Chlorzoxazone (CHZ), which is chemically named 5-chlorobenzo[d]oxazol-2(3H)-one (Fig. 1), is a centrally acting medication intended to treat muscular spasms. It suppresses reflexes in the spinal cord [1]. When treating muscular spasms caused by musculoskeletal disorders, it usually works when combined with analgesics. Ibuprofen (IBU) is chemically named 2-(4-isobutylphenyl) propanoic acid (Fig. 1). Nowadays, IBU is among the most commonly used NSAIDs. It is used as an anti-inflammatory to reduce fever and as a pain reliever [2].

There are several approaches for determining CHZ, including spectrofluorimetric [3,4], spectrophotometric [5,6], electrochemical [7,8], gas chromatographic [9], and liquid chromatographic approaches [[10], [11], [12], [13], [14], [15], [16]]. IBU has been determined using a variety of approaches, among them chromatographic [[17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30]], spectrophotometric [[31], [32], [33], [34]], spectrofluorimetric [[35], [36], [37]], capillary electrophoretic [38,39], IR spectroscopic [40], and electrochemical approaches [[41], [42], [43], [44]].

The combination of a non-steroidal anti-inflammatory drug (NSAID) such as ibuprofen (IBU) with a muscle relaxant like chlorzoxazone (CHZ) has been shown to be effective for the relief of pain and inflammation associated with conditions such as rheumatoid arthritis, sprains, ankylosing spondylitis, and postoperative pain. Moreover, this combination has demonstrated superior efficacy compared to ibuprofen alone in the management of acute low-back pain. The concurrent administration of these two medications underscores the need for accurate, practical, and selective analytical methods for their simultaneous quantification, particularly in biological matrices. Such methods are essential for therapeutic drug monitoring, pharmacokinetic studies, and bioequivalence assessments. There are several reported approaches for determination of CHZ and IBU combination, including spectrofluorimetric [[45], [46], [47]], spectrophotometric [[48], [49], [50], [51]], and liquid chromatographic approaches [52,53].

A comprehensive review of previously reported methods for the determination of chlorzoxazone (CHZ) and ibuprofen (IBU) reveals that most were limited to pharmaceutical dosage forms. The majority of spectrophotometric techniques employed mathematical resolution methods, which are generally unsuitable for biological matrices due to matrix interference and limited selectivity. While three spectrofluorimetric methods were developed, two were restricted to tablet analysis only, with only one applied to plasma samples. However, the latter involved liquid–liquid extraction using chloroform—a hazardous and non-environmentally friendly solvent—contravening the core principles of green analytical chemistry. Similarly, all reported HPLC methods for the simultaneous determination of CHZ and IBU were confined to tablet analysis and were associated with high solvent consumption and reliance on complex instrumentation.

On the other hand, the proposed approach is the first to simultaneously determine both medications in authentic human plasma by integrating a simple, efficient, and environmentally friendly salting-out assisted extraction technique with TLC-densitometric analysis. Due to its affordability and ease of use, thin-layer chromatography (TLC) is still a useful analytical technique for drug analysis. TLC can yield accurate quantitative data when paired with densitometric detection. Meanwhile, salting-out assisted liquid–liquid extraction (SALLE) represents a green sample preparation strategy that utilizes inorganic salts and water-miscible solvents to induce phase separation and protein precipitation, minimizing the use of organic solvents. This combined method offers a promising alternative to conventional chromatographic techniques, particularly for routine analysis in quality control laboratories.

A comparison of reported quantitation limits between HPLC, spectrophotometric, and spectrofluorimetric approaches was carried out. The lowest LOQ results for spectrophotometric techniques were 2 µg. mL-1 for both CHZ and IBU. For spectrofluorimetric approaches, it was found to be 0.09 µg. mL-1 for CHZ and 0.8 × 10 −3 µg. mL-1 for IBU. Regarding HPLC-reported approaches, the lowest LOQ values were 2.4 µg. mL-1 for CHZ and 1.6 µg. mL-1 for IBU. LOQ values were larger than those for the previously reported spectrofluorimetric and chromatographic approaches. However, without the need for additional enhancement, TLC technique coupled with SALLE was able to attain a sensitivity high enough to determine both IBU and CHZ in real human plasma samples.