Development of a capsule phase microextraction (CPME) protocol for the determination of four tetracyclines in human urine prior to HPLC–DAD analysis

In this work, a novel and green capsule phase microextraction method followed by high-pressure liquid chromatography coupled to diode array detector (CPME-HPLC-DAD) was developed for the selective determination of four tetracyclines, namely oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC) and doxycycline (DC), in human urine. Among five tested CPME devices, the sol–gel C₁₈ capsules exhibited the highest extraction efficiency. The main factors affecting the sample preparation procedure were optimized using the one-factor-at-a-time (OFAT) strategy, leading to the selection of 4 mL as the optimal sample volume. Following the optimization of CPME parameters, the method was validated in terms of linearity, selectivity, accuracy, sensitivity and precision. For all analytes, linearity was confirmed over the concentration range of 0.03–0.50 μg/mL (R² > 0.998), while method limits of detection (LODs) and quantification (LOQs) were equal to 9 ng/mL and 30 ng/mL, respectively. The relative standard deviation (%RSD) expressing intra-day and inter-day repeatability of the reported method were found to be lower than 6.7% and 8.7%, respectively. The accuracy of the optimized method was evaluated using spiked samples with analytical recoveries ranging from 93.9% to 110.8% (intra-day) and 90.8% to 112.4% (inter-day). The green character and practicality of the proposed method, as well as the level of innovation and analytical performance were assessed using advanced metric tools, including ComplexMoGAPI (Complex Modified Green Analytical Procedure Index), BAGI (Blue Applicability Grade Index), VIGI (Violet Innovative Grade Index) and RAPI (Red Analytical Performance Index). Overall, the developed CPME-HPLC–DAD protocol provides a simple, eco-friendly and highly reliable approach for the selective quantification of these four tetracyclines in human urine.