Evaporation or drying down is used widely in the cleanup of biological samples by liquid-liquid extraction (LLE), solid-phase extraction (SPE), or protein-precipitation methods. Though it is necessary in some cases, such as to change solvent and to concentrate an analyte, there are several problems associated with the evaporation procedure. In addition to environmental pollution, evaporation and the subsequent reconstitution and transfer steps are time-consuming. These extra steps are subject to more potential contamination. Most importantly, evaporative–adsorptive losses or chemical reaction/conversion can occur during evaporation. For example, in a typical mixed-mode strong cation exchange–based SPE method, the elution solution is usually a mixture of a base and an organic solvent — for example, 10% ammonium hydroxide in methanol. The analytes and the other co-extracted metabolites in this strong basic media are then evaporated under heating at about 50 °C for 30–50 min. The high pH and heating could result in the hydrolysis or dissociation of glucuronides, acetate, phosphate, or decomposition of other labile metabolites (1). Therefore, developing evaporation-free extraction methods is highly desirable (2,3).

Though not named as such, several ways have existed to achieve evaporation-free LLE and SPE. In LLE, the traditional LLE with back extraction is one way. Matsui and colleagues (3) developed a mild clean-up procedure by using this approach to prevent the interconversion of R-donepezil and S-donepezil during extraction. Briefly, the two analytes were first extracted from a plasma sample to an organic phase. Then, 0.2 mL of dilute hydrochloric acid was added to the collected organic phase to transfer the analytes back to aqueous phase, which was then injected without evaporation. Though the procedure is straightforward, it would be quite tedious and labor-intensive when a sample batch is large. In addition, it is difficult to automate an LLE method with back extraction. One step further, directly injecting organic layer from LLE onto a reversed-phase column (4) or a hydrophilic interaction liquid chromatography (HILIC) column (5), were reported. Despite its use in special cases, the wide application of this approach (direct injection of organic phase from LLE) is limited due to the strong smell and corrosive and highly evaporative nature of common organic solvents, as well as its immiscibility with commonly used aqueous mobile phases in reversed-phase liquid chromatography (LC).

In SPE-based methods, Zheng and colleagues (6) reported an evaporation-free SPE method based upon automatic dilution of high organic SPE eluate by using custom-designed sample storage tubes with pierceable caps. Sanchez and colleagues (7) proposed direct injection of SPE elaute to an HILIC column without dilution and evaporation. Alternatively, evaporation-free SPE can be achieved through on-line SPE and column switching techniques, where samples are extracted on-line and the elution is loaded directly onto an analytical column. This is a highly automatic approach. However, special equipment is usually necessary. In addition, as the samples usually are processed sequentially, all the samples of one batch have to be present and kept at either room temperature or 4 °C for an unnecessarily extended period of time, which places stringent requirements on benchtop stability of analytes in matrix.