Ion-pair High-performance Liquid Chromatography Research Articles

Influence of the Lipophilicity of an Ion‐Pairing Reagent on Metal Ion Separation using Ion‐Pair HPLC

Tetraalkyl ammonium salts (TAAS) of a wide range of lipophilicities are used as ion‐pairing reagents for simultaneous separation of proposed anionic metal complexes [M (H2PO4) 3]− of U(VI), Fe(III), and neutral Cu(II) using reversed‐phase ion pairing high performance liquid chromatography (RPIP‐HPLC). Tetramethylammonium bromide, tetraethyl ammonium chloride, and tetrapropyl ammonium bromide, due to their low hydrophobicities, are unable to separate U(VI) and Fe(III). Tetrabutyl ammonium perchlorate has separated the three metal ions. Tetrahexyl ammonium bromide, tetraheptyl ammonium bromide, and tetraoctyl ammonium bromide have enhanced resolution for the metal ions, but analysis time was prolonged due to the strong retention of nitrate ion. Also, equilibration time with the stationary phase increases with the increase in carbon atoms in the IPR. The potential of varied carbon containing IPRs has been compared for the analysis of U(VI) in simulated standards. The detection limit and RSD for Cu, Fe, and U are 2, 0.5, and 2 ppm and 4, 5, and 3.3%, respectively.

The efficacy and relationship between peak concentration and toxicity profile of fixed-dose-rate gemcitabine plus carboplatin in patients with advanced non-small-cell lung cancer

To investigate the efficacy and relationship between plasma concentrations at the end of infusion (C(end of infusion)) and toxicity profile of fixed-dose-rate gemcitabine plus carboplatin in chemotherapy-naive patients with advanced non-small-cell lung cancer (NSCLC). Patients were given gemcitabine by 120 min infusion [at a fixed dose rate (FDR) of 10 mg/m(2)/min] on days 1 and 8 of a 21-day cycle, immediately followed by carboplatin AUC 5 by 4 h infusion on day 1. C (end of infusion) of gemcitabine was determined by ion-pair reversed-phase high-performance liquid chromatography (HPLC). By the close-out date, in our study population, the estimated median time to tumor progression (TTP) was 7 months (95% CI 4-10 months), median overall survival (OS) was 12 months (95% CI 11.2-12.8 months). The mean value of C (end of infusion) of 21 eligible patients was 16.48 +/- 8.07 micromol/l (range 27.43-2.87 micromol/l). The main hematological toxicities were transient grade 3-4 thrombocytopenia. The mean percentages of reduction of WBC, NEC, PLTC and Hb of 21 eligible patients were 38.3 +/- 38.1%, 31.3 +/- 73.6%, 31.8 +/- 53.5% and 12.0 +/- 12.2%, respectively. The analysis of the C(end of infusion) of gemcitabine and the percentage of reduction in WBC showed a significant correlation (r(2) = 0.4575; p < 0.05). A significant correlation (r(2) = 0.5671; p < 0.05) was also observed between the percentage of reduction of PLTC and C(end of infusion) of gemcitabine infusion. The clinical data in this trial supports the further evaluation the regimen in advanced NSCLC patients, due to its predictable kinetic behavior and less severe toxicity profile than expected.

Classification and dating of black gel pen ink by ion-pairing high-performance liquid chromatography

A novel approach for classification and dating of the black gel pen ink entries on document was developed based on ion-pairing high-performance liquid chromatography (IP-HPLC). Ninety-three black gel pens were collected and divided into two groups, dye-based and pigment-based, by preliminary solubility test. The chromatographic conditions for separation of the dye-based black gel pen inks were optimized and the dye components in inks were satisfactorily separated by using 40 mmol/L tetrabutylammonium bromide as ion-pairing reagent. According to the number and the chromatographic retention times of the main dye components, the 50 dye-based inks were categorized into four classes. The inks within a class can be further identified by the percentage of each dye component. The compositional changes of the dye components in the black gel pen ink entries on paper were investigated in light and natural aging conditions and it has been found that the dye components in the ink entries underwent obvious decomposition, and the decomposing extent of the dye components was related to the aging time. The results can provide scientific evidences for dating of the suspicious black gel pen ink entries on documents.

Improved method for the determination of biogenic amines and polyamines in vegetable products by ion-pair high-performance liquid chromatography

Here, we report on the optimisation and validation of a liquid chromatographic method for the determination of 12 biologically active amines from vegetal food products in a single 40-min run. The suitability of the method was checked in five vegetal products of distinct matrix: spinach (leaves), hazelnut (high protein and fat content), banana, potato (high starch content), and milk chocolate (processed). Sample preparation consisted of a 0.6 M perchloric acid extraction from a minced homogeneous aliquot. For samples with high starch content, a previous mild hydrolytic treatment was required to prevent gel formation. The range of linearity was from 0.1 to 10 mg/l, except for serotonin and spermine (from 0.5 to 10 mg/l), and the correlation coefficient was higher than 0.997 ( P < 0.001) for all standard curves. The detection limits and the determination limit were below 0.07 and 0.2 mg/l, respectively, except for spermine, which was 0.14 and 0.4 mg/l. The precision of the method was satisfactory; the relative standard deviation obtained for each amine in each product was acceptable according to Horwitz. Recovery was between 77 and 110% for all amines, irrespective of the product.

On-line pre-reduction of Se(VI) by thiourea for selenium speciation by hydride generation

In this study, thiourea (TU) was novelly developed as a reduction reagent for on-line pre-reduction of selenium(VI) before conventional hydride generation (HG) by KBH 4/NaOH–HCl. After TU on-line pre-reduction, the HG efficiency of Se(VI) has been greatly improved and because even higher than that of the same amount of Se(IV) obtained in the conventional HG system. The possible pre-reduction mechanism is discussed. The detection limit (DL) of selenate reaches 10 pg mL − 1 when using on-line TU pre-reduction followed by HG atomic fluorescence detection. When TU pre-reduction followed by HG is used as an interface between ion-pair high performance liquid chromatography and atomic fluorescence spectrometry, selenocystine, selenomethionine, selenite and selenate can be measured simultaneously and quantitatively. The DLs of these are 0.06, 0.08, 0.05 and 0.04 ng mL − 1 , respectively, and the relative standard deviations of 9 duplicate runs for all the 4 species are less than 5%. Furthermore, it was successfully applied to Se speciation analysis of cultured garlic samples, and validated by determination of total selenium and selenium species in certified reference material NIST 1946.

Direct molecular haplotyping of multiple polymorphisms within exon 4 of the human catechol-O-methyltransferase gene by liquid chromatography–electrospray ionization time-of-flight mass spectrometry

The applicability of ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS) for the haplotyping of five SNPs (rs769223, rs4818, rs4986871, rs8192488, rs4680) located within exon 4 of the human catechol-O-methyltransferase (COMT, EC 2.1.1.6) gene is demonstrated. Two differently sized products of polymerase chain reaction--a 71-bp amplicon partially covering the sequence of a 124-bp amplicon--were used to determine unequivocally the allelic states of the single nucleotide polymorphisms linked on both chromosomes. The two amplicons were co-loaded onto the chromatographic column and simultaneously analyzed within a single gradient run. Using the described strategy, 101 individuals representing an Austrian population sample were typed. The obtained haplotype frequencies will serve as reference values in future association studies to examine the impact of the COMT gene on neuropsychiatric disorders. Additionally, two newly discovered polymorphic sites within the sequence of the COMT gene are described (a synonymous C>T mutation at the third position of the amino acid codon 99 in the soluble COMT protein or 149 in the membrane-bound COMT protein; a non-synonymous G>A substitution at the second position of the amino acid codon 95 in the soluble COMT protein or 145 in the membrane-bound-COMT protein).

Studies on the degradation of blue gel pen dyes by ion-pairing high performance liquid chromatography and electrospray tandem mass spectrometry

Ion-pairing high performance liquid chromatography (IP-HPLC) was utilized to monitor the composition changes of blue gel pen ink entries on paper stored in different light conditions and natural environment. The chromatographic conditions were optimized by comparing the separation efficiencies of the blue gel pen inks using a series of ion-pairing reagents, including ammonium carbonate, ammonium acetate, triethylamine acetate, tributylamine acetate, tetrabutylammonium bromide and dihexylammonium acetate. It has been found that tributylamine acetate was a suitable ion-pairing reagent for separation of the inks on the common C 18 column. The analysis results of the ink entries on paper in different aging conditions showed that the tendency of composition change in natural aging condition was similar with those in fluorescent light and UV light conditions, respectively. One main component dye of the blue gel pen ink, Acid Blue 9, and its degradation products were identified by ion-pairing high performance liquid chromatography coupled with electrospray tandem mass spectrometry. The results showed that the main degradation products originated from the Acid Blue 9. It gave a reasonable explanation for the changing rules of the relative content of the dyes in the blue gel pen ink. The results obtained can provide scientific evidences for dating of the blue gel pen ink entries on documents.

Selenium Speciation in Biological Samples Using a Hyphenated Technique of High-performance Liquid Chromatography and Inductively Coupled Plasma Mass Spectrometry

A hyphenated technique for chemical speciation analysis of selenium in biological samples is established. A reversed-phase ion-pair high-performance liquid chromatography is introduced for separating seleno-compounds, and meanwhile, an inductively coupled plasma mass spectrometry is combined for online elemental determination. Several standard compounds are used for the qualitative and quantitative analysis of the seleno-compounds, whereas seven selenium-containing compounds, such as SeO 4 2−, SeO 3 2−, TmSe +, SeCys, SeUr, SeMet, and SeCM, can be individually detected. Their detection limits are in the range of 0.1–1.5 μg/l, and the linear calibration for the Se concentrations is in the range of 1 to 100 μg/l. The technique is also applied to analyze human liver cytosolic extracts, human serum, and human urine. Only 50 μl of the samples are needed, and the recoveries of the spiked seleno-compounds are between 93% and 117%. The hyphenated method possesses the merits of simple operation, high sensitivity, and good reproducibility and is a feasible method for speciation analysis of small molecular seleno-compounds in different biological samples.

Comparison of Nanogel Drug Carriers and their Formulations with Nucleoside 5′-Triphosphates

The aim of the study is to synthesize and characterize nanogel carriers composed of amphiphilic polymers and cationic polyethylenimine for encapsulation and delivery of cytotoxic nucleoside analogs 5'-triphosphates (NTPs) into cancer cells. Nanogels were synthesized by a novel micellar approach and compared with carriers prepared by the emulsification/evaporation method. Complexes of nanogels with NTP were prepared; particle size and in vitro drug release were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed by ion-pair high-performance liquid chromatography. Binding to isolated cellular membranes, cellular accumulation and cytotoxicity were compared using breast carcinoma cell lines CL-66, MCF-7, and MDA-MB-231. In vivo biodistribution of the 3H-labeled NTP encapsulated in different types of nanogels was evaluated in comparison to the injected NTP alone. Nanogels with a particle size of 100-300 nm in the unloaded form and less than 140 nm in the NTP-loaded form were prepared. An in vitro release of NTP was >50% during the first 24 h. Nanogel formulations ensured increased NTP drug stability against enzymatic hydrolysis as compared to the drug alone. Pluronic-based nanogels NG(F68), NG(F127), NG(P85), and NGM(P123) demonstrated 2-2.5 times enhanced interaction with cellular membranes and association with various cancer cells compared to NG(PEG). Among them, NG(F68) and NG(F127) exhibited the lowest cytotoxicity. Injection of nanogel-formulated NTP significantly modulated the drug accumulation in different mouse organs. Nanogels composed of Pluronic F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy.

Determination of polyunsatured fatty acid monoepoxides by high performance liquid chromatography–mass spectrometry

Despite the implication of polyunsaturated fatty acid monoepoxides in a large panel of biological effects, few methods allowing their separation in a single run are available. We describe here a simple method based on reversed-phase ion-pair high-performance liquid chromatography (RP-HPLC) and developed to successfully separate the various monoepoxides of eicosatrienoic, arachidonic, eicosapentaenoic and docosahexaenoic acids. These compounds were easily identified by liquid chromatography–mass spectrometry (LC–MS) with atmospheric pressure chemical ionisation owing to the volatility of counter-ion species. Compared to established methods, this new protocol proved its ability to totally resolve, in a single run, all of the different regioisomeric epoxides. In the long run, this method will demonstrate its efficacy to give insights into the cytochrome P450-dependent metabolism of polyunsatured fatty acids (PUFAs) and the generation of physiologically active epoxy-derivatives.

Simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water samples by ion-pair high-performance liquid chromatography

A simple, fast, sensitive and accurate reversed-phase ion-pair HPLC method for simultaneous determination of nitrite and nitrate in atmospheric liquids and lake waters has been developed. Separations were accomplished in less than 10 min using a reversed-phase C 18 column (150 mm × 2.00 mm i.d., 5 μm particle size) with a mobile phase containing 83% 3.0 mM ion-interaction reagent tetrabutylammonium hydroxide (TBA-OH) and 2.0 mM sodium phosphate buffer at pH 3.9 and 17% acetonitrile (flow rate, 0.4 mL/min). UV light absorption responses at 205 nm were linear over a wide concentration range from 100 μg/mL to the detection limits of 10 μg/L for nitrite and 5 μg/L nitrate. Quantitation was carried out by the peak area method. The relative standard deviation for the analysis of nitrite and nitrate was less than 3.0%. This method was applied for the simultaneous determination of nitrite and nitrate in dew, rain, snow and lake water samples collected in southeast Massachusetts. Nitrate was found being present at 4.79–5.99 μg/mL in dew, 1.20–2.63 μg/mL in rain, 0.32–0.60 μg/mL in snow and 0.12–0.23 μg/mL in lake water. Nitrite was only a minor species in dew (0.62–0.83 μg/mL), rain (<0.005–0.14 μg/mL), snow (0.021–0.032 μg/mL) and lake water (0.12–0.16 μg/mL). High levels of nitrite and nitrate observed in dew water droplets may constitute an important source of hydroxyl radicals in the sunny early morning.

Evaluation of CD detection in an HPLC system for analysis of DHEA and related steroids

The biological importance of dehydroepiandrosterone (DHEA) is reflected by the fact that DHEA is a crucial precursor of the biosynthesis of the steroidal sex hormones. Simultaneous separation of DHEA, dehydroepiandrosterone sulfate (DHEA-S), pregnenolone, androstenedione and testosterone has been accomplished by reversed-phase ion-pair high-performance liquid chromatography (RP-IP-HPLC) based on isocratic elution applying circular dichroism (CD) detection at 295 nm. Addition of tetrabutylammonium hydrogensulfate to the mobile phase increases the retention of DHEA-S on the C8-silica column by an apparent ion-pairing mechanism without affecting the retention of the other (non-ionic) steroids. CD spectroscopy provides highly selective detection of compounds possessing optically active absorption bands and the separation is even more selective in the higher wavelength range applied. The linearity of the steroid concentration (c, mg mL(-1)) versus peak area was tested in the concentration range of 0.5-2 mg mL(-1) (injected quantities were 10-40 microg). The relative standard deviation (RSD) values for DHEA and DHEA-S indicated a good intra-assay and inter-assay precision of the method.

Determination of risedronate in rat plasma samples by ion-pair high-performance liquid chromatography with UV detector

In the present work, an analytical method for determination of risedronate, a member of bisphosphonates, is described for the routine analysis in rat plasma. Sample pre-treatment involves protein precipitation, co-precipitation with calcium at alkaline pH, hydrolysis of possible derivatives of pyrophosphate and reprecipitation. A good separation was obtained by using a reversed-phase column (Hypersil ODS-2 C 18, 4.6 mm × 250 mm, 5 μm). The mobile phase was an aqueous solution of buffer (contained 1.5 mM EDTA-2Na, 1 mM sodium etidronate, 11 mM sodium phosphate and 5 mM tetrabutylammonium bromide as ion-pair reagent) – methanol (88:12, v/v) adjusted to pH 6.75 using 1 M NaOH. The flow rate was 1 ml min −1. UV detection ( λ = 262 nm) was used to quantitate risedronate in the concentration range of 10–500 ng ml −1. The limit of detection and quantitation for risedronate were 7 and 10 ng ml −1, respectively. The method was applied successfully to plasma samples from Wistar rats undergoing oral administration of risedronate mini-pills. Precision, extraction recoveries, as well as accuracy results, were satisfactory and no interference was found at the retention time of risedronate. Hence, the method is suitable for monitoring risedronate in rat plasma.

The isolation and purification of glucoraphanin from broccoli seeds by solid phase extraction and preparative high performance liquid chromatography

Plant foods contain not only essential nutrients, e.g. protein, amino acids, vitamins and minerals, but also phytochemicals that have added health benefits. One such class of phytochemicals are the glucosinolates. Glucosinolates, particularly glucoraphanin, are predominant in plants of the Brassica genus, most notably in vegetables such as broccoli. There is a growing interest in the role glucoraphanin plays in chemoprotection and as a result there is a requirement to accurately determine the levels of glucoraphanin in vegetable products. Reverse phase ion pair high performance liquid chromatography (HPLC) is the method of choice; however, this work has been hindered by the lack of available standard reference materials. Broccoli seeds, which are particularly rich in glucoraphanin (20–50 mg/g), have proved to be ideal for the isolation of glucoraphanin on the preparative scale. A novel preparative scale HPLC method with simple compound recovery has been developed to meet the need for a glucoraphanin standard.

Accurate determination of allelic frequencies in mitochondrial DNA mixtures by electrospray ionization time-of-flight mass spectrometry

The mitochondrial locus 16519T/C was used as a model for the evaluation of the benefits of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS assay) for the determination of allelic frequencies of single nucleotide polymorphisms. This marker has gained interest in forensic science owing to its ability to increase the discrimination power of mitochondrial DNA testing as a consequence of its high variability across various populations. In a first set of experiments, artificial mitochondrial DNA mixtures prepared from all four theoretically possible 16519 alleles served as samples. Any allele occurring at a frequency of as low as 1-5% was unequivocally detectable irrespective of the kind of allelic mixture. Measured and expected allelic frequencies correlated well following correction of observed experimental bias, which was most probably attributable to differential PCR amplification and/or preferential ionization. For thirteen different T/C mixtures with C contents in the range 1.0-99.0%, an average error of 1.2% and a maximum error of 2.2% were observed. Furthermore, ICEMS was applied to the quantitative genotyping of eight selected individuals of which four were heteroplasmic with C contents in the range 1.9-34.1%. To check the reliability of these results, allelic proportions were additionally determined by a cloning assay. The results of the two assays correlated well (R (2)=0.9971). In all cases, deviations were obtained that were smaller than 5.4%. The overall observed assay performance suggests that the described mass spectrometric technique represents one of the most powerful assays for the determination of allelic frequencies available today.

Reversed phase ion-pairing HPLC-ICP-MS for analysis of organophosphorus chemical warfare agent degradation products

The separation and analysis of three organophosphorus chemical warfare degradation products is described. Ethyl methylphosphonic acid (EMPA, the major hydrolysis product of VX), isopropyl methylphosphonic acid (IMPA, the major hydrolysis product of Sarin (GB)), and methylphosphonic acid (MPA, the final hydrolysis product of both) were the analytes and were separated by reversed phase ion-pairing high-performance liquid chromatography (RP-IP-HPLC) with the use of myristyl trimethylammonium bromide as ion-pairing reagent and an ammonium acetate–acetic acid buffer system (pH 4.85). An Agilent 7500ce inductively coupled plasma mass spectrometer (ICP-MS) equipped with collision/reaction cell technology was coupled to the chromatographic system for detection of 31P and 47PO+. Historically, ICP-MS detection of phosphorus has been limited due to its high first ionization potential (10.5 eV) and the presence of severe nitrogen polyatomic interferences (such as 14N16O1H+ and 15N16O+) overlapping its only isotope at m/z = 31. Implementation of an octopole reaction cell with helium as the cell gas allowed for removal of the nitrogen polyatomic interferences and reduction of background signal. Detection limits for EMPA, IMPA, and MPA were found to be 263, 183 and 139 pg mL−1, respectively, with separation in less than 15 min. The developed method was successfully applied to the analysis of spiked environmental water and soil samples.

Enzymatic Redesigning of Biologically Active Heparan Sulfate

Heparan sulfate carries a wide range of biological activities, regulating blood coagulation, cell differentiation, and inflammatory responses. The sulfation patterns of the polysaccharide are essential for the biological activities. In this study, we report an enzymatic method for the sulfation of multimilligram amounts of heparan sulfate with specific functions using immobilized sulfotransferases combined with a 3'-phosphoadenosine 5'-phosphosulfate regeneration system. By selecting appropriate enzymatic modification steps, an inactive precursor has been converted to the heparan sulfate having three distinct biological activities, associated with binding to antithrombin, fibroblast growth factor-2, and herpes simplex virus envelope glycoprotein D. Because the recombinant sulfotransferases are expressed in bacteria, and the method uses a low cost sulfo donor, it can be readily utilized to synthesize large quantities of anticoagulant heparin drug or other biologically active heparan sulfates.

Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages

To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.

Determination of cyclamate in foods by high performance liquid chromatography–electrospray ionization mass spectrometry

A high sensitive method for determination of cyclamate in foods by ion-pair high-performance liquid chromatography–electrospray ionization mass spectrometry was developed and validated. The separation was achieved on a C 8 column with 5 mM tris(hydroxymethyl)aminomethane aqueous solution (pH 4.5, adjusted by acetic acid) as mobile phase with an isocratic mode. The quantification of target compound was completed using a selected ion recording (SIR) at m/ z 178 obtained from ESI-mode. Tiopronin was used as internal standard for the quantification of cyclamate. The correlation coefficient of the calibration curve were better than 0.996, in the range of 50–5000 ng/mL. The limit of detection is 5 ng/mL, the limit of quantification is 20 ng/mL. The inter- and intra-day accuracy, precision were investigated in detail. The method can be used to monitor effectively the content of the artificial sweetener in foods. The method has obvious merits such as high sensitivity, specificity and simply versus other methods reported.

Selenium speciation in Agaricus bisporus and Lentinula edodes mushroom proteins using multi-dimensional chromatography coupled to inductively coupled plasma mass spectrometry

In this study, selenium species from Se containing proteins in mushrooms ( Agaricus bisporus and Lentinula edodes) were investigated with size-exclusion liquid chromatography coupled to UV and inductively coupled plasma mass spectrometry (ICP-MS). Different protein extraction protocols were investigated. Variability of the fractionation patterns with three extraction media (0.1 M NaOH, 30 mM Tris–HCl, and enzymatic digestions) was evaluated for both mushroom types. A 24 h Tris–HCl extraction followed by acetone addition was found to be optimal for protein precipitation. Presumably protein bound selenoamino acids were released using enzymes (proteinase K, protease XIV and trypsin). The selenium speciation of the proteolytic extract of the water soluble proteins fraction was carried out by using reversed-phase ion-pairing high performance liquid chromatography (RP-HPIPC) coupled on-line to ICP-MS for selenium specific detection. Selenocystine, selenomethionine, methylselenocysteine and inorganic selenium were established in both samples utilizing retention time standards and standard additions to the sample.