###### Abstract  
A salt-induced homogeneous liquid-liquid microextraction (SI-HLME) protocol combined with high-performance liquid chromatography-diode array detection is presented for the first time for the determination of piroxicam and meloxicam in human urine. The main parameters affecting the performance of the sample preparation protocol were optimized by means of a two-step experimental design (i.e., 2-level fractional factorial design and Box-Behnken design). Following its optimization, the proposed method was thoroughly validated in terms of the total error concept in order to take into consideration the random and systematic errors. For the target analytes, accuracy profiles were constructed, and they were used as graphical decision-making tools. In all cases, the \(\beta\)-expectation tolerance intervals complied with the acceptance criteria of \(\pm\)15%, proving that 95% of future results will fall within the defined bias limits. The limits of detection were 0.02 \(\upmu\)mL\({}^{-1}\) and 0.03 \(\upmu\)mL\({}^{-1}\) for piroxicam and meloxicam, respectively. The relative standard deviations were lower than 4.4% in all cases, and the mean relative biases ranged between \(-\)5.7 and 3.4% for both drugs. The proposed scheme is simple and rapid, while it is characterized by high sample throughput. Moreover, SI-HLME requires reduced sample and reagent consumption, according to the requirements of Green Analytical Chemistry. piroxicam; meloxicam; homogeneous liquid-liquid microextraction; urine; salt-induced; experimental design; HPLC-DAD +
Footnote †: journal: Applied Sciences  
with the relatively low concentration of the target analytes in these samples, a sample preparation step is generally required before their analytical determination [7]. Solid-phase extraction (SPE) and liquid-liquid extraction (LLE) are the major conventional sample preparation techniques that are nowadays considered well-established in bioanalysis [8]. However, both techniques show some inherent disadvantages including the increased consumption of hazardous solvents, as well as having time-consuming and labor intensive steps [9]. After the introduction of the Green Analytical Chemistry (GAC) principles [10], increasing attention has been devoted towards the replacement of these techniques with the so-called \"green\" microextraction techniques. Following this trend, a wide variety of novel sample preparation techniques have been developed and applied in bioanalysis, aiming to replace the less environmentally friendly SPE and LLE approaches. Typical examples of green extraction techniques used for the determination of drugs in biological matrices include sorbent-based approaches such as solid-phase microextraction [11], fabric phase sorptive extraction (FPSE) [12], capsule phase microextraction (CPME) [13], magnetic solid-phase extraction (MSPE) [14], stir bar sorptive extraction (SBSE) [15], pipette tip-based microextraction [16] and liquid-based approaches such as dispersive liquid-liquid microextraction (DLLME) [17], electromembrane extraction (EME) [18], homogeneous liquid-liquid microextraction (HLLME), etc. [19]. The advances in sample preparation techniques for bioanalytical purposes has recently been reviewed [20]. HLLME typically utilizes small volumes of hydrophilic solvents as extractants that are miscible with water [21]. Due to the infinitely large surface area among the different phases, increased accessibility of this solvent to the target analytes can be achieved, resulting in a high extraction efficiency, high mass and rapid equilibrium [22; 23]. Moreover, HLLME overcomes the need for analyte back-extraction due to the compatibility of the solvent with most chromatographic systems [21]. Common types of HLLME include the salt-induced HLLME (SI-HLLME) [24] and the sugar-induced HLLME [25], in which phase separation is achieved by the addition of salts and sugars, respectively.