# New Challenges in (Bio)Analytical Sample Treatment Procedures for Clinical Applications
## 2 Clinical Applications  
### Anticancer Drugs  
[PERSON], [PERSON], and [PERSON] (2004) reported the use of a mixture of ethyl acetate and acetonitrile (4:1, \(v/v\)) after sample acidification with orthophosphoric acid to simultaneously extract capecitabine, 5\({}^{\prime}\)-DFUR (5\({}^{\prime}\)-deoxy- 5-fluorouracilidine), 5-FU (5-fluorouracilacil), and 5-FUH2 (5,6-dihydro-5-fluorouracil) from plasma. A similar extraction method is also reported using LLE with a mixture of ethyl acetate and acetonitrile as the extractant (8:3, \(v/v\)) [6].  
[PERSON] et al. (2014) have reported a similar extraction method using a mixture of ethyl acetate and acetonitrile (4:1, \(v/v\)) but without sample acidification. Acidification was not deemed necessary because the assay was developed to determine only capecitabine concentrations (no metabolites) [6].  
[PERSON], [PERSON], and [PERSON] [7] used LLE, specifically using a mixture of ethyl acetate and acetonitrile (4:1, \(v/v\)) after acidification of samples to extract capecitabine, 5\({}^{\prime}\)-dFUR, 5-FU, and 5-FUH2 from human plasma. [PERSON] et al. [8] reported a similar LLE method using a mixture of ethyl acetate and acetonitrile (4:1, \(v/v\)), but without sample acidification, because they wanted to find only capecitabine and not its metabolites.  
Table 1 provides a summary of sample-pretreatment procedures and the analytical figures of merit for capecitabine and its metabolites from different biological samples.  
Many studies reported the use of protein precipitation-like sample treatment for bioanalytical analysis. [PERSON] et al. (2014) and [PERSON] et al. (2015) used methanol as the solvent to precipitate the proteins from human plasma and human serum. Furthermore, [PERSON] et al. (2017), with the aim of not losing the drug, added 10% \(\
u/\
u\) of trichloroacetic acid in water.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Analy** & **Sample** & **Treatment** & **LOD/LOQ** & **Ref.** \\ \hline \hline \multicolumn{4}{c}{\(\mathcal{S}\)-DFUR} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{5-FUZ} \\ \multicolumn{4}{c}{Capeticahine} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{5-FU} \\ \multicolumn{4}{c}{FBAL} \\ \multicolumn{4}{c}  
Another protocol used to extract anticancer drugs, specifically osmeritinib, involved LLE. [PERSON] et al. used tert-butyl methyl ether for simultaneous extraction and concentration of it. This method was developed to follow treatment for patients with non-small cell lung carcinoma with epidermal growth factor receptor activating mutation [20].  
[PERSON] et al. studied nine kinase inhibitors, including cobimetinib, dasatinib, birtuinib, imatinib, nilotinib, palbociclib, ruxolitinib, sorafenib, and veumarafenib; the following two active metabolites of them: N-desmethyl imatinib, N-oxide sorafenib; the following two anti-androgen drugs: abiraterone and enzalutamide. They used a single-step protein precipitation with 100 uL of aqueous Zinc Sulfate Monohydrate ZnSO\({}_{4}\cdot\)H\({}_{2}\)O (10% \(w/v\) pH 5.4), adding this to 50 uL of human plasma followed by vortexing and centrifugation [21].  
[PERSON] et al. used whole blood, saliva, and urine samples. The difficulty, especially for whole blood, was to minimize the matrix interferets that can mess up the analysis. Usually, the first step is protein precipitation to convert whole blood into plasma. In this case, the authors directly used whole blood without converting it into plasma, thanks to the extraction protocol using fabric phase sorptive extraction (FPSE) to avoid the possible loss of analytical information related to the quantification of anastrozole, letrozole, and exemestane used in the treatment of metastatic breast cancer [22].