High performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry for V and Ni quantification as tetrapyrroles

A method was developed for the determination of V and Ni as tetrapyrroles by High Performance Liquid Chromatography hyphenated to Inductively Coupled Plasma Mass Spectrometry (HPLC-ICP-MS) using reversed phase and elution gradient. Chlorinated solvents and tetrahydrofuran were investigated as regard to separation time and ICP-MS detection efficiencies. The final elution gradient program started from pure methanol to a mixture of 20:80 (v/v) chloroform:methanol. External quantification of V and Ni with inorganic standards by flow injection ICP-MS, used online with HPLC, resulted in 95% of recoveries. The Limits of Detection for V during methanol elution and for Ni during the 20% chloroform gradient elution were evaluated by their minimum detectable concentrations, which were, respectively, 5 μg L^− 1 and 8 μg L^− 1. The methodology was applied to polar and resin fractions separated from a Brazilian crude oil and a sediment extract from an oil-polluted area in the Guanabara Bay, Rio de Janeiro, Brazil. Vanadium as tetrapyrroles represented the totality of V content in the polar fraction, whereas Ni was in different polar forms in the resin and sediment extract.     

Investigation of mercury-containing proteins by enriched stable isotopic tracer and size-exclusion chromatography hyphenated to inductively coupled plasma-isotope dilution mass spectrometry

In order to investigate trace mercury-containing proteins in maternal rat and their offspring, a method of enriched stable isotopic tracer (¹⁹⁶Hg and ¹⁹⁸Hg) combined with size-exclusion chromatography (SEC) coupled to inductively coupled plasma-isotope dilution mass spectrometry (ICP-IDMS) was developed. Prior to the analysis, ¹⁹⁶Hg- and ¹⁹⁸Hg-enriched methylmercury was administrated to the pregnant rats. Then the mercury-containing proteins in serum and brain cytosol of the dam and pup rats were separated by size-exclusion columns and the mercury was detected by ICP-MS. The ICP-MS spectrogram of the tracing samples showed significantly elevated ¹⁹⁶Hg and ¹⁹⁸Hg isotopic signals compared with the natural ones, indicating that the detection sensitivity could be increased by the tracer method. The contents of mercury in chromatographic fractions of the dam and pup rat brain cytosol were quantitatively estimated by post-column reverse ID-ICP-MS. The quantitative speciation differences of mercury in brain cytosol between the dam and pup rats were observed, indicating that such studies could be useful for toxicological estimation. Additionally, the isotopic ratio measurement of ¹⁹⁸Hg/²⁰²Hg in the tracing samples could be used to identify the artifact mercury species caused in the analytical procedure. The study demonstrates that the tracer method combined with high-performance liquid chromatography (HPLC)-ICP-IDMS could provide reliably qualitative and quantitative information on mercury-containing proteins in organisms.     

Method optimization and quality assurance in speciation analysis using high performance liquid chromatography with detection by inductively coupled plasma mass spectrometry

Achievement of optimum selectivity, sensitivity and robustness in speciation analysis using high performance liquid chromatography (HPLC) with inductively coupled mass spectrometry (ICP-MS) detection requires that each instrumental component is selected and optimized with a view to the ideal operating characteristics of the entire hyphenated system. An isocratic HPLC system, which employs an aqueous mobile phase with organic buffer constituents, is well suited for introduction into the ICP-MS because of the stability of the detector response and high degree of analyte sensitivity attained. Anion and cation exchange HPLC systems, which meet these requirements, were used for the seperation of selenium and arsenic species in crude extracts of biological samples. Furthermore, the signal-to-noise ratios obtained for these incompletely ionized elements in the argon ICP were further enhanced by a factor of four by continously introducing carbon as methanol via the mobile phase into the ICP. Sources of error in the HPLC system (column overload), in the sample introduction system (memory by organic solvents) and in the ICP-MS (spectroscopic interferences) and their prevention are also discussed. The optimized anion and cation exchange HPLC-ICP-MS systems were used for arsenic speciation in contaminated ground water and in an in-house shrimp reference sample. For the purpose of verification, HPLC coupled with tandem mass spectrometry with electrospray ionization was additionally used for arsenic speciation in the shrimp sample. With this analytical technique the HPLC retention time in combination with mass analysis of the molecular ions and their collision-induced fragments provide almost conclusive evidence of the identity of the analyte species. The speciation methods are validated by establishing a mass balance of the analytes in each fraction of the extraction procedure, by recovery of spikes and by employing and comparing independent techniques. The urgent need for reference materials certified for elemental species is stressed.     

Coupling nanoliter high‐performance liquid chromatography to inductively coupled plasma mass spectrometry for arsenic speciation

Nanoliter high‐performance liquid chromatography shows low consumption of solvents and samples, offering one of the best choices for arsenic speciation in precious samples in combination with inuctively coupled plasma mass spectrometry. A systematic investigation on coupling nanoliter high‐performance liquid chromatography to inductively coupled plasma mass spectrometry from instrument design to injected sample volume and mobile phase was performed in this study. Nanoflow mobile phase was delivered by flow splitting using a conventional high‐pressure pump with reuse of mobile phase waste. Dead volume was minimized to 60 nL for the sheathless interface based on the previously developed nanonebulizer. Capillary columns for nanoliter high‐performance liquid chromatography were found to be sensitive to sample loading volume. An apparent difference was also found between the mobile phases for nanoliter and conventional high‐performance liquid chromatography. Baseline separation of arsenite, arsenate, monomethylarsenic, and dimethylarsenic was achieved within 11 min on a 15 cm C₁₈ capillary column and within 12 min on a 25 cm strong anion exchange column. Detection limits of 0.9–1.8 μg/L were obtained with precisions variable in the range of 1.6–4.2%. A good agreement between determined and certified values of a certified reference material of human urine (GBW 09115) validated its accuracy along with good recoveries (87–102%).     

Arsenic and its speciation analysis using high-performance liquid chromatography and inductively coupled plasma mass spectrometry

It is known that arsenic has different toxicological properties dependent upon both its oxidation state for inorganic compounds, as well as the different toxicity levels exhibited for organic arsenic compounds. The field of arsenic speciation analysis has grown rapidly in recent years, especially with the utilization of high-performance liquid chromatography (HPLC) coupled to inductively coupled plasma mass spectrometry (ICP-MS), a highly sensitive and robust detector system. Complete characterization of arsenic compounds is necessary to understand intake, accumulation, transport, storage, detoxification and activation of this element in the natural environment and living systems. This review describes the essential background and toxicity of arsenic in the environment, and more importantly, some currently used chromatographic applications and sample handling procedures necessary to accurately detect and quantify arsenic in its various chemical forms. Applications and work using only HPLC-ICP-MS for arsenic speciation of environmental and biological samples are presented in this review.     

Multicapillary gas chromatography coupled to inductively coupled plasma-time-of-flight mass spectrometry for rapid mercury speciation analysis

A simple, rapid and accurate method on the basis of multicapillary gas chromatography (MCGC) combined with inductively coupled plasma-time-of-flight mass spectrometry (ICP-TOFMS) was developed for speciation analysis of methylmercury (MeHg⁺) and inorganic mercury (Hg²⁺). The potential of the ICP-TOFMS for transient multi-isotope detection of very short signals (peak width of 0.4 s at half peak height) was evaluated. Two injection systems (purge-and-trap (PTI) and split (SI) injections) were compared in terms of species separation resolution and transient signal profile. Using purge-and-trap injection, after in situ derivatization of the ionic mercury species with sodium tetraethylborate, a baseline separation of MeHg⁺ and Hg²⁺ was achieved within a chromatographic run of <35 s. To correct for matrix-induced ion signal variation and instrumental drift, propylmercury (PrHg⁺) was used as internal standard. Detection limits of 16 and 257 fg g⁻¹ for MeHg⁺ (as Hg) and Hg²⁺, respectively, were achieved. The analytical precision (R.S.D. (%)) for 10 successive injections of a standard mixture containing 10 pg MeHg⁺ (as Hg) and Hg²⁺ was 1.2% for MeHg⁺ and 4.1% for Hg²⁺. The method was validated by analysis of two biological certified reference materials (CRM): a dogfish muscle (DORM-2) and a freeze-dried tuna fish (CRM 464).     

Arsenic speciation by coupling high-performance liquid chromatography with inductively coupled plasma mass spectrometry

Summary An ICP-MS detector in combination with HPLC has been evaluated for the analysis of six arsenic compounds. The influence of the presence of an organic modifier in the mobile phase on arsenic response and the quality parameters of the analysis are discussed. Detection limits for arsenic species under study range from 10 to 30 pg. The determination of arsenic compounds in solutions simulating fish or sediment extracts has been used to demonstrate the applicability of this technique.     

高效色谱-电感耦合等离子体质谱法分析海藻类海产品中砷的形态

采用阴(Hamilton PRP-X100柱)阳(Dionex Ionpac CS-10柱)离子交换色谱-电感耦合等离子体质谱联用技术,分别以pH 10.3的20 mmol/L NH4HCO3和pH 2.0的5 mmol/L吡啶溶液为流动相,建立了As(Ⅲ)、As(Ⅴ)、一甲基砷酸(MMA)、二甲基砷酸(DMA)、砷甜菜碱AsB、砷糖PO4、砷糖OH、砷糖SO3、砷糖SO4砷形态的分析方法。采用微波消解法和超声溶剂提取法对不同海域10种紫菜和海带产品进行前处理,对As含量及其化学形态进行分析。实验表明,样品总砷的质量分数为1.7～38.7 mg/kg,样品萃取物中,As糖PO4和As糖OH为As的主要形态,其含量分别占可提取As的6.5%～67.7%和12.9%～86.2%,海带样品萃取物中还有As糖SO3和DMA被检测,其含量分别占可提取As的13.0%～52.1%和5.9%～17.4%。在紫菜和海带海藻类产品中,含As的化合物主要是毒性较低的有机砷。     

Functional speciation of metal-dissolved organic matter complexes by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry and deconvolution analysis

High performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (HP-SEC–ICP-MS), in combination with deconvolution analysis, has been used to obtain multielemental qualitative and quantitative information about the distributions of metal complexes with different forms of natural dissolved organic matter (DOM). High performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry chromatograms only provide continuous distributions of metals with respect to molecular masses, due to the high heterogeneity of dissolved organic matter, which consists of humic substances as well as biomolecules and other organic compounds. A functional speciation approach, based on the determination of the metals associated to different groups of homologous compounds, has been followed. Dissolved organic matter groups of homologous compounds are isolated from the aqueous samples under study and their high performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry elution profiles fitted to model Gaussian peaks, characterized by their respective retention times and peak widths. High performance size exclusion chromatography coupled to inductively coupled plasma mass spectrometry chromatograms of the samples are deconvoluted with respect to these model Gaussian peaks. This methodology has been applied to the characterization of metal–dissolved organic matter complexes in compost leachates. The most significant groups of homologous compounds involved in the complexation of metals in the compost leachates studied have been hydrophobic acids (humic and fulvic acids) and low molecular mass hydrophilic compounds. The environmental significance of these compounds is related to the higher biodegradability of the low molecular mass hydrophilic compounds and the lower mobility of humic acids. In general, the hydrophilic compounds accounted for the complexation of around 50% of the leached metals, with variable contributions of humic and fulvic acids, depending on the nature of the samples and the metals.     

盐酸提取-液相色谱-原子荧光联用技术检测水产品中甲基汞等汞化合物

建立了HCl提取,高效液相色谱与原子荧光联用技术测定水产中无机汞、甲基汞、乙基汞形态的分析方法。对前处理方法和液相色谱的最佳参数进行优化,实验表明,3种汞化合物的线性范围为0~100μg/L,相关系数(r)均优于0.9990,检出限在0.3~0.6μg/L之间,汞化合物各形态的RSD均小于5%,加标回收率在78.8%~116.8%之间,标准物质(GBW10029),(GBW09101B)中汞形态的测定值均在标准值范围内,参加甲基汞FAPAS国际比对,测定结果的Z比分数为1.0,故本方法适用于水产品中汞化合物形态的分析测定。     

Species analysis of metallothionein isoforms in human brain cytosols by use of capillary electrophoresis hyphenated to inductively coupled plasma-sector field mass spectrometry

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.     

New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry

This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of ³¹P may be used to quantify these compounds at the level of 80 μg L⁻¹, while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L⁻¹. The limit of detection was in the range of 0.07 and 0.13 mg L⁻¹. Accuracy varied with concentration, but was in the range of 3%.     

Selenium speciation in animal tissues after enzymatic digestion by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry

A procedure is described for the enzymatic digestion of tuna and mussel samples that allows the determination of selenium species by high-performance liquid chromatography in conjunction with inductively coupled plasma mass spectrometry. The species were extracted by two-step enzymatic hydrolysis with a non-specific protease (subtilisin). The selenium species were separated on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions, namely phosphate buffers at pH 2.8 and pH 6.0 as mobile phases. The method determines organic (trimethylselenonium, selenocystine, selenomethionine and selenoethionine) and inorganic selenium species (selenite and selenate), but only organic selenium species were found in the samples. The sum of identified selenium species in the sample was about 30% of the total selenium present in the enzymatic extract despite the fact that recoveries of total hydrolysed selenium were 93-102%. Trimethylselenonium ion and selenomethionine were found in both tuna and mussel samples and an unknown selenium species was also found in tuna samples.     

HPLC-ICP/MS联用同时分析中药材中的多种形态砷

利用高效液相色谱-电感耦合等离子体质谱(HPLC-ICP/MS)对中药材中的6种砷的形态(三价砷(As(Ⅲ))、五价砷(As(Ⅴ))、二甲基砷酸(DMA)、甲基砷酸(MMA)、砷甜菜碱(AsB)和砷胆碱(AsC))进行了同时分析.采用1.2mol/,L HCl浸提,Hamilton PRP-X100阴离子交换色谱柱分离...     

高效液相色谱-电感耦合等离子体质谱联用检测食品中的五种硒形态

建立了高效液相色谱-电感耦合等离子体质谱(HPLC-ICP-MS)联用检测硒酸盐(SeVI)、亚硒酸盐(SeIV)、硒代蛋氨酸(SeMet)、硒代胱氨酸(SeCys2)和硒代乙硫氨酸(SeEt)的方法。采用Hamilton PRP X-100色谱柱（250 mm×4.6 mm, 5 μm）,使用5 mmol/L的柠檬酸溶液(pH 4.5)作为流动相,电感耦合等离子体质谱(ICP-MS)检测,在21 min内可以完全分离5种硒形态。各形态硒的线性相关系数均大于0.9995, SeVI、SeIV、SeMet、SeCys2、SeEt的检出限分别为0.4、0.4、5.6、0.9、1.2 μg/L。探讨了不同提取方法的提取效果,鲜蘑菇和猪肉样品加标回收实验表明,对水溶性良好的无机硒和硒代蛋氨酸而言,采用柠檬酸溶液提取的效果非常好,SeIV和SeVI的回收率均在100%左右,SeMet的回收率为85.0%～95.3%;用蛋白酶水解提取,SeCys2和SeEt的回收率为79.9%～91.5%。该方法可完全满足食品中这5种硒形态的准确定量分析。     

Online anion exchange column preconcentration and high performance liquid chromatographic separation with inductively coupled plasma mass spectrometry detection for mercury speciation analysis

A hyphenated method for mercury speciation analysis by the coupling of high performance liquid chromatography and inductively coupled plasma mass spectrometry with the online strong anion exchange column (SAX) preconcentration was developed. The Hg analytes (Hg⁺, MeHg, EtHg and Hg²⁺) were absorbed on the SAX column preconditioned with sodium 3-mercapto-1-propanesulfonate, and then rapidly eluted (less than 16 s) by 5 μL 3% (v/v) 2-mercaptoethanol. The enrichment factors of 1025 for Hg⁺, 1084 for MeHg, 1108 for EtHg and 1046 for Hg²⁺ were obtained using 6 mL sample in a 1.5-min enrichment procedure. Rapid separation of the four mercurial compounds was achieved within 5 min on a 50-mm C₁₈ column using 0.5% (v/v) 2-mercaptoethanol as the mobile phase. The detection limits for Hg⁺, MeHg, EtHg and Hg²⁺ were 0.015, 0.010, 0.009 and 0.016 ng L⁻¹, each, and the relative standard deviations of peak height and peak area (5 ng L⁻¹ for each Hg species) were all below 5%. Mercury speciation in three freshwater, two drinking water and two seawater samples were then analyzed by the proposed method. MeHg and Hg²⁺ concentrations down to 0.14 and 0.56 ng L⁻¹ were detected in the drinking waters.     

Speciation of the bio-available iodine and bromine forms in edible seaweed by high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry

A bioavailability study based on an in vitro dialyzability approach has been applied to assess the bio-available fractions of iodine and bromine species from edible seaweed. Iodide, iodate, 3-iodo-tyrosine (MIT), 3,5-diiodo-tyrosine (DIT), bromide and bromate were separated by anion exchange chromatography under a gradient elution mode (175 mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase, and flow rates within the 0.5–1.5 mL min⁻¹ range). Inductively coupled plasma-mass spectrometry (ICP-MS) was used as a selective detector for iodine (¹²⁷I) and bromine (⁷⁹Br). Low dialyzability ratios (within the 2.0–18% range) were found for iodine species; whereas, moderate dialyzability percentages (from 9.0 to 40%) were obtained for bromine species. Iodide and bromide were the major species found in the dialyzates from seaweed, although MIT and bromate were also found in the dialyzates from most of the seaweed samples analysed. However, DIT was only found in dialyzates from Wakame, Kombu, and NIES 09 (Sargasso) certified reference material; whereas, iodate was not found in any dialyzate. Iodine dialyzability was found to be dependent on the protein content (negative correlation), and on the carbohydrate and dietary fibre levels (positive correlation). However, bromine dialyzability was only dependent on the protein amount in seaweed (negative correlation).     

Environmental application of elemental speciation analysis based on liquid or gas chromatography hyphenated to inductively coupled plasma mass spectrometry—A review

In recent years the number of environmental applications of elemental speciation analysis using inductively coupled plasma mass spectrometry (ICP-MS) as detector has increased significantly. The analytical characteristics, such as extremely low detection limits (LOD) for almost all elements, the wide linear range, the possibility for multi-elemental analysis and the possibility to apply isotope dilution mass spectrometry (IDMS) make ICP-MS an attractive tool for elemental speciation analysis. Two methodological approaches, i.e. the combination of ICP-MS with high performance liquid chromatography (HPLC) and gas chromatography (GC), dominate the field. Besides the investigation of metals and metalloids and their species (e.g. Sn, Hg, As), representing “classic” elements in environmental science, more recently other elements (e.g. P, S, Br, I) amenable to ICP-MS determination were addressed. In addition, the introduction of isotope dilution analysis and the development of isotopically labeled species-specific standards have contributed to the success of ICP-MS in the field. The aim of this review is to summarize these developments and to highlight recent trends in the environmental application of ICP-MS coupled to GC and HPLC.     

Determination of selenium species in human urine by high performance liquid chromatography and inductively coupled plasma mass spectrometry

A method developed to determine organic and inorganic selenium species in human urine samples is presented in detail. After a simple sample treatment based on elimination of non-charged organic compounds, selenium species were separated by high performance liquid chromatography (HPLC) on a Spherisorb 5 ODS/AMINO column using two different chromatographic conditions: phosphate buffers at pH 2.8 and 6.0. Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS). Trimethylselenonium ion and two unknown selenium species in urine samples were found. Selenium species were shown to have stability problems, with the maximum allowed storage time of 1 week.