**2. Methods Used to Analyze Antibiotics**

Various methods have been developed to detect and quantify antibiotics in various types of samples. The referred methods include chromatographic, spectrophotometric, and electrochemical methods [34]. High performance liquid chromatography (HPLC) is, by far, the most extensively utilized instrumental method in pharmaceuticals analysis [35].

#### *2.1. Chromatographic Methods*

Pharmaceuticals and their metabolites have been analyzed using a variety of chromatographic methods. Such methods can be used alone or hyphenated with mass spectrometry. Mass spectrometry-based approaches, particularly liquid chromatography, coupled with tandem mass spectrometry (LC/MS/MS) can reach extraordinarily high degrees of specificity compared with immunoassay or even chromatographic detection utilizing detectors such as UV or fluorescence. The specificity and sensitivity of a chromatographic method are controlled by chromatographic conditions such as choice of mobile phase and analytical column, detector, and sample preparation [36]. For antibiotic analysis, the analytical method is selected based on the characteristics of the analyzed antibiotic, which includes solubility in water and organic solvents or acid-base properties [37].

Thin-layer chromatography (TLC) is one of the most important analytical methods used to determine the qualitative and semiquantitative levels of pharmaceuticals in various types of samples [34]. TLC is usually applied as the quick, easy, and straightforward procedure. The effective separation is determined by the sample's properties as well as the properties of the stationary and mobile phases [38]. TLC can successfully be used for preliminary screening of the pharmaceutical compounds. It is commonly employed in contemporary analysis as a separation method to determine the presence or absence of antibiotics over a predetermined concentration level [38,39]. It can also be used to evaluate and categorize pure and impure antibiotic preparations as well as assay antibiotics quantitatively in bulk or pharmaceutical preparations [40,41].

Gas chromatography (GC) is a commonly used analytical technique that combines separation chromatographic stage with measurement capacity. GC employs the gas as the mobile phase and coating inside the long capillary column or, less typically, the tiny particles of a solid material packed in a column as the stationary phase. The sample in GC should be able to evaporate so that it flows with the gaseous mobile phase. The temperature gradient to which the chromatographic column is subjected is frequently utilized to speed up the elution of less volatile substances that would otherwise take a long time to elute. The detector signals for the sample's eluting components are used for quantitative and qualitative analysis [42]. GC is a useful technique for evaluating pharmaceutically relevant substances [43] and impurities [44]. Many pharmaceutical chemicals, however, cannot be gas chromatographed in their natural state and must be transformed into stable and volatile

derivatives in order to accomplish successful GC elution and separation. The derivatives are sometimes created in order to attain the appropriate sensitivity, selectivity, or specificity for a given separation [44,45].

High-performance liquid chromatography (HPLC) is a chromatographic technique that can separate a mixture of substances, and it is used in biochemistry and analytical chemistry to identify, quantify, and purify different components of the mixture [46]. HPLC employs various types of the stationary phases, and the pump that drives the mobile phase(s) and analyte through the column and detector to provide a characteristic retention time for the analyte. The retention period of an analyte varies according to the strength of its interactions with the stationary phase, solvent(s) ratio/composition utilized, and flow rate of the mobile phase [47]. HPLC has a number of advantages, including low organic solvent utilization, minimal sample volume, quick analysis, and high chromatographic resolution [48]. Apart from conventional HPLC, other sophisticated HPLC-based techniques have been widely applied for the determination of pharmaceuticals, including antibiotics in various samples. The referred methods include liquid chromatography—mass spectrometry (LC-MS) [49,50], ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) [51–53], and liquid chromatography linked to tandem mass spectrometry (LC-MS/MS) [54,55].

#### *2.2. Spectrophotometric Methods*

Spectrophotometric methods are based on the creation of the complex between the pharmaceutical and the reagent [12]. The intensity of the color is used to calculate pharmaceutical concentration. The complex generated by the pharmaceutical and reagent can either be charge transfer or ion-pair in nature. The charge transfer complex, also known as the electron donor-acceptor complex, transfers a fraction of electrical charge between molecules. Coulomb attraction holds oppositely charged ions together in solution in the ion-pair complex [56]. Some antibiotics have been analyzed using spectrophotometric methods including amoxicillin [40], azithromycin [41,57], tetracycline, doxycycline [58], and cefixime trihydrate [59]. They have also been used to analyze gentamicin sulfate [60], cefadroxil, ceftazidime, cefazolin sodium, cefoperazone sodium, cefaclor, cephaprin sodium, cefotaxime sodium, and cefuroxime sodium [61].
