Elsevier

Analytica Chimica Acta

Volume 421, Issue 2, 29 September 2000, Pages 147-153
Analytica Chimica Acta

Speciation analysis of selenium in garlic by two-dimensional high-performance liquid chromatography with parallel inductively coupled plasma mass spectrometric and electrospray tandem mass spectrometric detection

https://doi.org/10.1016/S0003-2670(00)01039-4Get rights and content

Abstract

Speciation of selenium in garlic harvested in naturally seleniferous soil was investigated. The sample was leached with water and the aqueous extract was fractionated by preparative size-exclusion chromatography. Selenium was found in only one (low-molecular mass) fraction. The chromatographic purity of the fraction was verified by reversed-phase chromatography with inductively coupled mass spectrometric detection. Again, one major signal accounting for more than 95% of the total selenium was observed. The heartcut fraction containing this compound produced an intense peak in an electrospray mass spectrum with the selenium pattern centered at m/z 313 (80Se). Protonated molecular ions corresponding to the four Se isotopes gave rich fragmentation patterns by collision induced dissociation that allowed the identification of the selenium species to be γ-glutamyl-Se-methylselenocysteine without the need for an authentic standard.

Introduction

Selenium is an essential component of glutathion peroxidase which is an important enzyme for processes that protect lipids in polyunsaturated membranes from oxidative degradation. Several lines of scientific inquiry suggest that an increased risk of cancer occurs as a result of low concentrations of selenium in the diet [1], [2]. This has triggered interest in selenium-accumulating plants, garlic or yeast, resulting in several reports on their cancer preventing activity [2], [3].

The absorption, tissue distribution, and body retention of selenium is dependent on the chemical species of the element present in food and food supplements [4]. For this reason, speciation of selenium has become a hot topic in analytical chemistry of natural products. Most of studies focused on Allium plants (garlic, onion) [5], [6], [7], [8], [9], [10], selenized yeast [6], [7], [8], [9], [10], [11], [12], [13], [14], phytoremediation samples [7], [9], and fresh [15] and cooked [16] fish. Since most of selenium species present are non volatile, the coupling of HPLC with inductively coupled plasma mass spectrometry (ICP MS) has been the primary analytical tool in these investigations [17].

The major problem in analytical chemistry of naturally-occurring selenium species remains their identification. Indeed, even when Se-specific detection is used, chromatograms contain a large number of peaks of which the identity cannot be properly established. The two major reasons are (i) the unknown chromatographic purity of signals recorded and (ii) the lack of standards necessary to match the retention times of analyte signals. The approaches developed to alleviate these difficulties have included the synthesis of a large number of Se analogues of amino acids supposed to exist in these plants for peak matching [7], [8], [9], increasing the chromatographic resolution by the choice of ion-pairing reagents which extend the range of elution of selenocompounds and, thus, improve the separation efficiency [8], [9], and the use of electrospray mass spectrometry [8], [9], [10], [14], [16], [18]. All of them have represented a major contribution to the knowledge of selenium speciation in natural samples despite many drawbacks. They enabled identification of selenomethionine, Se-methylselenocysteine, γ-glutamyl derivatives of Se-methylcysteine and selenomethionine, and Se-adenosyl/homocysteine in garlic and yeast [8], [9], [10], [14], [18].

Indeed, synthesis of standards is cumbersome and the choice of compounds to be synthesized lacks sound premises. The unambiguous identification on this basis requires the certitude about the chromatographic purity of a signal which can be obtained only by the use of a number of orthogonal chromatographic approaches and spiking experiments. In the majority of studies (all except one [14]) mass spectrometric data were obtained by the comparison of mass spectra of sample (chromatographic peak) with the reference spectra of previously synthesized standards. Mass spectrometry of protonated molecular ions provides information about the molecular mass but, since two compounds may have the same molecular mass, the use of a complementary HPLC technique (retention time standard is required) was advised. Also, mass spectrometry in the scanning mode lacks sensitivity. The use of more sensitive single ion monitoring and multiple reaction monitoring data acquisition modes in routine chromatography is hampered due to unknown molecular masses and the lack of knowledge of the fragmentation patterns of the analyte compounds.

In our former study, we proposed an approach based on the isolation of the major selenocompound in selenized yeast followed by its identification on the basis of the fragmentation of protonated molecular ions for the two adjacent most abundant selenium isotopes [14], and further confirmed using an authentic sulfur analogue. Electrospray collision induced dissociation (CID) MS was applied below to identify the major selenocompounds in garlic grown naturally in a seleniferous area of China. It is part of an ongoing study to establish a library of mass spectrometric data for naturally occurring organoselenium compounds. The modified purification procedure was based the use of two-dimensional (size-exclusion-reversed-phase) chromatography.

Section snippets

Instrumentation and materials

For preparative size-exclusion chromatography a 700 mm×16 mm column (Pharmacia, Uppsala, Sweden) was filled with G-75 Sephadex Gel (Pharmacia) according to the manufacturer’s protocol. The sample and mobile phase was pumped with a peristaltic pump (Minipuls3, Gilson, France). Fractions were collected using an automatic fraction collector (Dynamax FC-2). Selenium in the effluent was monitored (off-line) using the PE ELAN 6000 (PE-SCIEX, Ontario) ICP mass spectrometer fitted with a cross-flow

Extraction of selenocompounds

A sample of 0.2 g was extracted in 5 ml deionized water by stirring for 1 h at 85–90°C, centrifuged (4000 rps, 30 min) and filtered (0.45 μm).

Preparative size-exclusion chromatography

A sample of 5 ml of the extract was filtered for the removal of particles and passed on the Sephadex G75 column. The eluent was 1% (v/v) acetic acid (pH 2.97) pumped at a rate of 0.7 ml min−1. The eluate was collected every 2 min giving the equivalent of about 1.4 ml in each fraction. The duration of the preparative chromatography run was 3.20 min. Selenium was

Purification of selenocompounds in garlic

In contrast to yeast, water soluble selenium in garlic accounts for ca. 85–95% of the total concentration of this element. Size-exclusion chromatography was chosen as the technique for the preliminary screening of the extract for the presence of selenium species based on the literature reports for fish [15] and yeast [13] that showed good recovery (percent of selenium injected that left the column) in this type of chromatography. Fig. 1 shows that soluble selenium in garlic is present almost

Conclusions

Tandem mass spectrometry is an attractive technique allowing the identification of selenocompounds in natural products without standards available. In the case of garlic, the use of orthogonal size-exclusion (to isolate amino acid fraction) and reversed-phase (to remove salts) chromatographic mechanisms leads to a remarkable purity of γ-glutamyl-Se-methylselenocysteine for the characterization by MS/MS. The knowledge of fragmentation patterns may allow in future the use of multiple reaction

Acknowledgements

W. Yang gratefully acknowledges post-doctoral grant from the Region Centre. The authors thank Dr. Hubert Chassaigne for help with ES MS/MS measurements.

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