiratory disorder syndrome. Chemically it is trans-4-{(2-amino-3, 5-dibromobenzyl) amino} cyclohexanol hydrochloride. Literature survey reveals HPLC, RP-HPLC and UV-spectrophotometric method for determination of ambroxol hydrochloride in tablets¹².

It is an expectoration improver and a mucolytic agent used in the treatment of acute and chronic disorders characterized by the production of excess or thick mucous. It has been successfully used for decades in the form of its hydrochloride as a secretion-releasing expectorant in a variety of respiratory disorders¹³.

It stimulates the transportation of the viscous secretions in the respiratory organs and reduces the stand stillness of the secretions. It is official in IP1. Few methods have been reported in the literature for the determination of AMBRO individually or in combination with other drugs¹⁴.

Fig 2: Chemical Structure of Ambroxol¹⁵

MATERIALS AND METHODS:

Roxithromycin (Pure) from Sura labs, Ambroxol (Pure) from Sura labs, Water and Methanol for HPLC from LICHROSOLV (MERCK), Acetonitrile for HPLC from Merck.

HPLC METHOD DEVELOPMENT:

TRAILS:

Preparation of standard solution:

Accurately weigh and transfer 10mg of Roxithromycin and Ambroxol working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

Pipette out 0.3ml of Roxithromycin and 0.75ml of Ambroxol stock solutions was take in a 10ml of volumetric flask dilute up to the mark with diluent.

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Orthophosphoric acid and Phosphoric acid (pH 3): Acetonitrile and Methanol: ACN with varying proportions. Finally, the mobile phase was optimized to Buffer: Methanol: ACN in proportion 65:25:10v/v respectively.

Optimization of Column:

The method was performed with various C18 columns like ODS and Zodiac column. Altima C18 (4.6×150mm, 5µ) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

Validation methods procedures followed as per ICH guidelines.

RESULTS AND DISCUSSION:

Optimized Chromatogram (Standard):

Mobile phase : Buffer: Methanol: ACN (65:25:10v/v)

Column : Altima C18 (4.6×150mm, 5.0 µm)

Flow rate : 1ml/min

Wavelength : 215nm

Column temp : 38ºC

Injection Volume : 10µl

Run time : 5 minutes

Fig 3: Optimized Chromatogram

Table 1: - peak results for optimised

S. No	Peak name	R_t	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Ambroxol	2.488	1308595	247456		1.2	5835.5
2	Roxithromycin	4.866	124505	19187	6.0	1.1	5745.2

Optimized Chromatogram (Sample)

Figure 4: Optimized Chromatogram (Sample)

Table 2: Optimized Chromatogram (Sample)

S. No	Peak name	R_t	Area	Height	USP Resolution	USP Tailing	USP plate count
1	Ambroxol	2.487	1307139	246586		1.2	5565.5
2	Roxithromycin	4.865	124452	19117	6.0	1.1	5355.2

Assay (Standard):

Table 3: Peak results for assay standard

S. No.	Name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count	Injection
1	Ambroxol	2.488	1308945	247282		1.3	5568.0	1
2	Roxithromycin	4.838	124336	19189	6.0	1.2	5359.2	1
3	Ambroxol	2.492	1309481	247456		1.0	5565.5	2
4	Roxithromycin	4.846	124505	19187	6.0	1.3	5355.2	2
5	Ambroxol	2.493	1317926	247578		1.0	5545.5	3
6	Roxithromycin	4.844	124903	19210	6.0	1.3	5352.1	3

Assay (Sample):

Table 4: Peak results for Assay sample

S. no.	Name	Rt	Area	Height	USP Resolution	USP Tailing	USP plate count	Injection
1	Ambroxol	2.494	1307139	246586		1.3	6568.0	1
2	Roxithromycin	4.840	124452	19117	6.0	1.1	5359.2	1
3	Ambroxol	2.491	1308903	248422		1.3	5565.5	2
4	Roxithromycin	4.842	124632	19178	6.0	1.2	5355.2	2
5	Ambroxol	2.491	1325993	248924		1.3	5391.1	3
6	Roxithromycin	4.834	126697	19237	6.0	1.2	5564.0	3

%ASSAY =

Sample area Weight of standard Dilution of sample Purity Weight of tablet

___________ × ________________ × _______________×_______×______________×100

Standard area Dilution of standard Weight of sample 100 Label claim

The % purity of Ambroxol and Roxithromycin in pharmaceutical dosage form was found to be 100.3%.

LINEARITY:

Chromatographic Data for Linearity Study:

Ambroxol:

Concentration Level (%)	Concentration mg/ml	Average Peak Area
33.3	2.5	47510
66.6	5	85701
100	7.5	124802
133.3	10	162731
166.6	12.5	199732

Figure 5: calibration graph for Ambroxol

Roxithromycin:

Concentration Level (%)	Concentration mg/ml	Average Peak Area
33	10	518934
66	20	956781
100	30	1413873
133	40	1863458
166	50	2267084

Figure 6: calibration graph for Roxithromycin

ACCURACY:

Table 6: The accuracy results for Ambroxol

%Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	716072.7	15	14. 9	99.3	99.6%
100%	1404258	30	30.1	100.3
150%	2064609	45	44.7	99.3

Table 7: The accuracy results for Roxithromycin

%Concentration (at specification Level)	Area	Amount Added (ppm)	Amount Found (ppm)	% Recovery	Mean Recovery
50%	63467	3.75	3.72	100.8	100.4%
100%	124353.3	7.5	7.57	100. 9
150%	178607.7	11.25	11.20	99.5

Limit of Detection:

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

LOD= 3.3 × σ / s

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

RESULT:

Ambroxol:

=3.3 × 16724.53/45217

=1.2µg/ml

Roxithromycin:

=3.3 × 662.3965/15811

=0.13µg/ml

Limit of Quantitation

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined.

LOQ=10×σ/S

Where

σ = Standard deviation of the response

S = Slope of the calibration curve

RESULT:

Ambroxol:

=10×16724.53/45217

= 3.6 µg/ml

Roxithromycin:

=10 × 662.3965/15811

= 0.41 µg/ml

Robustness:

Table 8: Results for Robustness

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Actual Flow rate of 1.0 mL/min	1308495	2.344	5568.2	1.3
Less Flow rate of 0.9 mL/min	1300148	2. 244	5922.2	1.2
More Flow rate of 1.1 mL/min	1306476	2.243	5868.8	1.2
Less organic phase	1304520	2.345	5836.2	1.2
More organic phase	1207845	2.344	5282.6	1.1

Ambroxol:

Table 9: Results for Robustness

Roxithromycin:

Parameter used for sample analysis	Peak Area	Retention Time	Theoretical plates	Tailing factor
Actual Flow rate of 1.0 mL/min	124505	3.286	6098.1	1.2
Less Flow rate of 0.9 mL/min	156550	3.181	5999.1	1.2
More Flow rate of 1.1 mL/min	122702	3.181	5989.2	1.1
Less organic phase	122626	3.278	6387.2	1.1
More organic phase	1207845	3.015	4417	1.1

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Roxithromycin and Ambroxol in bulk drug and pharmaceutical dosage forms.

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

Roxithromycin and Ambroxol was freely soluble in ethanol, methanol and sparingly soluble in water.

Buffer: Methanol: ACN (65:25:10v/v) was chosen as the mobile phase. The solvent system used in this method was economical.

The %RSD values were within 2 and the method was found to be precise.

The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

This method can be used for the routine determination of Roxithromycin and Ambroxol in bulk drug and in Pharmaceutical dosage forms.

ACKNOWLEDGEMENT:

Thе Authors arе thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

REFERENCES:

1. R Anusha Naik, Ajay Kumar D., M. Venkatesh. Method Development and Validation of Taurine and Acetyl Cysteine by Using RP -HPLC Method. Asian J. Res. Pharm. Sci. 2018; 8(4):223-235. doi: 10.5958/2231-5659.2018.00038.3.

2. Govinda Rao Kamala, Sowjanya Vadrevu, Malipeddi Haripriya. Method Development and Validation for Simultaneous Estimation of Omeprazole and Domperidone by RP-HPLC. Asian J. Pharm. Ana. 5(4): 2015; 195-205. doi: 10.5958/2231-5675.2015.00031.9

3. Mohd. Yaqub Khan, Maryada Roy, Mohammad Ahmad, Mohammad Imtiyaz Ahmad, Irfan Aziz. A Review- Acute Respiratory Infections- the Forgotten Killer of Children. Asian J. Res. Pharm. Sci. 5(1): Jan.-March 2015; Page 49-54. doi: 10.5958/2231-5659.2015.00008.9

4. Anaghya V Naik, Hebsiba Joseph, Nidhina Mathew, Sumanth Shetty, Rajesh K S, Bharath Raj KC, Juno J Joel1, Gururaj MP, Nandakumar UP. Drug Prescription Pattern in Paediatric Respiratory Tract Infections. Research J. Pharm. and Tech. 2019; 12(3): 1280-1284. doi: 10.5958/0974-360X.2019.00214.2

5. Kaushal Patidar. A Study to Assess the Effectiveness of Self Instructional Module on Knowledge Regarding Prevention of Acute Respiratory Tract Infection Among the Mother of Under Five Year Children in Bhandu Village, Mehsana District.. Asian J. Nursing Education and Research. 2018; 8(1): 05-06. doi: 10.5958/2349-2996.2018.00002.2

6. Nomitha Baby, Chandrashekar M, Sheela Williams. A Comparative Study to Assess the Effect on Respiratory Status of Under Five Children with Lower Respiratory Tract Infection in Prone and Lateral Position in Selected Hospitals at Mysore. Asian J. Nur. Edu. and Research 5(4): Oct.- Dec.2015; Page 537-541. doi: 10.5958/2349-2996.2015.00110.X

7. Surbhi Choursiya, Arpana Indurkhya. Development and Evaluation of Hard Candy Lozenges Containing Roxithromycin for Treatment of Oral Infection. Asian J. Pharm. Ana. 2020; 10(3):150-154. doi: 10.5958/2231-5675.2020.00027.7

8. Shilpa Jagtap, Smrutidevi Sonavane, Shrikrishna Baokar, Vinod Pawar, Patil R. N. In Vitro New Dissolution Method for the Evaluation of Roxithromycin using pH 6.0 Phosphate Buffer and Determination of its Content by Validated UV Spectrophotometric Method. Research J. Pharma. Dosage Forms and Tech. 2013; 5(5): 277-281.

9. K Ravi Sankar, NL Prasanthi, SS Manikiran, N Rama Rao. Effect of Solid Dispersion Technique on Improving the Solubility of Roxithromycin. Research J. Pharma. Dosage Forms and Tech. 2010; 2(2):184-188.

10. https://go.drugbank.com/structures/DB00778/.

11. Hisham A. Abbas. Modulation of antibiotic activity against Pseudomonas aeruginosa by N-acetylcysteine, ambroxol and ascorbic acid. Asian J. Res. Pharm. Sci. 2(4): Oct.-Dec. 2012; Page 123-128

12. Potdar VH, Kuchekar BS, Patil SS. Selective Visible Spectrophotometric Estimation of Ambroxol Hydrochloride in Presence of Cetirizine Hydrochloride in Tablets. Asian J. Research Chem. 2(3): July-Sept. 2009 page 278-279.

13. Chhotaram Seervi, Kundan Pawar, PN Dhabale, ID Gonjari, Chandrakant Raut, Deepali Gharge. Development and Validation of UV Spectrophotometric Method of Ambroxol Hydrochloride in Bulk and Pharmaceutical Formulation. Asian J. Research Chem. 2(4): Oct.-Dec. 2009 page 547-549.

14. Sohan S. Chitlange, Sneha R. Tawargeri, Kaushalendra K. Chaturvedi. Simultaneous Determination of Amoxicillin Trihydrate and Ambroxol Hydrochloride in Solid Dosage Form By Spectrophotometric and Stability Indicating RP-HPLC Method. Asian J. Research Chem. 4(6): June, 2011; Page 1025-1030.

15. https://go.drugbank.com/structures/DB06742/

Received on 23.10.2022 Modified on 26.11.2022

Research J. Science and Tech. 2023; 15(1):1-7.

DOI: 10.52711/2349-2988.2023.00001