Journal of Pharmaceutical and Biomedical Analysis 67–68 (2012) 159–162 Contents lists available at SciVerse ScienceDirect Journal of Pharmaceutical and Biomedical Analysis jou rn al h om epage: www.elsevier.com/locate/jpba Short communication Survey of several methods deproteinizing human plasma before and within the chloroformate-mediated treatment of amino/carboxylic acids quantitated by gas chromatography Petr Huˇ sek a,b,∗ , Zdenˇ ek ˇ Svagera a,c , Dagmar Hanzlíková a , Petr ˇ Simek b a Institute of Clinical Biochemistry, Faculty Hospital, 17. listopadu 1790, CZ-70852 Ostrava, Czech Republic b Biology Centre, Czech Academy of Sciences, Braniˇ sovská 31, CZ-370 05 ˇ Ceské Budˇ ejovice, Czech Republic c Faculty of Medicine, University of Ostrava, Dvoˇ rákova 7, 701 03 Ostrava 1, Czech Republic a r t i c l e i n f o Article history: Received 20 February 2012 Received in revised form 19 April 2012 Accepted 19 April 2012 Available online 7 May 2012 Keywords: Plasma (lipo)protein precipitation Chloroformate-treated supernatant S-amino acids GC–MS assay a b s t r a c t Trichloroacetic acid, perchloric acid, phosphotungstic acid, acetonitrile, methanol and some other organic solvents were evaluated for their ability to provide protein and lipid-free plasma supernatants. The residual proteins, total cholesterol and triacylglycerols were assayed in the supernatant on a Beckman Analyzer instrument. The free cholesterol and the neutral lipids were further analyzed by means of high-temperature GC analysis. The conditions for the deproteinizing step were optimized for minimal lipoprotein disruption. A substantial difference regarding contamination by the lipids was found if the plasma supernatant or the whole serum were treated with an alkyl chloroformate reagent. Three plasma sulfur amino acids and the aromatic ones were chosen as model compounds to evaluate compatibility of the precipitation methods with a subsequent methyl chloroformate-mediated derivatization and GC–MS analysis. The results of the total homocysteine assay matched well with that obtained using a commercial immunoassay. Precipitation with trichloroacetic acid has proven to be a method of choice for the analysis of the acido-basic analytes by GC–MS via chloroformates. © 2012 Elsevier B.V. All rights reserved. 1. 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 accom- plished 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 usu- ally measure the rate of lipoprotein disruption that is followed by the release of lipids into the plasma supernatant with a pos- sible 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 ∗ Corresponding author at: Institute of Clinical Biochemistry, Faculty Hospital, 17. listopadu 1790, CZ-70852 Ostrava, Czech Republic. Tel.: +420 597374092/387775286; fax: +420 387775287. E-mail addresses: petr.husek@fno.cz, husek@bclab.eu (P. Huˇ sek). co-extracted together with the analytes of interest into the organic phase amenable to GC analysis. As a result, a progressive contam- ination 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 [4–6]. 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 con- tamination 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 pre- cipitation 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 pre- viously applied perchloric acid (PCA) [10], and an aqueous solution of phosphotungstic acid-magnesium chloride (PTM) [11]. Zinc sul- fate was abandoned since yields of aminothiols in particular were low or even zero in its presence. In order to assess compatibility 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.04.027