Currently, there is still a need for broad-spectrum antibiotics. The new cephalosporin antibiotics include, among others, ceftobiprole, a fifth-generation gram-positive cephalosporin, active against Staphylococcus aureus methicillin agonist (MRSA). The main focus of the work was to optimize the conditions of ceftobiprole qualitative determination and to validate the developed procedure according to ICH guidelines. As a result of the optimization process, HPTLC Cellulose chromatographic plates as a stationary phase and a mixture consisting of ethanol:2-propanol: glacial acetic acid: water (4:4:1:3, v/v/v/v) as a mobile phase were chosen. The densitometric detection was carried out at maximum absorbance of ceftobiprole (λ = 232 nm). Next, the validation process of the developed procedure was carried out. The relative standard deviation (RSD) for precision was less than 1.65%, which proves the high compatibility of the results, as well as the LOD = 0.0257 µg/spot and LOQ = 0.0779 µg/spot values, which also confirm the high sensitivity of the procedure. The usefulness of the developed method for the stability studies of ceftobiprole was analyzed. Study was carried out under stress conditions, i.e., acid and alkaline environments, exposure to radiation imitating sunlight and high temperature (40–60 °C). It was found that cefotbiprole is unstable in an alkaline environment and during exposure to UV-VIS radiation. Moreover, the lipophilicity parameter, as a main physicochemical property of the biologically active compound, was determined using experimental and computational methods.

A quick and accurate chromatographic–densitometric method for the determination of ceftobiprole in biological material (whole blood and urine) was developed. Preparation of the test sample required extraction of the drug from the matrix and was carried out by testing methanol or acetone as extracting agents, which were successfully used to isolate ceftobiprole from biological material. Under optimization of the procedure, various stationary and mobile phases were tested. Lastly, HPTLC cellulose plates and a mixture containing ethanol, 2-propanol, glacial acetic acid, and water in the ratio 4:4:1:3 (v/v/v/v) were chosen. Densitometric detection was made at a maximum absorbance of 316 nm. The developed method was validated; a linear function of the ceftobiprole concentration was obtained in the range of 2.4–72 µg/mL (r > 0.99) for both methanol and acetone solutions. The average accuracy of the devised method was measured at nearly 100%; nevertheless, the limit of the quantification was at 8.92 for methanol and 9.14 µg/mL for acetone solution. Therefore, the above method can be successfully used to ceftobiprole in biological material.

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