| Selenium is recognized as a necessary nutritional element, which plays important physiological and biochemical roles in the body, such as anti-oxidation, improvement of immunity, anti-aging etc. Both selenium deficiency and excess have adverse effects on human health. The safety dose of selenium is narrow and its toxicity and bioavailability depend largely on the chemical speciation. So the establishment of methods for the actual chemical form of Se speciation analysis is of great significance. The concentration of Se speciation is very low in the human body, which requires methods of high sensitivity and accuracy. In this study, a method for quantification of the total Se by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)and a method for analysis of Se speciation in human serum and urine by high performance liquid chromatography (HPLC)-dynamic reaction cell(DRC)-inductively coupled plasma mass spectrometry (ICP-MS) were developed and validated. The information of the methods as follows:PART I:A method for detection of total selenium in human serum and urine by ICP-MS is developed. Three different pre-treatment methods are compared: microwave digestion, the direct dilution and water bath digestion. In addition, the differences between standard mode (STD) and dynamic reaction cell mode (DRC) are given. It is shown that selecting the microwave digestion for serum samples while diluting for urine samples as pre-treatment methods and Iridium as an internal standard element and82Se as the determination isotope in the STD mode. The correlation coefficient of the calibration curves is0.999in0.00-10.0ng/mL. The detection limit of serum and urine are0.24ng/mL and0.23ng/mL, the recoveries are in the range of81～123%and78～121%and RSD are less than7.0%and5.0%respectively. When in the DRC mode, choosing78Se as the determination isotope and Indium as the internal standard element, the correlation coefficient of the calibration curves is0.999in0～10ng/mL concentration range. The detection limit of total Se in serum and urine are0.021ng/mL and0.014ng/mL, the recoveries are in the range of86%～115%and82～96%, the RSD are less than6.0%and4.0%respectively. The results show that the quantification of Se in serum and urine samples in standard mode by ICP-MS is accurate and reliable, which can meet the requirements of determination. Dynamic reaction cell mode is more accurate and reliable, using O2as the reaction gas could effectively eliminate the interference of polyatomic molecular ions on the determination of78Se, results in ten times lower detection limit than normal model and precision raise. So DRC is expected to be applied in the field of determination of selenium speciation.PART Ⅱ:A hyphenated technique is developed and optimized to simultaneously determining four different selenium species,including Se(Ⅳ); Se(Ⅵ); selenocystine(SeCys) and selenomethionine(SeMet), by high performance liquid chromatography-inductively coupled plasma mass spectrometry. The operating parameters of chromatography and mass spectrometry are optimized to get the best working conditions. Ammonium citrate and methanol are used as mobile phase. Anion exchange column is HamiltonPRP-X100. Mobile phase A is composed of0.50mM ammonium citrate-2%(Ⅴ/Ⅴ) methanol(pH3.70), and mobile phase B is composed of20mM ammonium citrate-2%(Ⅴ/Ⅴ) methanol (pH8.00). When gradient elution programm(0minl00%A→1.5min100%A→2.0min100%B→8.0min100%B→10.0min100%A) is used, the four Se species can be separated in6min. Ultra-filtration and direct filtration are applied to serum and urine samples as pre-treatment respectively. The results show that the working curve is linear in the0.00-50.Ong/mL range and the correlation coefficient is greater than0.999. The detection limits for Se(Ⅳ); Se(Ⅵ); SeCys and SeMet are0.17;0.24;0.43;0.49ng/mL while limit of quantification are0.56;0.80;1.4;1.6ng/mL respectively. The recoveries of spiked selenium speciation in serum samples are between82%and117%and the RSD are less than8%. The recoveries of spiked selenium speciation in urine samples are ranged from81to121%and the RSD are less than7%. This method is applied to analyze serum and urine samples. The concentrations of Se(IV) in serum samples are approximately2.6ng/mL. The method is simple, sensitive, repeatable and suitable for selenium speciation analysis in human serum and urine samples.
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