Journal of Pharmaceutical and Biomedical Analysis
Short communicationSurvey of several methods deproteinizing human plasma before and within the chloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography
Highlights
► Plasma protein precipitation non-destructive to lipoproteins examined. ► Five deproteinants studied, trichloroacetic acid found as the most suitable one. ► Methyl chloroformate-mediated derivatization found compatible with the approach. ► GC–MS determination of sulfur and aromatic acids in plasma supernatant. ► High-temperature GC uncovered lipids in extracts of earlier approaches.
Introduction
Plasma is a complex body fluid containing a large proportion of proteins. These proteins often interfere with the determination of small analytes and have to be eliminated so as not to harm any subsequent instrumental assay. This task has mainly been accomplished by protein precipitation, a process normally performed with organic solvents, strong acids or by a combination of both [1], [2].
However, the studies focused on precipitation do not usually measure the rate of lipoprotein disruption that is followed by the release of lipids into the plasma supernatant with a possible contamination of both GC and LC instrumentation. Using the increasingly popular derivatization with alkyl chloroformates (RCF), which is based on a combined action of alcohol, pyridine and RCF directly on aqueous fluid [3], the plasma lipoproteins are to a certain extent disrupted. Then, liberated neutral lipids such as cholesterol esters (CHE) and triacylglycerols (TAG) are co-extracted together with the analytes of interest into the organic phase amenable to GC analysis. As a result, a progressive contamination of the capillary column with the non-eluted species might require an early column replacement. However, plasma can be advantageously deproteinized with an alcohol corresponding to the alkyl of the reagent, the supernatant basified to retain carboxylic acids as salts and neutral lipids are extracted off with hexane; the delipidated supernatant is then subjected to the RCF treatment [4], [5], [6]. On the other hand, treating serum directly with e.g. ethyl [7] or propyl [8] chloroformate in a combination with corresponding alcohols results in a massive liberation of neutral lipids and contamination of the GC system [9]. Although this phenomenon was not discussed in the mentioned papers we will bring evidence of the present lipid species by means of the corresponding GC record. In addition, the lipids and the proteins in the supernatant were also assayed by the Beckman spectrophotometric analyzer.
In this study, alternative agents of plasma (lipo)protein precipitation were tested, the impact of which on lipolysis was none or negligible. Two popular agents, acetonitrile (ACN) and trichloroacetic acid (TCA) were accompanied by methanol, the previously applied perchloric acid (PCA) [10], and an aqueous solution of phosphotungstic acid-magnesium chloride (PTM) [11]. Zinc sulfate was abandoned since yields of aminothiols in particular were low or even zero in its presence. In order to assess compatibility of the precipitants with the RCF assay, sulfur-containing amino acids and the aromatic ones were chosen as model compounds. At the same time, a complete method for their GC–MS quantification was devised including the assay of total homocysteine (tHcy); 2,3-dimercaptopropanesulfonate (DMPS) reductant favorably replaced the commonly used dithiothreitol (DTT) because of affording significantly cleaner extracts [10].
Section snippets
Materials and equipments
Fasting plasma samples consisted of aliquots of clinical material remaining after routine CLI-Hcy assays (Immulite 2500, DPC, LA, CA) performed in a clinical laboratory. Venous blood samples were collected in EDTA (±NaF) or heparin containing vials, separated by centrifugation at 2000 × g and stored at −20 °C. Sigma–Aldrich (Prague, Czech Republic) supplied all the chemicals. TCA was purchased as a 6 M aqueous solution and diluted 10-fold to a 10% solution, and ≤72% PCA was diluted to a 4%
Results and discussion
The supernatant composition after the precipitation step was evaluated in three ways: (i) by the determination of residual proteins, (ii) by the assay of total cholesterol and TAG and, (iii) using the GC–MS analysis of seven model amino acids present in low and middle μM range in plasma.
Conclusions
When small polar analytes in plasma are determined by GC following derivatization with chloroformates, the elimination of proteins is not always a prerequisite. On the other hand, more attention should be devoted to a possible disruption of the plasma lipoproteins in course of the precipitation process as a release of the high-molecular weight neutral lipids might interfere in the downstream instrumental analysis. The precipitation agents investigated here turned out to disrupt lipoproteins
Acknowledgment
This study was financially supported by project no. NS9755-4/2008 of the Internal Grant Agency of the Ministry of Health of the Czech Republic, and by the Czech Science Foundation, project P206/10/2401.
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