Coupling of acetonitrile deproteinization and salting‐out extraction with acetonitrile stacking for biological sample clean‐up and the enrichment of hydrophobic compounds (porphyrins) in capillary electrophoresis

Abstract

A new sample pretreatment approach in CE was developed for concurrent biological sample clean-up and the concentration of hydrophobic compounds based on the combination of ACN deproteinization with salting-out extraction. Further enhancement in concentration detection sensitivity was achieved by coupling (offline) salting-out extraction with an online CE sample enrichment technique known as "ACN stacking". By optimizing the pH of salting-out extraction, a number of model compounds (hydrophobic porphyrins with clinical significances), i.e. zinc-protoporphyrin, protoporphyrin, and coproporphyrin (CP) III and I, can be efficiently extracted from the aqueous sample into a smaller volume organic solvent (ACN) phase and an enrichment factor of ca. 100 can be obtained. The pressure injection of the enriched ACN phase (containing ca.1% NaCl) into the CE capillary at 10% capillary volume resulted in additional concentration of the various hydrophobic porphyrins, allowing for a combined enrichment factor of ca.1000 to be obtained. Calibration curves obtained for the determination of a pair of positional isomers with significant diagnostic value, urinary CPIII and CPI, were found to be linear between 10-300 ng/mL (with R2 = 0.999), and LODs (absorbance detection at 400 nm) were ca. 0.8 ng/mL (1.1 nmol/L of CPIII or CPI). Based on a single salting-out extraction, intraday precisions (nine consecutive injections) for both CPIII and CPI (at spiked concentrations of 10-300 ng/mL into urine) in terms of migration time and peak area were found to be within the range of 0.2-0.5 and 0.8-2.9%, respectively.

Salting‐out assisted liquid/liquid extraction with acetonitrile: a new high throughput sample preparation technique for good laboratory practice bioanalysis using liquid chromatography–mass spectrometry

“…Although the SALLE approach with acetonitrile was applied in the LC-UV analysis of biological samples first time in 1989 (Rustum 1989), only very limited applications of the method have been reported so far. (Yoshida et al, 2004;Buratti et al, 2006;Li and Huie, 2006;Kankaanpaa et al, 2007). These applications have aimed at determination of specific target analyte.…”

Analysis of Urinary Porphyrins by High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry

“…MP (200 nM) as IS was added to the samples because it was not present in human urine. [27][28][29]40] The peaks of UP, HEPTAP, PENTAP, and CP in the human urine samples were identified with the detection of the corresponding molecular ions and with the comparison of chromatographic retention times with those of the authentic standards shown in Figure 3. HEXAP was not detected in the urine samples from the healthy human.…”

“…More recently, a number of separation methods have been developed and applied for the analysis of porphyrins in biological samples, including gas chromatography (GC), [21] flow-injection analysis (FIA), [22] thin-layer chromatography (TLC), [23,24] high performance liquid chromatography (HPLC), [25][26][27] and capillary electrophoresis (CE). [28,29] HPLC has obtained considerable attention for the porphyrin separation [30] with various detection techniques including immunoassay, [31] inductive coupled plasma-atomic emission spectrum (ICP-AES), [32] electrochemical mode, [33,34] and the conventional ultraviolet visible (UV-Vis) detector. [25,35,36] More sensitive detection such as fluorometry [37][38][39] and mass spectrometry (MS) [27,40] have also been reported.…”

Salting-out assisted liquid–liquid extraction with the aid of experimental design for determination of benzimidazole fungicides in high salinity samples by high-performance liquid chromatography

“…This method coupled sample clean-up (e.g. acetonitrile deproteinization) with enrichment (via salting-out extraction) has been reported for water [10,11], biological [12][13][14], food [15,16], swine muscle [17], and drug [18] sample extraction processes. Also its prospective use for high salinity sample preparation is booming.…”