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ANALYSIS OF LYSOZYME IN HUMAN SALIVA USING BY OFF-LINE COMBINATION OF PREPARATIVE
ISOTACHOPHORESIS AND MASS SPECTROMETRY
MONIKA KONDEKOVÁ*, ANDREA STAOVÁ, JOZEF MARÁK
Department of Analytical Chemistry, Faculty of Natural Sciences, Comenius University in Bratislava
Mlynská Dolina CH-2, SK-842 15 Bratislava, Slovak Republic
e-mail: kondekova@fns.uniba.sk
INTRODUCTION
In the recent years, there has been visible in a literature an increased interest in the study of saliva. This body fluid
contains a vast number of protein species, e.g., the salivary peptidome of low molecular weight, comprising approximately
40-50% of the total secreted proteins, in addition to peptides generated by proteolysis of proteins of different sources. Due to
the presence of other components, in particular mucins and enzymes, some distinctive requirements and precautions related
to the sample collection, time of analysis, sample preservation and treatment are necessary to take into account for the
successful analysis of salivary peptides. More than 2000 peptides compose the salivary peptidome, from which only 400-600
are directly derived from salivary glands, suggesting an important qualitative peptide contribution of other sources, namely of
epithelial cells. The interest in saliva analysis has been growing for the clinical purposes, as it is an alternative sample to
other traditional body fluids (blood, urine) since it involves an easy and noninvasive collection [1]. For example, the study of
lysozyme secretion rates among patients with mild and severe psoriasis was realized. Lysozyme is a low-molecular-weight
cationic protein that is synthesized in and continuously released from monocytes or macrophages, and widely distributed in
human tissues and secretion glands [2].
The analysis of high molecular weight compounds present in complex biological samples is a very complicated
analytical task. There is the large number of separated analytes present in complex matrices and, therefore, also the
possibility of the occurrence of two or more substances with close retention or migration characteristics. Even using the most
efficient analytical separation systems, there is a peak overlap, which prevents reliable qualitative and quantitative analysis of
the compound of interest. This fact means that the successful analytical procedure requires the using of combination of
powerful separation technique with the sufficiently sensitive and/or selective detection technique, and very often, also using
an efficient sample preparation. Sample pretreatment techniques play a key role in a trace analysis of analytes present in
complex biological matrices.
Capillary isotachophoresis (cITP) is one of the basic modes of capillary electrophoresis (CE) using discontinuous
electrolytes system, i.e., leading electrolyte (LE) and terminating electrolyte (TE), which determine the mobility interval for
the migration of analytes from the sample. Unlike other CE techniques, isotachophoretic separation provides the selfsharpening
effect at the zone boundaries and the inherent concentration capability [3]. After reaching an isotachophoretic
steady state, the zones of separated analytes are migrating according their effective mobilities with a constant speed. The
concentration of the analyte in its zone is given by Kohlrausch regulation function and does not depend on the concentration
of the analyte in the injected sample. Mainly due to this fact ITP is usually used as an on-line sample pretreatment technique
before CZE or another (usually electrophoretic) separation technique [4,5]. Preparative capillary isotachophoresis (pITP) was
proved to be a powerful sample pretreatment technique [6]. One of the possibilities of using isotachophoresis as a sample
pretreatment technique is an off-line mode while using micro-preparative valve in single or column coupling arrangement
[7,8]. Main advantages of this technique are rapid simplification and/or reduction of complex ionic matrices, increasing the
concentration(s) of the analyte(s) in the collected fraction, well-defined sample pretreatment conditions in ITP separation
mode, isolation of analyte into well-defined fraction with known composition when discrete spacer technique is used, easily
obtainable compatibility of the whole analytical separation system when final non-CE analytical techniques is used. These
key steps offer, for example, a very suitable analytical tool for some trace analytes present in complex biological samples.
The potential of preparative isotachophoresis (pITP) in single column as a sample pretreatment before high performance
liquid chromatography (HPLC) for analysis of herbicides present in soil [9] and urine matrices [10] and determination of
flavonoids in plant extract [11] was studied.
The above facts indicate the use of the combination of highly-efficient separation techniques with sensitive and/or
selective detection. Liquid chromatography with mass spectrometry (LC-MS) or liquid chromatography with tandem mass
spectrometry (LC-MS/MS) provides unique opportunities for pharmaceutical analysis. LC/MS and LC/MS/MS methods can
be applied to a broad group of pharmaceutically important substances mainly due to the significant levels of analytical
performance parameters (sensitivity, selectivity, speed and cost-effectiveness analysis) [12]. The liquid chromatographyelectrospray
ionization source in combination with hybrid ion trap and high-resolution time-of-flight mass spectrometry
(LC-ESI-IT-TOF-MS) appears to be an effective analytical technique for the separation and identification of biologically
important substances (therapeutic drugs and their metabolites) present in various complex biological matrices. The main
advantages of this system are the high separation efficiency, short analysis time, high selectivity and sensitivity of detection
and a small amount of sample needed for analysis. LC-ESI-IT-TOF-MS is an excellent technique to obtain the chemical and
Zborník príspevkov
z 18. medzinárodnej vedeckej konferencie
"Analytické metódy a zdravie loveka", ISBN 978-80-969435-7-9
- 124 -
hotel Falkensteiner, Bratislava
11. - 14. 10. 2010