Research Article

Content Detection of Acetylcysteine Mixed with Combivent® (Containing Salbutamol and Ipratropium Bromide) by Hplc

Rui Zhang^1,2, Haihua Guo¹, Liangjun Deng¹, Sha Li¹ and Wen Tan^2,3*

¹Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, China

²Post-Doctoral Innovation Site, Jinan University Affiliation, China

³Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, Malaysia

Submission: February 4, 2022; Published: February 15, 2022

*Corresponding author: Rui Zhang, Institute of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, China

How to cite this article: Rui Zhang, Haihua Guo, Liangjun Deng, Sha Li and Wen Tan. Content Detection of Acetylcysteine Mixed with Combivent® (Containing Salbutamol and Ipratropium Bromide) by Hplc. Glob J Oto, 2022; 25 (1): 556153. DOI: 10.19080/GJO.2022.25.556153

Abstract

Clinically, Combivent® (Compound ipratropium bromide solution which contains salbutamol and ipratropium bromide) and acetylcysteine inhalant are often mixed and administered at the same time, so as to achieve the effects of antiasthmatic and expectorant. After mixing, its content and inhalation performance shall be investigated by High Performance Liquid Chromatography (HPLC). The specificity, linearity, recovery, precision and stability of salbutamol and ipratropium bromide and acetylcysteine were tested to prove the reliability of developed HPLC method. The developed HPLC method had high specificity, with linear R2≥0.999, recovery RSD, precision RSD and stability RSD less than 2.0% at 8 time points. The HPLC methodology developed in this study can be used for the determination of salbutamol and ipratropium bromide mixed with acetylcysteine. To provide reference for the determination of its content after mixing and provide data support for its clinical medication.

Keywords: Inhalant; Salbutamol; Ipratropium Bromide; Acetylcysteine; HPLC

Introduction

Inhalant refers to the preparation that delivers drugs to the affected area through gas or aerosol for treatment [1-3]. Due to its characteristics, it’s often used in the treatment of respiratory diseases. Inhalant can provide high local concentration in the respiratory system. Moreover, it can deliver the drug into the microcirculation through large surface areas such as throat mucosa and alveoli, so that the bioavailability of the drug is higher, and there is no first-pass effect [4]. At the same time, taking advantage of the characteristic that the absorption site of the inhalant is in the throat mucosa and alveolar, the inhalant has a faster onset time than the oral preparation in the treatment of respiratory diseases such as respiratory infection or asthma [5]. Compound ipratropium bromide solution is a compound preparation composed of salbutamol and ipratropium bromide. Salbutamol is a short acting β2 adrenoceptor agonist can inhibit the release of histamine and alleviate bronchospasm. Ipratropium bromide is smooth muscle M receptor blocker, which can reduce bronchoconstriction and dilate bronchus. Studies have shown that the anti-asthma effect of double target is more effective [6,7]. Acetylcysteine is often used as an expectorant. It reduces the viscosity of sputum through sulfhydryl group and facilitates sputum excretion. It has been reported that inhaled acetylcysteine is used as an adjuvant for resolving phlegm in patients with moderate and severe COPD [8]. Clinically, to alleviate spasm symptoms and reduce sputum, compound ipratropium bromide solution and acetylcysteine are commonly mixed in a nebulizer for atomization [9-12]. However, after the two drugs are mixed, their content changes will affect the drug inhalation effect [13-15]. Therefore, this study developed a HPLC method for the determination of salbutamol and ipratropium bromide mixed with acetylcysteine, which provides a methodological basis for the determination of the content of the two inhalants after mixing and provides a reference for their clinical use.

Materials

Methanol (Thermo Fisher Scientific Co., Ltd., lot No.: 203195, purity 99.9%, chromatographic grade); Acetonitrile (Thermo Fisher Scientific Co., Ltd., lot No.: 197164, purity 99.95%, chromatographic grade); Potassium dihydrogen phosphate (Shanghai Aladdin Biochemical Technology Co., Ltd., lot No.: b1912076, purity 99.5%, chromatographic grade); Ipratropium bromide (National Institute of Control of Pharmaceutical and Biological Products, lot No: 100522-200601, content 100%); Salbutamol sulfate (National Institute of Control of Pharmaceutical and Biological Products, lot No.: 100328-200703, content 99.3%); Budesonide (National Institute of Control of Pharmaceutical and Biological Products, lot No.: 100989-201502, content 98.9%); Beclomethasone propionate (National Institute of Control of Pharmaceutical and Biological Products, lot No.: 10019- 201504, content 99.0%); N-acetyl-L-cysteine (Shanghai Yuanye Biotechnology Co., Ltd., lot No.: l22j8x40528, content: 99%).

Method

HPLC conditions

HPLC: waters e2695, Chromatographic column: Agilent TCC18( 150 × 4.6 mm, 5mm), wavelength: 210 nm, Flow rate: 1.0 ml/ min, Injection volume: 20 μl, Column temperature: 25oc, Mobile phase: take potassium dihydrogen phosphate solution (2.50 g of potassium dihydrogen phosphate was dissolved in 800 ml water, adjust the pH value to 3.20 ± 0.05 with phosphoric acid solution and made to a constant volume of 1 L) as mobile phase A and acetonitrile as mobile phase B. The gradient conditions are shown in (Table 1.1).

Solution preparation

10 mg of ipratropium bromide was weighed accurately into a 10 ml volumetric flask and diluted with 50% methanol (methanol: water=1:1) to 10 ml, shake well, made a solution containing 1 mg of ipratropium hydrobromide in each ml of solvent as the stock solution; Salbutamol and N-acetyl-L-cysteine were prepared as the stock solution by the same method.

Method validation results

Specificity

Took N-acetyl-L-cysteine (NAC, 200 μg/ml), salbutamol (SAL, 10μg/ml), ipratropium bromide (IPRA, 10 μg/ml) and blank solvent each 20 μl for HPLC analysis. Record the chromatogram (Figure 1). The results show that the above components are completely separated and do not affect each other’s detection. The retention times were 2.668, 4.951 and 11.739 respectively.

Linear range

Take each sample stock solution, IPRA, Sal and NAC mixed with methanol: potassium dihydrogen phosphate buffer (1:1) and diluted to contain IPRA, Sal: 40, 30, 25, 20, 15, 10, 5, 1 and 0.5 μg/ ml，NAC: 800, 600, 500, 400, 300, 200, 100, 20, 10 μg/ml mixed solutions of different concentrations. 20 μl at each concentration level was took for HPLC analysis. Taking the Concentration(C) as the Abscissa (x) and the corresponding peak Area(A) as the Ordinate (y), the concentration peak area linear regression equations of the three samples were obtained. The results show that the peak area of each sample has a good linear relationship with the concentration in the above concentration range (Table 2). 4.3 recovery. Take each sample stock solution, IPRA, Sal and NAC mixed with methanol: potassium dihydrogen phosphate buffer (1:1) and diluted to contain IPRA and sal: 16.0 μg/ml (80%) 20.0 μg/ ml (100%) and 24.0 μg/ml (120%) NAC: 320.0 μg/ml (80%) 、400.0 μg/ml(100%) and 480.0 μg/ml(120%) of mixed solutions of different concentrations, 20 μl at each concentration level was took for HPLC analysis. The concentration of each sample was calculated by linear equation, and the recovery was calculated by the ratio of theoretical amount to the actual amount (Table 3-5). The RSD of the three concentration levels is less than 2%, indicating that the method has good accuracy. 4.4 precision Take each sample stock solution, IPRA, Sal and NAC mixed with methanol: potassium dihydrogen phosphate buffer (1:1) and diluted to contain IPRA and sal: 10.0 μg/ml, NAC: 200.0 μg/ml mixed solution. 20 μl of the mixed solution was took for HPLC analysis, repeated injection 6 times, and calculated the RSD of peak area (Table 6). According to the data in the table, the RSD of each sample is less than 2.0%, indicating that the precision of this method makes the grade

Stability

Take each sample stock solution, IPRA, Sal and NAC mixed with methanol: potassium dihydrogen phosphate buffer (1:1) and diluted to contain IPRA and sal: 10.0 μg/ml, NAC: 100.0 μg/ ml mixed solution. 20 μl of the mixed solution was took for HPLC analysis, and detected again at 2, 4, 6, 8, 10, 12 and 14 h after the first injection, and calculate the RSD of the peak area. Within 14 h, the RSD of each sample was less than 2.0% (Table 7). It shows that this method has good stability.

Conclusion

Through the HPLC methodology test, it is verified that the specificity, linearity, recovery, precision, and stability of the liquid phase method meet the requirements [1] and can be used for the content determination of compound ipratropium bromide mixed with acetylcysteine. At the same time, this study also provides data reference for the clinical use of the above two drugs.

Disclaimer

Any views expressed in this paper are those of the authors and do not reflect the official policy or position of the Department of Defense.

Acknowledgments

We thank the Boehringer Ingelheim company for providing the medicine in this study.

References

1. (2020) National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China. China Medical Science and Technology Press.
2. Hickey AJ (2013) Back to the future: inhaled drug products. Journal of pharmaceutical sciences 102(4): 1165-1172.
3. Zhang X, Cui Y, Liang R (2020) Novel approach for real-time monitoring of carrier based DPIs delivery process via pulmonary route based on modular modified Sympatec HELOS. Acta Pharmaceutica Sinica B 10(7): 1331-1346.
4. Telko M J, Hickey A J (2005) Dry powder inhaler formulation. Respiratory care 50(9): 1209-1227.
5. Zhang Rui, Zhou Haoluan, Zhang Xuejuan (2020) Preparation and formulation optimization of R-bambuterol inhalation solution. Journal of Guangdong Pharmaceutical University 36(3): 322-328.
6. Wan Hualin, Zhou Yuzhen, Dai Xinjian (2015) Efficacy and safety of doxofylline combined with combivent against acute exacerbation of bronchial asthma in 60 patients. Chinese Hospital Pharmacy Journal 35(16): 1485-1487.
7. Dalby R, Suman J (2003) Inhalation therapy: technological milestones in asthma treatment. Advanced drug delivery reviews 55(7): 779-791.
8. Van Overveld F J, Demkow U, Gorecka D (2005) New developments in the treatment of COPD: comparing the effects of inhaled corticosteroids and N-acetylcysteine. Journal of physiology and pharmacology 56: 135.
9. Sanguinetti C M (2015) N-acetylcysteine in COPD: why, how, and when. Multidisciplinary respiratory medicine 11(1): 1-11.
10. Kamin W, Schwabe A, Krämer I (2006) Inhalation solutions which one are allowed to be mixed? Physico-chemical compatibility of drug solutions in nebulizers. Journal of cystic fibrosis 5(4): 205-213.
11. Bonasia P, Cook C, Cheng Y (2007) Compatibility of arformoterol tartrate inhalation solution with three nebulized drugs. Current medical research and opinion 23(10): 2477-2483.
12. Gordon A, Young M, Bihler E (2021) COPD Maintenance Pharmacotherapy. Critical Care Nursing Quarterly 44(1): 19-25.
13. Chen Y, Du S, Zhang Z (2020) Compatible Stability and Aerosol Characteristics of Atrovent^®(Ipratropium Bromide) Mixed with Salbutamol Sulfate, Terbutaline Sulfate, Budesonide, and Acetylcysteine. Pharmaceutics 12(8): 776.
14. Bonasia P J, McVicar W K, Williams B (2008) Chemical and physical compatibility of levalbuterol inhalation solution concentrate mixed with budesonide, ipratropium bromide, cromolyn sodium, or acetylcysteine sodium. Respiratory care 53(12): 1716-1722.
15. Zhang R, Hu J, Deng L (2020) Aerosol Characteristics and Physico-Chemical Compatibility of Combivent^®(Containing Salbutamol and Ipratropium Bromide) Mixed with Three Other Inhalants: Budesonide, Beclomethasone or N-Acetylcysteine. Pharmaceutics 12(1): 78.