# Recent Applications of Solid Phase Microextraction Coupled to Liquid Chromatography
## 2 Applications  
### Biological  
#### 2.1.1 Fluids and Tissues  
Liquid chromatography can be considered as the technique of choice currently used for drugs and metabolites analysis in biological fluids and tissues. However, biological matrices, such as serum, plasma, and tissues, are complex mixtures incompatible with a direct injection into conventional LC supports and a sample preparation step that limits the number of interferents co-extracted with the analytes is always needed, especially when working with LC coupled to electrospray mass spectrometry (ESI-MS) to prevent ionization issues. The commonly used extraction techniques (SPE, LLE, ultrafiltration, dialysis) generally work off-line, therefore being complex and tedious, and a substantially time-limiting step in the analytical process. Furthermore, they often require protein precipitation procedures that can induce analyte losses due to the known phenomenon of co-precipitation. SPME-LC has proven to be a good fast and sensitive approach for the determination of semi- or non-volatile analytes in biological samples, also for its ability to determine free and/or total drug concentration, depending on the extraction conditions in the sample under investigation [31]. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of the antifibrinolytic drug tranexamic acid in plasma samples extracted by SPME, using a commercial biocompatible C18 coating, was accomplished by [PERSON] et al. [32]. Good accuracy and precision were obtained, and the matrix effect was minimized making the sample compatible with ESI detection. A comparison between the new method and standard techniques of protein precipitation and ultrafiltration showed no significant differences among the performances of the approaches. The potential of the procedure was demonstrated by the successful analysis of clinical plasma samples, determining the analyte at concentration levels that exceeded the capability of current assays employing sample preparation using protein precipitation followed by LC/MS analysis. In another study, the SPME-LC-UV simultaneous determination of clenbuterol, salbutamol, and ractopamine residues using polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibers and on-line desorption was reported by [PERSON] et al. [33], and its application to real samples demonstrated by the analysis of pig samples (muscle and liver), while [PERSON] and co-workers proposed a one-step simple extraction (using two fibers, carboxen-polydimethylsiloxane (CAR/PDMS) and polyacrylate (PA)) of BTEX (benzene, toluene, ethylbenzene, and xylenes and its metabolites) from spiked tooth followed by SPME-HPLC or HS-SPME-GC/MS determination [34]. In particular, the SPME-LC procedure showed the typical advantages of SPME (simple, rapid, accurate, solvent-free), good precision, linearity, and sensitivity, even if limits of detection (LODs) were slightly lower than those obtained with alternative approaches due to the limitation of the UV detector. The method was successfully applied in human teeth as environmental biomarker of BTEX and metabolites. SPME (biocompatible C18 fibers) coupled to LC-MS/MS (triple quadrupole, QqQ) was also used to determine selected antibiotics and their metabolites in human blood and tissue taken from afflicted areas [35]. Samples of 25 patients under treatment were successfully analyzed to estimate pharmacokinetic profiles. Interestingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS), coupled off-line with LC, was also used for identification and quantification of the analytes, showing results comparable to those obtained with LC-MS/MS. Another pharmacokinetic study in plasma for methylphenidate, a stimulant for the human central nervous system, was successfully performed coupling SPME (PDMS fibers) and thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) [36]. The applicability of the approach was demonstrated by the validation of the relevant results by means of LC-MS/MS.