204 © 2025 The Author(s). Published by College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License Spectrophotometric Investigations for Simultaneous Analysis of Certain Antibacterial: A Brief Review Farah Nouri Jasim1 and Nahla A. Alassaf 2* 1,2Department of Chemistry, College of Education for Pure Science (Ibn Al-Haitham), University of Baghdad, Baghdad, Iraq. *Corresponding Author. Received: 5 April 2023 Accepted: 25 July 2023 Published: 20 April 2025 doi.org/10.30526/38.2.3385 Abstract Antibacterial substances belong to a group of compounds that attack dangerous microorganisms. Therefore, killing bacteria or reducing their metabolic activity will lessen their adverse effects on a biological system. They originated from either synthetic materials, microbes, or mold. Many of these medications treat the gram-negative bacteria from the critical precedence group, such as pseudomonas, carbapenem-resistant acinetobacter, and enterobacterales. This study aims to investigate the simultaneous analysis of specific antibacterial spectrophotometrically. The WHO maintains this list of priority infections with antibiotic resistance. Drug combinations in single dosage forms are becoming increasingly popular in the pharmaceutical industry. This has created a significant issue for pharmaceutical administrators, particularly in combating fake medications and pharmaceutical analysts and developing reliable and accurate methodologies with minimal overlapping effects on quantification. The basics of several spectrophotometric methods utilized to conduct multi- component analysis are collected in the current work, and the validation criteria that are an essential component of the approaches are also described. Numerous analytical techniques, including high-performance liquid chromatography, electrochemical methods, flow injection techniques, gas chromatography, spectrofluorometric techniques, capillary electrophoresis, and spectrophotometric techniques, have been reported to determine antibacterials. This study conducts a concise narrative evaluation of the many spectrophotometric methods that have been published for the simultaneous investigation of Levofloxacin, Sulfamethoxazole, Metronidazole, and Rifampicin in their pure forms, pharmaceutical dosage forms, and biological samples due to their sensitivity, simplicity, and cost-effectiveness. Keywords: Antibacterial agents, Levofloxacin, Metronidazole, Rifampicin, Spectrophotometric techniques, Sulfamethoxazole. https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0009-0003-9486-6365 mailto:fnouri244@gmail.com https://orcid.org/0000-0003-2803-8003 mailto:nahla.a.a@ihcoedu.uobaghdad.edu.iq IHJPAS. 2025, 38(2) 205 1. Introduction Streptomycin was discovered in 1943 by Selman A. Waksman and Albert Schatz. Waksman introduced the term "antibiotic," which refers to a substance created by an organism to eradicate or stop the growth of other species (1). Pathogenic bacteria are those that cause bacterial infections and sickness. When they enter the body, start reproducing, displace beneficial bacteria, or develop into typically sterile tissues, they cause illnesses and ailments. Antibacterial medications, considered the most promising chemotherapeutic agents, have been discovered by researchers to treat infectious diseases (2). Although antibacterials are life-saving medications, improper use can result in antibiotic resistance, a significant public health risk. The rise of antibiotic-resistant bacterial infections is one of the major factors contributing to increased morbidity and mortality, especially in the elderly and individuals with medical conditions (3,4). Antibacterials, a subclass of antibiotics, have previously been categorized in several ways; however, to make it more straightforward, antibacterial agents can be divided into five categories (5). Based on their mode of action, antibacterials can usually be divided into bacteriostatic (like Sulfamethoxazole) and bactericidal (i.e., Levofloxacin, Metronidazole, and Rifampicin). While bactericidal antibacterials aim to kill germs by attacking their cell wall or membranes, bacteriostatic antibacterials work to slow or prevent bacterial growth (6). A subclass of antibiotics known as antibacterials can be produced synthetically, chemically altered from naturally existing substances, or spontaneously from fungal sources (7). Classification by activity spectrum is another way of classifying antibiotics or antimicrobials based on target specification. In this category, antibiotics are either narrow-spectrum (Metronidazole) or broad-spectrum (Levofloxacin, Rifampicin, Sulfamethoxazole). Generally, a narrow-spectrum antibiotic is favored over a broad-spectrum antibiotic, which is ideal. This is because narrow-spectrum antibiotics are less likely to result in superinfections than broad- spectrum antibiotics. After all, they do not entirely eradicate the body's usual microbes. Bacteria become less immune as a result (8). A variety of skeletal antibiotics display various curative behaviors. As a result, it is crucial to categorize antibiotics according to their molecular structure. This classification is also vital because comparable structural units share similar toxicity, potency, and other related properties. Antibiotics are typically split structurally into β-lactams (group A) and aminoglycosides (group B). However, β-lactam/β-lactamase, β-lactams inhibitor mixtures, amine glycosides, quinolones, macrolides, and fluoroquinolones are more specific antibiotic categories (5). Function refers to a substance's mode of operation or mode of activity. One of the most crucial aspects of antimicrobial agents is this. Cell membrane function, cell wall synthesis, nucleic acid synthesis, protein synthesis, etc., are the primary processes or activities in bacterial growth. Antibiotics can be used to treat any one of these mechanisms. Antibiotics can be divided into four categories based on how they interfere with or disrupt these processes: inhibitors of cell wall synthesis, protein synthesis inhibitors (Metronidazole, etc.), membrane function inhibitors, and nucleic acid synthesis inhibitors (Levofloxacin, Rifampicin, Sulfamethoxazole, etc.) (9). This study aims to investigate the simultaneous analysis of specific antibacterial spectrophotometrically. IHJPAS. 2025, 38(2) 206 2. Materials and Methods 2.1. Levofloxacin Levofloxacin (Figure 1a), chemically is 7H-Pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-hydrate (2:1), (S)-; (−)- (S)-9-Fluoro-2,3-dihydro-3 methyl-10- (4-methyl-1-piperazinyl)- 7-oxo-7H-pyrido [1,2,3-de]- 1,4-benzoxazine-6-carboxylic acid, hemihydrate (10). It is a third-generation broad-spectrum fluoroquinolone antibiotic used to treat bacterial infections such as acute bacterial sinusitis, acute bacterial exacerbation of chronic bronchitis, acute bacterial prostatitis, and acute pyelonephritis. Levofloxacin was patented in 1987 and subsequently in the United States in 1996 for treating hospital-acquired pneumonia, community-acquired pneumonia, urinary tract infections, and skin or skin structure infections. Also, it is used to prevent and treat plaque caused by Yersinia pasties. Levofloxacin was FDA-approved to control the progression of inhaled anthrax (11,12). 2.2. Metronidazole As an antiprotozoal, antiamebic, and antibacterial medication, Metronidazole with chemical formula (2-(2-Methyl-5-nitro-1H-imidazol-1-yl) ethanol) (13) Figure 1b, is a bactericidal synthetic derivative of azomycin. Due to its effectiveness against anaerobic bacteria and protozoa, it has a wide range of applications. In the human liver, Metronidazole is converted into more polar metabolites. Metronidazole is the best option for colitis brought on by Clostridium difficile (14–16). 2.3. Rifampicin Rifampicin (Figure 1c) is a semi-synthetic antibiotic derived from rifamycin SV and is chemically ((2S,12Z,14E,16S,17S,18R,19R,20R,21S,22R,23S,24E)- 5,6,9,17,19-pentahydroxy- 23-methoxy-2,4,12,16,18,20,22- heptamethyl-8-[[(4-methylpiperazin-1-yl) imino] methyl]-1,11- dioxo-1,2-dihydro-2,7-(epoxypentadeca {1,11,13} trieneimino) naphtho[2,1-b] furan-21-yl acetate)) (13) active in vitro against microbacteria and gram-positive micro-organisms. Since its development, it has been used in veterinary and human medicine to treat tuberculosis and other mycobacterial infections (17,18). 2.4. Sulfamethoxazole Sulfamethoxazole is a sulfonamide antibiotic (Figure 1d) with the IUPAC name (4-amino-N- (5- methylisoxazol-3-yl)-benzenesulfonamide) (13). Sulfamethoxazole inhibits bacterial dihydro- folate synthesis due to its structural similarity to para-aminobenzoic acid, an endogenous substrate that interferes with folate synthesis in susceptible bacteria (19). The emergence of resistant strains has limited its use, and it is now mainly used as a mixture with trimethoprim (20). IHJPAS. 2025, 38(2) 207 (a) (b) (d) (c) Figure 1. Structures formula of (a) Levofloxacin, (b) Sulfamethoxazole, (c) Metronidazole and (d) Rifampicin. 3. Results and Discussion Several methods have been introduced to measure Levofloxacin, Sulfamethoxazole, Metronidazole, and Rifampicin separately or simultaneously. High-performance liquid chromatography, spectrophotometry, capillary electrophoresis, titration, spectrofluorometry, and voltammetry are a few examples. Simultaneous spectrophotometry is the most widely used due to its simplicity, sensitivity, and cost-effectiveness (21–23). The primary application of UV spectrophotometry techniques is multicomponent analysis, which minimizes the laborious process of isolating interferences, shortens analysis time and expenses, and enables the measurement of more analytes (24). The foundation of multicomponent UV spectrophotometry is the documentation of standard absorption spectra and its mathematical analysis. The benefits of spectrophotometry are as follows: It is broadly applicable to both organic and inorganic systems while avoiding traditional separation methods, such as extraction, occupant enrichment, and potentially essential purification steps; spectral data acquisition is easy to acquire; detection limits typically of 10-4-10-5 M; reasonable to high selectivity and good accuracy (25). Diverse spectrophotometry, Vierodt’s method, derivative spectrophotometry, dual wavelength method, absorbance ratio spectral method, differential ratio spectral method, absorption coefficient method, continuous ratio differential spectral method, Q-absorption ratio method, isosbestic points method, and other techniques are all examples of multicomponent spectrophotometry. In addition, chemometric methods have been used for multicomponent analysis, including multivariate regression models, principal component analysis, partial least squares, parallel factor analysis, and artificial neural networks (26). Table 1 illustrates a brief discussion of each method, including the combined drug(s), method(s), analytical results, and application remark. IHJPAS. 2025, 38(2) 208 Table 1. Spectrophotometric methods for simultaneous determination of Levofloxacin, Sulfamethoxazole, Metronidazole and Rifampicin. Drug Method(s) Analytical Results Application remark Year Ref Target Combined(s) L ev o fl o x a ci n Azithromycin Area under curve method using DDQ and p-CA λ= (390-650) nm and (400- 700) nm, Linearity range: 14.0-140.0 μg/mL and 30- 300.0μg/mL, R2 0.999 for both, Recovery 99.71- 102.64% and 99.38-101.76%, LOD 1.40 μg/mL and 3.00 μg/mL Pure mixture, Pharmaceutical binary dosage forms 2019 (27) Azithromycin Direct measurement of Abs in perchloric acid methanolic solution λ= 224nm, Linearity range: 2.5-20.0 μg/mL, R2 0.9991, Recovery100.95%, LOD 0.80 μg/mL Bulk powder, Laboratory- prepared tablets 2020 (28) First derivative peak-to-peak amplitudes λ= 280–253nm, Linearity range: 5.0-40.0 μg/mL, R2 0.9992, Recovery100.53%, LOD 1.50 μg/mL Atenolol, Paracetamol, and Hydrochlorot hiazide Extended derivative ratio λ= 282.8nm, Linearity range: 1.0-15.0 μg/mL, R2 0.9996, Recovery 100.15% LOD 0.25 μg/mL Dosage forms, Human urine 2020 (29) Multivariate curve resolution - alternating least squares λ= (220.0-290.0) nm, Linearity range: 1.0-7.0 μg/mL, R2 0.9999, Recovery 99.56% Ambroxol Absorbance ratio method λ= (244.6, 218,6)nm, Linearity range: 3.0-7.0 μg/mL, R2 0.9999, Recovery 99.0- 100.0%, LOD 0.18 μg/mL Tablets 2021 (30) Doxycycline Absorbance ratio method, Simultaneous equation method λ= 287nm, Linearity range: 2- 20 μg/mL, R2 0.9998, Recovery 99.66%, LOD 0.63 μg/mL Synthetic mixture, Combined pharmaceutical dosage forms 2022 (31) Ornidazole Area under curve method using DDQ and p-CA λ=(390-690)nm and (400-700) nm, Linearity range: 10.0- 200.0 μg/mL and 30-600.0 μg/mL, R2 0.998 and 0.999, Recovery 98.81-101.55% and 98.8-100.86%, LOD 3.3 μg/mL and 9.9μg/mL Pure mixture, Pharmaceutical binary dosage forms 2022 (32) M et ro n id a zo le Spiramycin Direct UV- spectrophotometry λmax= 311 nm., Linearity range: 5–25 μg/mL, R2 0.9997, Recovery 100.16%, LOD 0.3938μg/mL Pure form, Tablets 2010 (33) IHJPAS. 2025, 38(2) 209 Drug Method(s) Analytical Results Application remark Year Ref Target Combined(s) Ratio derivative spectrophotometry (1DD) λmax= 311nm, Linearity range: 5–25 μg/mL, R2 0.9997, Recovery 100.16%, LOD 0.3938μg/mL Mean centring of the ratio spectra (MCR) λ= (200-400)nm, Linearity range: 5–25 μg/mL, R2 1.0000, Recovery 101.1%, LOD 0.4967μg/mL Ciprofloxacin hydrochloride Second derivative ratio spectrophotometry technique (2DD) λ= 301nm, Linearity range: 4– 16 μg/mL, R2 0.9992, Recovery 100.21%, LOD 0.4900μg/mL Pure form, Tablets 2012 (34) Moxifloxacin Partial least squares regression (PLS), Principal component regression (PCR) λ= 200-500nm, Standard error of prediction (SEP): 0.036 (PLS), 0.043 (PCR) Prediction residual error sum- of squares (PRESS): 0.052 (PLS), 0.064 (PCR), Recovery:99.4%(PLS), 99.43% (PCR) Pharmaceutical tablets 2017 (35) Metronidazole benzoate 1st and 2nd derivative spectrophotometry λ= 200-350nm, Linearity range: 1–25 μg/mL, R2 0.9999, Recovery 99.75%- 101.25% Pharmaceutical preparations 2017 (36) Amoxicillin Trihydrate, Pantoprazole Artificial Neural Networks λ= 200-400 nm, 3 layers feed- forward neural networks with the lower backward-spread set of rules, RMSE: 0.34 Tablets 2022 (37) R if a m p ic in Piperine Q-Absorbance method λ= 387 nm, 337 nm, Linearity range: 5–40 μg/mL, R2 0.999, Recovery 98.36-99.53%, LOD 1.51μg/mL Combined capsule 2012 (38) Isoniazid Partial least squares regression (PLSR) λ= 449 nm, Linearity range: 8 -57 μg/mL, Recovery 92- 119%, RMSEP3LVs1.64 μg/mL Urine, Pharmaceutical formulations 2013 (39) Isoniazid Simultaneous equation λ= 338 nm, Linearity range: 5 -50 μg/mL, R2 0.992, Recovery 98.12-98.32%, LOD 3.50 μg/mL Co-formulated tablets 2017 (40) First-derivative spectrophotometry λ= 263 nm, Linearity range: 5- 50 μg/mL, R2 0.996, Recovery 98.80-99.15%,LOD2.30 μg/mL IHJPAS. 2025, 38(2) 210 Drug Method(s) Analytical Results Application remark Year Ref Target Combined(s) Isoniazid, Pyrazinamide First order derivative spectrophotometry λ= 322 nm, Linearity range: 1.5-7.5 μg/mL, R2 0.9927, Recovery 100.80%, LOD 0.03μg/mL Bulk form, Pharmaceutical dosage form 2020 (41) Isoniazid Vierordt's method λ= 335 nm, Linearity range: 6-36 μg/mL Simulated Lungs Alveolar Macrophages Fluid 2022 (42) S u lf a m et h o x a zo le Trimethoprim First derivative method λ= 288 nm, Linearity range: 4- 20 μg/mL, R2 0.9999, Recovery 108.64% Tablets, Syrups 1997 (43) Classical least squares (CLS) method λ= 200-350 nm, Linearity range: 4-20 μg/mL, Recovery 100.00% Sulfacetamide 1st derivative: Peak to baseline, Peak to peak, and Peak area λ= 219.75 nm, (219.0, 253.0) nm, and (270.2-322.5)nm, Linearity range: 1–8μg/mL, Recovery 98.9-101.4% Synthetic Sample and Urine 2014 (44) 2nd derivative: Peak to baseline, Peak to peak, and Peak area λ= 275.0nm, (232.0, 275.0) nm, and (215.2-227.4)nm, Linearity range: 1–8μg/mL, Recovery 98.1-102.3% PLS1, PLS2 λ=190-350 nm, Linearity range:1–8μg/mL, Recovery100.72%, 01.16% Trimethoprim Partial Least Square (PLS) regression λ= 200-400 nm, Linearity range: 1–16μg/mL, R2 0.99 RMSEC 0.167%, Recovery 100.72%, 101.16% Combined Tablets 2015 (45) Trimethoprim Mean Centering of Ratio Spectra (MCR) λ= 200-400 nm, Linearity range:3.5–9.5μg/mL, Recovery 103.31±0.34% Tablets 2018 (46) Trimethoprim Aea under peak of SIA λ= 540 nm and 264 nm, Linearity range: 0.1-10 μg/mL and 0.03-5 μg/mL, R2 0.9797 and 0.9928, LOD 0.01 and 0.03 μg/mL Tablets 2020 (47) 4. Conclusion Due to numerous benefits such patient compliance, timely dose administration, and fewer doses, multi-drug therapies and formulations are becoming increasingly important. Analytical chemists must pursue new directions and develop new, accurate, precise, cost-effective, time- effective, and simple methodologies to estimate pharmaceuticals in a multi-component system. Due to their inherent advantages, simultaneous analysis procedures are used more frequently for IHJPAS. 2025, 38(2) 211 drug estimation in multi-component pharmaceutical formulations. These advantages include avoiding time-consuming extraction and separation, being economical by using fewer expensive regents, and being equally accurate and precise. According to the International Conference on Harmonization recommendations, techniques must be validated using the same parameters. Acknowledgment This work was partially supported by the Department of Chemistry/ College of Education for Pure Science (Ibn Al- Haitham)/ University of Baghdad. Conflict of Interest The authors declare that they have no conflicts of interest. Funding No financial support was found. Ethical Clearance The study has been approved by the Committee of the University of Baghdad/ College of Education for Pure Science (Ibn Al-Haitham). References 1. Kresge N, Simoni RD, Hill RL. Selman waksman: The father of antibiotics. J Biol Chem. 2004; 279(48):e7. https://doi.org/10.1016/S0021-9258(20)67861-9. 2. Li D, Zhou B, Lv B. Antibacterial therapeutic agents composed of functional biological molecules. J Chem. 2020; 2020(6578579):1-13. https://doi.org/10.1155/2020/6578579 3. Mikhael EM, Hasan MK, Abdulridha SZ. Assessment of antibiotic knowledge among final year pharmacy students at Baghdad University. TOPHJ. 2019; 12(1):379-383. https://doi.org/10.2174/1874944501912010379 4. Miry SM, Al-Hayann HS. Antibacterial and anti-biofilm activities of iraqi propolis extracts against some antibiotic-resistant pathogenic bacteria. Iraqi J Biotechnol. 2022; 21(2):511-521. 5. Etebu E, Arikekpar I. Antibiotics: Classification and mechanisms of action with emphasis on molecular perspectives. Int J Appl Microbiol Biotechnol Res. 2016; 4(2016):90-101. 6. Aminov RI. A brief history of the antibiotic era: lessons learned and challenges for the future. Front Microbiol. 2010; 1:134. https://doi.org/10.3389/fmicb.2010.00134. 7. Oloke JK. Activity pattern of natural and synthetic antibacterial agents among hospital isolates. Microbios. 2000; 102(403):175-181. 8. Carbon C, Isturiz R. Narrow versus broad spectrum antibacterials: Factors in the selection of pneumococcal resistance to beta‐lactams. Drugs. 2002; 62(9):1289‐1294. https://doi.org/10.2165/00003495-200262090-00001. 9. Newton BA. Mechanisms of antibiotic action. Annu Rev Microbiol. 1965; 19(1):209-240. https://doi.org/10.1146/annurev.mi.19.100165.001233. 10. U.S. Pharmacopeia National Formulary (USP44-NF39), 2021. 11. Bush LM, Chaparro-Rojas F, Okeh V, Etienne J. Cumulative clinical experience from over a decade of use of levofloxacin in urinary tract infections: critical appraisal and role in therapy. Infect Drug Resist. 2011; 4:177-189. https://doi.org/10.2147/IDR.S15610. https://doi.org/10.1016/S0021-9258(20)67861-9 https://doi.org/10.1007/s12108-016-9307-z https://doi.org/10.2165/00003495-200262090-00001 https://doi.org/10.1146/annurev.mi.19.100165.001233 https://doi.org/10.2147/IDR.S15610 IHJPAS. 2025, 38(2) 212 12. Anderson VR, Perry CM. Levofloxacin: a review of its use as a high-dose, short-course treatment for bacterial infection. Drugs. 2008; 68(4):535-565. https://doi.org/10.2165/00003495-200868040-00011. 13. British Pharmacopoeia. British Pharmacopoeia. 2022; Vol. 2, p. 302, 820, 1042. 14. Anjana GV. Synthesis and antimicrobial evaluation of deuterated analogues of metronidazole. Iraqi J Pharm Sci. 2022; 31(2):297-303. https://doi.org/10.31351/vol31iss2pp297-303. 15. McDonald LC, Gerding DN, Johnson S, Bakken JS, Carroll KC, Coffin SE, Dubberke ER, Garey K W, Gould CV, Kelly C, Loo V, Sammons JS, Sandora TJ, Wilcox MH. Clinical practice guidelines for Clostridium difficile infection in adults and children: 2017 update by the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiology of America (SHEA). Clin Infect Dis. 2018; 66(7):e1-e48. https://doi.org/10.1093/cid/cix1085. 16. Dingsdag SA, Neil Hunter N. Metronidazole: An update on metabolism, structure–cytotoxicity and resistance mechanisms. J Antimicrob Chemother. 2018; 73(2):265-279. https://doi.org/10.1093/jac/dkx351. 17. Tawgozy F, Qarasnji BK. Molecular analysis of Rifampicin Resistance conferring mutations in mycobacterium tuberculosis. Baghdad Sci J. 2020; 17(2):444-451. https://doi.org/10.21123/bsj.2020.17.2.0444. 18. Al-Rubaye DS, Henihan G, Al-Abasly AKA, Seagar AL, Al-Attraqchi AAF, Schulze H, Hashim DS, Kamil JK, Laurenson IF, Bachmann TT. Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays. J Med Microbiol. 2016; 65(2):114-122. https://doi.org/10.1099/jmm.0.000203. 19. Khalil I, Ronn AM, Alifrangis M, Gabar HA, Satti GM, Bygbjerg IC. Dihydrofolate reductase and dihydropteroate synthase genotypes associated with in vitro resistance of Plasmodium falciparum to pyrimethamine, trimethoprim, sulfadoxine, and sulfamethoxazole. Am J Trop Med Hyg. 2003; 68(5): 586-589. https://doi.org/10.4269/ajtmh.2003.68.586. 20. Khalaf HS, Al-Haidari AM, Dikran SB, Mohammed AK. Spectrophotometric determination of sulfamethoxazole in pure and pharmaceutical preparations based on condensation reaction method. JUBPAS. 2017; 25(2):515-524. 21. Abrahem S, Abdul kader S, Ibrahim S. Different analytical methods for the determination of metronidazole–A Review. Int J Res Eng Innov. 2020; 4(2):86-90. https://doi.org/10.36037/IJREI.2020.4202. 22. Kotadiya RM, Patel FN. Analytical methods practiced to quantitation of Rifampicin: A captious survey. Curr Pharm Anal. 2021; 17(8):983-999. https://doi.org/10.2174/1573412916999200704144231. 23. Biancolillo A, Marini F. Chemometric methods for spectroscopy-based pharmaceutical analysis. Front Chem. 2018; 6:576. https://doi.org/10.3389/fchem.2018.00576. 24. Gupta D, Bhardwaj S, Sethi S, Pramanik S, Kumar Das D, Kumar R, Pratap Singh P, Kumar Vashistha V. Simultaneous spectrophotometric determination of drug components from their dosage formulations. Spectrochim Acta A Mol Biomol Spectrosc. 2022; 270:120819. https://doi.org/10.1016/j.saa.2021.120819. 25. Skoog DA, Holler FJ, Crouch SR. Principles of Instrumental Analysis; 7th ed., Cengage Learning, U.S, 2018. 26. Sowjanya G, Chandaka PK. Chemometrics approach to drug analysis–An overview. A J Pharm Tech Res. 2019; 9(1):1-13. https://doi.org/10.46624/ajptr.2019.v9.i1.001. 27. Vittal SS, Veeraiah T, Reddy ChVR. Simultaneous spectrophotometric determination of levofloxacin and azithromycin using Π-acceptors as analytical reagents. IOSR J Pharm. 2019; 9(1):50-61. https://doi.org/10.37285/ijpsn.2020.13.5.5. https://doi.org/10.2165/00003495-200868040-00011 https://doi.org/10.31351/vol31iss2pp297-303 https://doi.org/10.21123/bsj.2020.17.2.0444 https://doi.org/10.1099/jmm.0.000203 https://doi.org/10.4269/ajtmh.2003.68.586 https://doi.org/10.36037/IJREI.2020.4202 https://doi.org/10.2174/1573412916999200704144231 https://doi.org/10.3389/fchem.2018.00576 https://doi.org/10.1016/j.saa.2021.120819 https://doi.org/10.46624/ajptr.2019.v9.i1.001 https://doi.org/10.37285/ijpsn.2020.13.5.5 IHJPAS. 2025, 38(2) 213 28. El-Yazbi AF, Khamis EF, Youssef RM, El-Sayed MA, Aboukhalil FM. Green analytical methods for simultaneous determination of compounds having relatively disparate absorbance; application to antibiotic formulation of azithromycin and levofloxacin. Heliyon. 2020; 6(9):e04819. https://doi.org/10.1016/j.heliyon.2020.e04819. 29. Mostafa A. Spectrophotometric and multivariate calibration techniques for simultaneous determination of different drugs in pharmaceutical formulations and human urine: Evaluation of greenness profile. J Anal Methods Chem. 2020; 2020(1):8873003. https://doi.org/10.1155/2020/8873003. 30. Bodhankar V, Thoke ST, Kouthekar VR, Deshmukh SM, Jadhao UT. Simultaneous estimation of ambroxol hydrochloride and levofloxacin hemihydrate using absorbance ratio method. Eur J Pharm Med Res. 2021; 8(3):640-645. 31. Gholse YN, Chaple DR, Kasliwal RH. Development and validation of novel analytical simultaneous estimation based UV spectrophotometric method for Doxycycline and Levofloxacin determination. BRIAC. 2022; 12(4):5458-5478. https://doi.org/10.33263/BRIAC124.54585478. 32. Ravi M, Veeraiah T, Venkata RRCh. Simultaneous spectrophotometric estimation of Levofloxacin and Ornidazole using DDQ and p-CA as analytical reagents. J Pharm Sci & Res. 2022; 14(9):901- 907. 33. Khattab FI, Ramadan NK, Hegazy MA, Ghoniem NS. Simultaneous determination of metronidazole and spiramycin in bulk powder and in tablets using different spectrophotometric techniques. Drug Test Anal. 2010; 2(1):37-44. https://doi.org/10.1002/dta.83. 34. Alphonse M. Development and validation of a UV spectrophotometric method for the simultaneous determination of ciprofloxacin hydrochloride and metronidazole in binary mixture. J Chem Pharm Res. 2012; 4(11):4710-4715. 35. Ertokuş GP, Çelik UM. Chemometric analysis of Moxifloxacin and Metronidazole in binary drug combinations with spectrophotometric method. EAJS. 2017; 3(2):30-37. 36. Alsamarrai HAJ, Alsamarrai KF. Spectrophotometric determination of Metronidazole and Metronidazole benzoate via first and Second Derivative order spectroscopy. Baghdad Sci J. 2017; 14(2):320-327. https://doi.org/10.21123/bsj.2017.14.2.0320. 37. Hendawy H, Amin AS, Moalla SM, Aish MM. Artificial neural networks for the simultaneous spectrophotometric determination of Amoxicillin Trihydrate, Metronidazole and Pantoprazole in pharmaceutical formulations. Egypt J Chem. 2023; 66(6):119-127. https://doi.org/10.21608/ejchem.2022.151776.6582. 38. Khamar JC, Patel SA. Q-Absorbance ratio spectrophotometric method for the simultaneous estimation of Rifampicin and Piperine in their combined capsule dosage. J Appl Pharm Sci. 2012; 2(4):137-141. https://doi.org/10.7324/JAPS.2012.2416. 39. Stets S, Tavares TM, Peralta-Zamora PG, Pessoab CA, Nagata N. simultaneous determination of Rifampicin and isoniazid in urine and pharmaceutical formulations by multivariate visible spectrophotometry. J Braz Chem Soc. 2013; 24(7):1198-1205. http://dx.doi.org/10.5935/0103-5053.20130154. 40. Tilinca M, Hancu G, Mircia E, Iriminescu D, Rusu A, Vlad RA, Barabás E. Simultaneous determination of isoniazid and rifampicin by UV spectrophotometry. Farmacia. 2017; 65(2):219-224. 41. Khawas S, Parui S, Dey S, Mondal SK, Sarkar S. Simultaneous spectrophotometric estimation of Rifampicin, Isoniazid and Pyrazinamide in their pharmaceutical dosage form. Asian J Research Chem. 2020; 13(2):117-122. https://doi.org/10.5958/0974-4150.2020.00024.3. https://doi.org/10.1016/j.heliyon.2020.e04819 https://doi.org/10.1155/2020/8873003 https://doi.org/10.33263/BRIAC124.54585478 https://doi.org/10.1002/dta.83 https://doi.org/10.21123/bsj.2017.14.2.0320 https://doi.org/10.21608/ejchem.2022.151776.6582 https://doi.org/10.7324/JAPS.2012.2416 https://jbcs.sbq.org.br/audiencia_pdf.asp?aid2=3706&nomeArquivo=v24n7a14.pdf https://jbcs.sbq.org.br/audiencia_pdf.asp?aid2=3706&nomeArquivo=v24n7a14.pdf https://jbcs.sbq.org.br/audiencia_pdf.asp?aid2=3706&nomeArquivo=v24n7a14.pdf http://dx.doi.org/10.5935/0103-5053.20130154 https://doi.org/10.5958/0974-4150.2020.00024.3 IHJPAS. 2025, 38(2) 214 42. Bhoyar V, Belgamwar VS, Trivedi S. Simultaneous determination and validation of anti-tubercular drugs in simulated lungs alveolar macrophages fluid by ultraviolet-visible spectrophotometric method. J Appl Spectrosc. 2022; 89(5):892-897. https://doi.org/10.1007/s10812-022-01444-z. 43. Mohamed EM. Simultaneous spectrophotometric determination of Sulfamethoxazole and Trimethoprim in drugs. Anal Lett. 1997; 30(13):2341-2352. https://doi.org/10.1080/00032719708001746. 44. Samar A. Development and validation of new methods for simultaneous spectrophotometric determination of Sulfamethoxazole and Sulfacetamide drugs in their pure forms, Synthetic Samples and Urine, Doctoral dissertation, University of Baghdad, Baghdad, 2014. 45. Rohman A, Silawati D, Sudjadi, Riyanto S. Simultaneous determination of Sulfamethoxazole and Trimethoprim using UV spectroscopy in combination with multivariate calibration. J Med Sci. 2015; 15(4):178-184. https://doi.org/10.3923/jms.2015.178.184. 46. Muchlisyam, Pardede Tr, Satiawan R. Determination of simultaneous Sulfamethoxazole and Trimethoprim by ultraviolet spectrophotometry with mean centering of ratio spectra. Asian J Pharm Clin Res. 2018; 11(1):61. https://doi.org/10.22159/ajpcr.2018.v11s1.26569. 47. Nesmerak K, Kroiherova A, Baptistova A, Hranicek J. Spectrometric determination of trimethoprim and sulfamethoxazole in tablets by automated sequential injection technique. Monatsh Chem. 2020; 151(8):1311-1316. https://doi.org/10.1007/s00706-020-02631-4. https://doi.org/10.1007/s10812-022-01444-z https://doi.org/10.1080/00032719708001746 https://ascidatabase.com/author.php?author=Sugeng&mid=&last=Riyanto https://doi.org/10.3923/jms.2015.178.184 https://doi.org/10.22159/ajpcr.2018.v11s1.26569 https://doi.org/10.1007/s00706-020-02631-4