Int J App Pharm, Vol 16, Issue 1, 2024, 306-310Original Article

QUANTITATIVE ANALYSIS OF DEXTROMETHORPHAN-HBr, GUAIFENESIN, AND DIPHENHYDRAMINE-HCl IN TABLET DOSAGE FORM BY RATIO DIFFERENCE SPECTROPHOTOMETRY METHOD

RIDA EVALINA TARIGAN1, MUHAMMAD ANDRY1, ANNISA TIFANY ZULMI MARPAUNG1, MUHAMMAD AMIN NASUTION2, MUHAMMAD FAUZAN LUBIS3*

1Departement of Pharmaceutical Chemistry, Faculty of Pharmacy and Health, Institut Kesehatan Helvetia, Medan, Sumatera Utara-20124, Indonesia. 2Faculty of Pharmacy, Universitas Muslim Nusantara Al Washliyah, Medan, Sumatera Utara-20147, Indonesia. 3Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia
*Corresponding author: Muhammad Fauzan Lubis; *Email: fauzan.lubis@usu.ac.id

Received: 24 Aug 2023, Revised and Accepted: 23 Oct 2023

ABSTRACT

Objective: This study aims to develop a spectrophotometric method with the Ratio Difference method using ethanol pro analysis solvent to obtain the results of Dextromethorphan Hydrobromide (HBr) levels of Guaifenesin and Diphenhydramine Hydrochloride (HCl) in tablets.

Methods: The Ratio Difference Sprctrophotography method involves dividing the mixture spectrum by the standard spectrum of each analyte and reducing the ratio to obtain a spectrum that does not depend on the concentration of the analyte used as a divider and can directly determine the levels of Dextromethorphan HBr, Guaifenesin, and Diphenhydramine HCl in the range 200-400 nm wavelength using experimentally calculated absorbance.

Results: The maximum wavelengths of Dextromethorphan HBr, Guaifenesin, and Diphenhydramine HCl were obtained at 278 nm, 273 nm, and 252 nm, respectively. The average % accuracy obtained was 99.60% for Dextromethorphan HBr, 98.98% for Guaifenesin, and 100.32% for Diphenhydramine HCl in dosage forms.

Conclusion: This method was successfully applied with simultaneous estimation to determine Dextromethorphan HBr, Guaifenesin, and Diphenhydramine HCl levels in tablet preparations and met the validation requirements.

Keywords: Dextromethorphan HBr, Guaifenesin, Diphenhydramine HCl, Spectrophotometry, Ratio difference

© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2024v16i1.49218 Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

Cough medicine preparations are already widely available in the market in various variations by combining or combining two or more active substances in one preparation, one of which is by combining Dextromethorphan HBr (DEX), Guaifenesin (GUA), and Diphenhydramine HCl (DIF). Combining two or more active ingredients means the drug can be more effective in achieving therapeutic targets [1–3].

Coughing is a physiological reflex that occurs as lung protection from mechanical trauma or a natural defense process to protect the respiratory tract by preventing foreign bodies from entering the respiratory tract. One of the drugs that can treat cough is Gratusif Tablet from Graha Farma, which is used as a cough medicine and contains three components, Dextromethorphan HBr 15 mg, Guaifenesin 100 mg, and Diphenhydramine HCl 5 mg [4, 5].

Dextromethorphan hydrobromide (DEX) is an antitussive agent commonly used in cough and cold medicines. DEX is chemically known as morphinean 3-methoxy-17-methyl-(9α,13α,14α) hydrobromide monohydrate, combined with another ingredient, Guaifenesin (GUA) 3-(2-methoxyphenoxy)propane-1, 2-diol is a class of drugs called as expectorants and is mainly used to split up congestion and reduce mucus thickness and Diphenhydramine HCl (DIF), namely as a decongestant or as an anti-allergic, the combination of these three drugs is very effective for the treatment of cough [6–9].

Examination of active substance levels is a requirement that must be fulfilled in the process of making drugs and guaranteeing the quality of drug preparations. Excellent and appropriate drug preparations and ingredients will support the achievement of the expected drug therapeutic effect so that the drug is safe to use [10, 11]. The requirements that must be met for the quality of the ingredients of the drug are by the Indonesian Pharmacopoeia Edition VI of 2020, the requirements for the content of cough medicine tablets containing three-drug combinations, namely dextromethorphan HBr 98.0%, diphenhydramine HCI not more than 102.0%, for guaifenesin tablets not less than 98.0% and not more than 102.0% [12].

In previous studies, several techniques for determining drug levels could be carried out using LC-MS/MS [13, 14], UV spectrophotometry [15, 16], gas chromatography [17, 18], HPLC [19, 20], HPTLC [21, 22], Infrared Infrared Infrared [23, 24], potentiometric and voltammetry [25, 26], can also be carried out with a simultaneous UV spectrophotometer for analysis in single or combined preparations. However, no studies have used a combination of 3 drugs, DEX, GUA, and DIF, using the Ratio Difference (RD) method. 

RD method is used to analyze the difference in amplitude between two points on the spectrum, the ratio of the mixture is directly proportional to the concentration of the desired component, and the independence of the interfering components is the basic principle of the difference ratio method. This method can be chosen if a part of the extended spectrum has two wavelengths, which will be reduced by the amplitude of one of the analytes [27, 28]. Based on this explanation, the levels of the three-drug combinations DEX, GUA, and DIF in tablets can be determined using the RD spectrophotometry method.

MATERIALS AND METHODS

Materials

DEX, GUA and DIF raw materials were obtained from the Indonesian Food and Drug Supervisory Agency. Grantusif® tablets containing 15 mg Dextromethorphan HBr, 100 mg Guaifenesin, and 5 mg Diphenhydramine HCl (produced by Graha Farma, Surakarta, Indonesia) and pro-ethanol analysis (e-Merck).

Instrumentation

UV-Vis 1800 spectrophotometer (Shimadzu) and a set of Personal Computers (PC) equipped with UV-Probe 2.42 software.

Preparation of standard solution

Accurately weighed 50 mg of DEX, GUA and DIF standard was separately transferred into 50 ml volumetric flask and dissolved in ethanol to give solutions containing 1000 µg/ml DEX, GUA and DIF.

Selection of analytical wavelength

The solutions of DEX, GUA and DIF were prepared in diluent by appropriate dilution and spectrum was recorded. The maximum wavelength was selected by measuring at 200 to 400 nm with concentrations of DEX (28; 42; 56; 71; 85 μg/ml), GUA (16; 24; 32; 40; 48 μg/ml) and DIF (7; 10; 13; 16; 19 μg/ml). The ratio difference concentrations is calculated to selected wavelength range analysis.

Assay of tablet formulation by ratio difference spectrophotometry method

The content of 20 tablets was weighed accurately. A powder quantity equivalent to Dextromethorphan HBr 15 mg, Guaifenesin 100 mg and Diphenhydramine HCl 5 mg was accurately weighed and transferred into a volumetric flask of 50 ml capacity; solvent was transferred into this volumetric flask and sonicated for 10 min. The flask was shaken, and the volume was made up to the mark with diluent. Filtered the solution with whatman® filter paper no. 42, discarded the first 10 ml of filtrate. Pipette 0.4 ml of the filtrate into a 25 ml volumetric flask and add solvent to the marked line. The resulting solution was analyzed by the proposed method. The quantitation was carried out by keeping these values to the straight-line calibration curve equation. The absorbance was then measured according to the optimization results procedure using the Ratio Difference method.

Method validation

The methods was validated was validated based on linearity, accuracy, precision, LOD and LOQ referring to International Conference of Harmonization (ICH) guidelines (29–33).

RESULTS AND DISCUSSION

Selection of analytical wavelength

The maximum wavelength is chosen by looking for the assay value with the RD method of each spectrum with different concentrations. Based on fig. 1 and 2, DEX showed a linear response at λmax 278 nm, GUA 273 nm and DIF was measured at 252 nm, as shown in fig. 1 and 2.

Fig. 1: Absorption spectra of DEX (a), GUA (b), and DIF (c) at various concentrations

Based on Figure, DEX, GUA, and DIF were measured at 278 nm, 273 nm, and 252 nm, respectively; absorption spectra of DEX, GUA, and DIF with various concentrations show that these concentrations do not change the solvent; both DEX, GUA, and DIF obtained stable spectra. Ordinary spectrophotometric methods, such as ultraviolet spectrophotometry, cannot be used to determine the levels of DEX, GUA, and DIF in tablet mixtures because their spectra overlap, so the RD method is used.

The Ratio Difference Method is used to determine three compounds in a mixture simultaneously. The amplitude difference between two points on the mixture ratio spectrum is directly proportional to the concentration of the desired component, as in research conducted by Emam et al. 2018 [34]. The distribution concentration used in this study was guaifenesin 32µg/ml. The basis for choosing a division concentration is that there is no difference in the location of the maximum wavelength of a substance in the spectrum; the only difference is the resulting absorption value, and this concentration is the concentration for the maximum wavelength according to research by Mansour, 2018 [35].

Each spectrum is divided by its respective divider. After all the spectra were divided based on their respective spectra, a comparison of the DEX, GUA, and DIF spectra was obtained, which can be seen in fig. 2.

Fig. 2: Overlapping spectrum of RD method of DEX (), GUA (), and DIF ()

Table 1: Validation parameters for DEX, GUA and DIF

No Parameter DEX GUA DIF
1 Linearity 0.9984 0.9982 0.9994
2 Accuracy(%) 99.60% 98.98% 100.32%
3 Precision % 0.02757 0.59600 0.05230
4 LOD (μg/ml) 2.9502 2.5456 2.5178
5 LOQ (μg/ml) 5.3864 4.6475 4.5969

Method validation

The method was validated based on linearity, accuracy, precision, LOD and LOQ. The validation results are shown in table 1.

Table 1 shows that the linearity obtained meets the linearity requirements for method validation because there is a correlation coefficient value of ≤1. The test accuracy is measured in the percentage of the recovery. Since method validation is between 98% and 102%, the percentage recovery obtained is certified as meeting the accuracy standar (36). The precision results obtained have a value of less than 2 percent, which meets the precision requirements for the method Validation [37].

Application of the method in tablet dosage form

The proposed method was applied for the determination of DEX, GUA and DIF in their combined tablet and the results are shown in table 2.

Table 2: DEX, GUA and DIF contents in tablet

Component of drugs

Contents %

Level requirements (%)

DEX

100.4035±0.5104

98-102

GUA

99.2467±0.7499

98-102

DIF

99.3712±0.5225

98-102

Data are given as mean±SEM, n = 3.

The levels of DEX, GUA and DIF obtained can be seen in table 2 above and are still within the range of requirements in the Indonesian Pharmacopeia VI edition. This proves that the levels of DEX, GUA and DIF obtained meet the standard levels of the Indonesian Pharmacopoeia Edition VI (38).

CONCLUSION

The RD spectrophotometric method is a simple, accurate, precise, sensitive and easy-to-apply spectrophotometry method. This method can be applied to the analysis of DEX, GUA and DIF simultaneously in combination with tablet preparations and, meets the validation requirements and can be applied routinely DEX, GUA and DIF analysis.

ACKNOWLEDGEMENT

The authors acknowledge the facilities and scientific and technical support from laboratory at North Sumatra University’s Pharmacy Faculty available for this research and Institut Kesehatan Helvetia who have supported this research in the form of places and tools.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Gamil AM, Hamad MA. Validation of hplc method for simultaneous determination of pseudoephedrine HCL, guaifenesin, chlorpheniramine maleate and dextromethorphan HBR. Univ J Pharm Res. 2020;5(5)448. doi: 10.22270/ujpr.v5i5.488.

  2. Merey HA, Ramadan NK, Diab SS, Moustafa AA. Validated UPLC method for the determination of guaiphenesin, oxeladin citrate, diphenhydramine, and sodium benzoate in their quaternary mixture used in the treatment of cough, in the presence of guaiphenesin-related substance (guaiacol). Chem Pap. 2018;72(9):2247-54. doi: 10.1007/s11696-018-0454-2.

  3. Itagimatha N, Manjunatha DH. RP-HPLC-UV method development and validation for simultaneous determination of terbutaline sulphate, ambroxol HCl and guaifenesin in pure and dosage forms. Ann Pharm Fr. 2019;77(4):295-301. doi: 10.1016/j.pharma.2019.02.004, PMID 31027752.

  4. Wibowo A, Kerja M. Obat anti Batuk mechanism of action of anti-cough medicine. JK Unila. 2021;5(1):75-83.

  5. Imani LN, Lestari K, Mulyaningsih W. Kajian farmasi klinis penggunaan obat batuk ”X” dengan kandungan Bromheksin HCl untuk pengencer dahak pada anak. J Pharm Sci. 2023;6(1):315-21. doi: 10.36490/journal-jps.com.v6i1.69.

  6. Palur K, Archakam SC, Koganti B. Chemometric assisted UV spectrophotometric and RP-HPLC methods for simultaneous determination of paracetamol, diphenhydramine, caffeine and phenylephrine in tablet dosage form. Spectrochim Acta A Mol Biomol Spectrosc. 2020;243:118801. doi: 10.1016/j.saa.2020.118801, PMID 32827914.

  7. Huang SJ, Kannaiyan S, Venkatesh K, Cheemalapati S, Haidyrah AS, Ramaraj SK. Synthesis and fabrication of Ni-SiO2 nanosphere-decorated multilayer graphene nanosheets composite electrode for highly sensitive amperometric determination of guaifenesin drug. Microchem J. 2021;167:106325. doi: 10.1016/j.microc.2021.106325.

  8. Mohamed D, Hegazy MA, El-Sayed GM, Youssef SH. Greenness evaluation of different chromatographic approaches for the determination of dextromethorphan, phenylephrine and brompheniramine in their pharmaceutical formulation. Microchem J. 2020;157:104893. doi: 10.1016/j.microc.2020.104893.

  9. Tarigan RE, Muchlisyam SSM, Sinaga SM, Alfian Z. Development and validation of area under curve spectrophotometry method for ternary mixture of dextromethorphan HBr, doxylamine succinate and pseudoephedrine HCl in tablet dosage form. AIP Conf Proc. 2021;2342(6):6-9. doi: 10.1063/5.0045551.

  10. Benni I, Iga S. Uji Sifat fisik tablet salut enterik kalium diklofenak. J Penelit. Farmaco Indones. 2019;8(1):12-7. doi: 10.51887/jpfi.v8i1.588.

  11. Bachri M, Permata YM, Syahputra H. PLS calculation of FTIR and spectrophotometric methods for determination simultaneous of dextromethorphan HBr and glyceryl guaiacolate in tablet mixture. Rasayan J Chem. 2022;15(1):143-9. doi: 10.31788/RJC.2022.1516510.

  12. Cheng Y, Qin H, Acevedo NC, Jiang X, Shi X. 3D printing of extended-release tablets of theophylline using hydroxypropyl methylcellulose (HPMC) hydrogels. Int J Pharm. 2020;591:119983. doi: 10.1016/j.ijpharm.2020.119983, PMID 33065220.

  13. Chaudhari K, Wang J, Xu Y, Winters A, Wang L, Dong X. Determination of metformin bio-distribution by LC-MS/MS in mice treated with a clinically relevant paradigm. Plos One. 2020;15(6):e0234571. doi: 10.1371/journal.pone.0234571, PMID 32525922.

  14. Zilhadia Z, Supandi S, Alfarisi S. Simultaneous determination of metformin and glimepiride in fixed-dose combination tablets sold in Pramuka market using validated UV spectrophotometric method. J Sains Farm Klin. 2021;8(3):303. doi: 10.25077/jsfk.8.3.303-308.2021.

  15. Abafe OA, Spath J, Fick J, Jansson S, Buckley C, Stark A. LC-MS/MS determination of antiretroviral drugs in influents and effluents from wastewater treatment plants in KwaZulu-Natal, South Africa. Chemosphere. 2018;200:660-70. doi: 10.1016/j.chemosphere.2018.02.105, PMID 29524887.

  16. Tarigan RE, Sarumaha Y. Determination of isoniazid and pyridoxine hydrochloride levels in tablets with ultraviolet spectrophotometry by successive ratio derivative. Int Arch Sci Public Heal. 2022;3(1):95-101.

  17. Wozniak MK, Wiergowski M, Aszyk J, Kubica P, Namiesnik J, Biziuk M. Application of gas-chromatography–tandem mass spectrometry for the determination of amphetamine-type stimulants in blood and urine. J Pharm Biomed Anal. 2018;148:58-64. doi: 10.1016/j.jpba.2017.09.020, PMID 28957720.

  18. Buchalter S, Marginean I, Yohannan J, Lurie IS. Gas chromatography with tandem cold electron ionization mass spectrometric detection and vacuum ultraviolet detection for the comprehensive analysis of fentanyl analogues. J Chromatogr A. 2019;1596:183-93. doi: 10.1016/j.chroma.2019.03.011, PMID 30876740.

  19. Nezhadali A, Shapouri MR, Amoli Diva M, Hooshangi AH, Khodayari F. Method development for simultaneous determination of active ingredients in cough and cold pharmaceuticals by high-performance liquid chromatography. Heliyon. 2019;5(12):e02871. doi: 10.1016/j.heliyon.2019.e02871, PMID 31872108.

  20. Lotfy HM, Ahmed DA, Abdel Rahman MK, Weshahy SAF. Study of efficiency and spectral resolution for mathematical filtration technique using novel unlimited derivative ratio and classical univariate spectrophotometric methods for the multicomponent determination-stability analysis. Heliyon. 2019;5(5):e01669. doi: 10.1016/j.heliyon.2019.e01669, PMID 31193052.

  21. Abdelaleem EA, Abdelwahab NS. Green chromatographic method for analysis of some anti-cough drugs and their toxic impurities with comparison to conventional methods. Saudi Pharm J. 2018;26(8):1185-91. doi: 10.1016/j.jsps.2018.07.007, PMID 30532640.

  22. Alam P, Ezzeldin E, Iqbal M, Mostafa GAE, Anwer MK, Alqarni MH. Determination of delafloxacin in pharmaceutical formulations using a green RP-HPTLC and NP-HPTLC methods: a comparative study. Antibiotics (Basel). 2020;9(6):1-14. doi: 10.3390/antibiotics9060359, PMID 32630451.

  23. Voltammetric SW. Selective I. Anal Square Wave Voltammetric Ion Sel. 2022;14(6):621-37.

  24. Fan Z, Chang Y, Cui C, Sun L, Wang DH, Pan Z. Near-infrared fluorescent peptide nanoparticles for enhancing esophageal cancer therapeutic efficacy. Nat Commun. 2018;9(1):2605. doi: 10.1038/s41467-018-04763-y, PMID 29973582.

  25. Atole DM, Rajput HH. Ultraviolet spectroscopy and its pharmaceutical applications-a brief review. Asian J Pharm Clin Res. 2018;11(2):59-66. doi: 10.22159/ajpcr.2018.v11i2.21361.

  26. Yildiz T, Gu R, Zauscher S, Betancourt T. Doxorubicin-loaded protease-activated near-infrared fluorescent polymeric nanoparticles for imaging and therapy of cancer. Int J Nanomedicine. 2018;13:6961-86. doi: 10.2147/IJN.S174068, PMID 30464453.

  27. Gadhari NS, Gholave JV, Patil SS, Patil VR, Upadhyay SS. Enantioselective high-performance new solid contact ion-selective electrode potentiometric sensor based on sulphated γ-cyclodextrin‑carbon nanofiber composite for determination of multichiral drug moxifloxacin. J Electroanal Chem. 2021;882:114981. doi: 10.1016/j.jelechem.2021.114981.

  28. Proma Mukherjee, Debarupa Dutta Chakraborty, Prithviraj Chakraborty, Bhupendra Shrestha, Bhuyan NR. Different ultraviolet spectroscopic methods: a retrospective study on its application from the viewpoint of analytical chemistry. Asian J Pharm Clin Res 2021;14(9):1-11. doi: 10.22159/ajpcr.2021.v14i9.42172.

  29. Emam AA, Abdelaleem EA, Naguib IA, Abdallah FF, Ali NW. Successive ratio subtraction as a novel manipulation of ratio spectra for the quantitative determination of a mixture of furosemide, spironolactone and canrenone. Spectrochim Acta A Mol Biomol Spectrosc. 2018;192:427-36. doi: 10.1016/j.saa.2017.11.034, PMID 29202387.

  30. Bachri M. Simultaneous assays of metformin HCl and glibenclamide mixture using two analytical methods of spectrophotometry. Int J App Pharm. 2019;11(6):139-46. doi: 10.22159/ijap.2019v11i6.34022.

  31. Tarigan RE, Muchlisyam SSM, Sinaga SM, Alfian Z. Development and validation of area under curve spectrophotometry method for ternary mixture of dextromethorphan HBr, doxylamine succinate and pseudoephedrine HCl in tablet dosage form. AIP Conf Proc. 2021;2342(6):1-4. doi: 10.1063/5.0045551.

  32. Bachri M, Reveny J, Metri Permata YM, Situmorang CEA. Validation of intersection absorption spectrum for simultaneous determination of betamethasone valerate and neomycin sulfate in cream. Rasayan J Chem. 2019;12(1):232-9. doi: 10.31788/RJC.2019.1215013.

  33. Bachri M, Sitohang E. Simultaneous assay of metformin and glibenclamide in combined dosage form by mean centered of spectra ratio methods. Rasayan J Chem. 2019;12(3):1509-17. doi: 10.31788/RJC.2019.1235208.

  34. Mansour FR. A new innovative spectrophotometric method for the simultaneous determination of sofosbuvir and ledipasvir. Spectrochim Acta A Mol Biomol Spectrosc. 2018;188:626-32. doi: 10.1016/j.saa.2017.07.066, PMID 28783604.

  35. Sinaga SM, Pertiwi NN, Muchlisyam. Quantitative analysis of amlodipine besylate and valsartan in tablet dosage form by absorption factor spectrophotometry method. Rasayan J Chem. 2022;15(2):1386-9. doi: 10.31788/RJC.2022.1526611.

  36. Gamil AM, Hamad MA. Validation of hplc method for simultaneous determination of pseudoephedrine HCL, guaifenesin, chlorpheniramine maleate and dextromethorphan HBr. Univ J Pharm Res. 2020;5(5):448. doi: 10.22270/ujpr.v5i5.488.

  37. Anunthawan T, Rimlumduan T. Antioxidant activity, anti-tyrosinase activity, and lipstick pigments of Oryza sativa Linn. and carissa carandas linn. extracts. J Appl Pharm Sci. 2023;13:1-8. doi: 10.7324/JAPS.2023.145623.

  38. Sen AK, Ghodasara S, Sen DB, Maheshwari RA, Zanwar AS, Dumpala RL. Evaluation of remogliflozin, vildagliptin, and metformin in tablet dosage form using modern and cost-effective UV spectroscopic methods. J Appl Pharm Sci. 2023;13(09):121-32. doi: 10.7324/JAPS.2023.136050.