## 1 Introduction  
High-performance liquid chromatography (HPLC) is the most widely used analytical tool for pharmaceutical analysis. Indeed, it is the most important technique involved in the quality control of bulk drugs and pharmaceutical formulations (i.e., analysis of active pharmaceutical ingredients (API), characterization of impurities, determination of degradation products to test product stability, and determination of enantiomeric purity), and also in the determination of drugs and metabolites in biological samples [1]. Most HPLC methods developed in pharmaceutical laboratories are based on the reversed-phase (RP) mode, using a hydrophobic stationary phase and a polar mobile phase. In quality control, the primarily used detection mode is ultraviolet (UV)/Visible detector. Therefore, mobile phase compatibility with this detection is a parameter often taken into account in pharmaceutical analysis development. The mobile phase of RP-HPLC is usually a mixture of water (containing additives to adjust pH and ionic strength) and organic solvent, such as acetonitrile (ACN) and methanol (MeOH) [2]. These two solvents are by far the preferred organic solvents used in RP-HPLC because of their remarkable combination of properties favorable for RP-HPLC applications. Among them include complete miscibility with water, relatively low viscosity of their aqueous solutions (especially in the case of ACN), low UV cut-off wavelength (190 nm and 205 nm for ACN and MeOH, respectively), availability in the high purity required for HPLC, and low chemical reactivity with most sample species, as well as with HPLC instrument and column surfaces [2; 3]. Despite these remarkable properties, ACN and MeOH present some issues in terms of environmental impact and health safety. Indeed, ACN is flammable, volatile, and toxic. Even if MeOH is less toxic and more easily biodegradable than ACN, it is also ranked as a hazardous solvent due to its inherent toxicity and the great requirements of its waste disposal [4; 5]. Unfortunately, the amount of waste generated by RP-HPLC analyses cannot be neglected. In fact, one continuously operating liquid chromatograph equipped with a conventional LC column (15-25 cm length, 4.6 mm i.d., packed with 5 \(\upmu\)m particles) and a mobile phase flow rate of 1 mL/min produces about 1.5 L of waste per day, meaning about 500 L of effluent per year [3]. Although this volume of waste is small compared to the amount of waste generated by large industrial manufacturing companies, some big pharmaceutical companies use hundreds of liquid chromatographs in their research and development, and quality control laboratories, resulting in thousands of liters of toxic waste produced every day. Moreover, the use of HPLC is becoming more and more intensive due to the technological advances allowing high-throughput analysis, which also increases, at the same time, the amount of waste produced. These HPLC waste streams containing ACN and MeOH must be disposed as chemical waste, which is costly and adds to the environmental waste-disposal burden of the laboratory. Regarding the health and environmental issues of organic solvents commonly used in RP-HPLC, the greening of RP-HPLC methods has received great interest in the analytical community, whose aim is to search for new alternatives to replace polluting analytical methods with cleaner ones. Green analytical chemistry (GAC) emerged from green chemistry in the 2000s [6; 7], and has gained increasing attention and acceptance among researchers. Its concept refers to eliminating or reducing hazardous chemicals from analytical processes to improve environmental and health friendliness, without compromising method performance [8]. Based on the 12 principles of green chemistry, some strategies are commonly implemented to achieve greener liquid chromatography methods.