Synthesis and characterization of isotopically enriched methylmercury (CH3201Hg+)

A simple procedure for the synthesis of an important standard, isotopically enriched methylmercury, which is not commercially available, has been established successfully. The isotopically enriched standard synthesized is utilized in conventional isotope dilution mass spectrometry (IDMS), as well as in speciated IDMS (SIDMS), for determination of the true concentration of methylmercury in environmental samples. The CH₃²⁰¹Hg⁺ standard has been synthesized from commercially available ²⁰¹HgO and tetramethyltin. The synthesis time required is 1 h at 60°C. The product is highly pure, yielding more than 90% as ²⁰¹Hg in CH₃²⁰¹Hg⁺. Hazardous dimethylmercury does not occur during this synthesis procedure. The product synthesized was analyzed using high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (ICP‐MS) and ICP‐MS alone in order to determine its concentration, isotopic composition and purity. The stability of the product was also evaluated for over 6 months and found to be stable at 4°C in the dark. The isotopically enriched methylmercury synthesized can be used in SIDMS and IDMS analyses as a standard. Copyright © 2003 John Wiley & Sons, Ltd.     

同位素内标-液相色谱-串联质谱法同时测定粮食及其制品中的5种真菌毒素

建立了同位素内标-液相色谱-串联质谱快速测定粮食及其制品中玉米赤霉烯酮(ZON)、雪腐镰刀菌烯醇(NIV)、脱氧雪腐镰刀菌烯醇(DON)及其衍生物3-乙酰基脱氧雪腐镰刀菌烯醇(3-ACDON)和15-乙酰基脱氧雪腐镰刀菌烯醇(15-ACDON)5种真菌毒素的分析方法。以乙腈-水(84∶16,v/v)为提取液,采用多功能净化柱净化,同位素内标法定量。5种真菌毒素在各自的线性范围内线性关系良好,相关系数(r2)均大于0.99,检出限(LOD,S/N=3)为5~20μg/kg。大麦、小麦、燕麦、玉米等9种代表性粮食及其制品在3个不同添加水平下的加标回收率为84.2%~114.5%,相对标准偏差(RSD)为0.4%~9.9%(n=6)。该法操作简单,成本低,准确可靠,灵敏度高,可同时检测粮食及其制品中的5种真菌毒素。     

High yield synthesis and characterization of isotopically enriched monoethylmercury chloride (C2H5201HgCl)

An important but commercially unavailable compound isotopically enriched monoethylmercury chloride (C₂H₅²⁰¹HgCl), has been synthesized from commercially available ²⁰¹HgO (98.11% enriched isotopic purity) and tetraethyltin. The required synthesis time is 1 h at 90 °C, and the product is the single product of monoethylmercury chloride, yielding more than 95% as ²⁰¹Hg in C₂H₅²⁰¹Hg⁺ (98.19 ± 0.22% enriched isotopic purity). The synthesized product was analyzed with high‐performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC‐ICP‐MS) to determine its concentration, isotopic composition and purity. The synthetic isotopically enriched monoethylmercury synthesized can be used in speciated isotope dilution mass spectrometry (SIDMS) and isotope dilution mass spectrometry (IDMS) analyses as a standard. Copyright © 2005 John Wiley & Sons, Ltd.     

Trace iodine quantitation in biological samples by mass spectrometric methods: The optimum internal standard

Accurate quantitation of iodine in biological samples is essential for studies of nutrition and medicine, as well as for epidemiological studies for monitoring intake of this essential nutrient. Despite the importance of accurate measurement, a standardized method for iodine analysis of biological samples is yet to be established. We have evaluated the effectiveness of ⁷²Ge, ¹¹⁵In, and ¹²⁹I as internal standards for measurement of iodine in milk and urine samples by induction coupled plasma mass spectrometry (ICP-MS) and of ³⁵Cl¹⁸O₄⁻, ¹²⁹I⁻, and 2-chlorobenzenesulfonate (2-CBS) as internal standards for ion chromatography-tandem mass spectrometry (IC-MS/MS). We found recovery of iodine to be markedly low when IC-MS/MS was used without an internal standard. Percent recovery was similarly low using ³⁵Cl¹⁸O₄ as an internal standard for milk and unpredictable when used for urine. 2-Chlorobenzebenzenesulfonate provided accurate recovery of iodine from milk, but overestimated iodine in urine samples by as much as a factor of 2. Percent recovery of iodine from milk and urine using ICP-MS without an internal standard was ∼120%. Use of ¹¹⁵In predicted approximately 60% of known values for both milk and urine samples. ⁷²Ge provided reasonable and consistent percent recovery for iodine in milk samples (∼108%) but resulted in ∼80% recovery of iodine from urine. Use of ¹²⁹I as an internal standard resulted in excellent recovery of iodine from both milk and urine samples using either IC-MS/MS and ICP-MS.     

Isotope dilution-thermal ionisation mass spectrometric analysis for tin in a fly ash material

Isotope dilution-thermal ionisation mass spectrometry (ID-TIMS) analysis has been applied to the determination of tin in a fly ash sample supplied by the EC Joint Research Centre (Ispra, Italy). The proposed procedure includes the silica gel/phosphoric acid technique for tin thermal ionisation activation and a strict heating protocol for isotope ratio measurements. Instrumental mass discrimination factor has been previously determined measuring a natural tin standard solution. Spike solution has been prepared from ¹¹²Sn-enriched metal and quantified by reverse isotope dilution analysis. Two sample aliquots were spiked and tin was extracted with 4.5 M HCl during 25 min ultrasound exposure time. Due to the complex matrix of this fly ash material, a two-step purification stage using ion-exchange chromatography was required prior TIMS analysis. Obtained results for the two sample-spike blends (10.11 ± 0.55 and 10.50 ± 0.64 μmol g⁻¹) are comparable, both value and uncertainty. Also a good reproducibility is observed between measurements. The proposed ID-TIMS procedure, as a primary method and due to the lack of fly ash reference materials certified for tin content, can be used to validate more routine methodologies applied to tin determination in this kind of samples.     

A multiple-reaction-monitoring mass spectrometric method for simultaneous quantitative analysis of five plasma apolipoproteins

A multiplexed targeted proteomic assay using a mTRAQ-MRM/MS-based approach was developed and assessed to systematically quantify the relative expressions of five candidate plasma apolipoproteins that have been previously shown to be dysregulated in neuropsychiatric disorders and cognitive dysfunction:apolipoprotein H(APOH),apolipoprotein J(APOJ),apolipoprotein A4(APOA4),apolipoprotein E(APOE),and apolipoprotein D(APOD).The peptides and transitions of each APO were carefully selected according to the tandem MS signals acquired on a TripleTOFTM 5600,followed by optimization of the declustering potential and collision energy voltages for transitions on a QTRAP 5500.Our results showed that the collision energies of mTRAQ-labeled peptides were approximately 15%–20%higher than corresponding non-labeled peptides.Through optimized transitions and parameters,we analyzed the relative abundances of the five APOs in human plasma with and without depletion of high abundant proteins.The results indicated that the MRM signals of four target APOs were significantly increased after depletion,while the MRM signal of one APO,APOD,was decreased.Furthermore,the relative abundances of the five target APOs in healthy human plasma were stable,and the ranking of these proteins according to their MS responses changed slightly.Therefore,we deduced that the rank order of the MS signals for these target proteins can be developed as a diagnostic signature for diseased plasma.     

同位素稀释液相色谱-串联质谱法测定化妆品中的N-亚硝基二乙醇胺

建立了化妆品中N-亚硝基二乙醇胺(NDELA)的同位素稀释液相色谱-串联质谱分析方法。水溶性化妆品样品以水为提取溶剂提取,样品提取液经高速离心处理后,上清液过Oasis HLB固相萃取柱净化。脂溶性化妆品样品用二氯甲烷和水混合溶剂进行液-液分配萃取。NDELA经Waters Atlantis T3色谱柱(150 mm×2.1 mm,3 μm)分离后在多反应监测模式下进行串联质谱定性及定量分析,以d8-NDELA为内标定量。NDELA的方法定量限为50 μg/kg;在50-250 μg/kg的3个添加水平范围内的平均回收率为89.1%～98.2%,日内精密度均小于9%,日间精密度均小于11%。该方法能够满足化妆品中NDELA的检测要求。     

Evaluation of online carbon isotope dilution mass spectrometry for the purity assessment of synthetic peptide standards

We present a novel method for the purity assessment of peptide standards which is applicable to any water soluble peptide. The method is based on the online ¹³C isotope dilution approach in which the peptide is separated from its related impurities by liquid chromatography (LC) and the eluent is mixed post-column with a continuous flow of ¹³C-enriched sodium bicarbonate. An online oxidation step using sodium persulfate in acidic media at 99 °C provides quantitative oxidation to ¹²CO₂ and ¹³CO₂ respectively which is extracted to a gaseous phase with the help of a gas permeable membrane. The measurement of the isotope ratio 44/45 in the mass spectrometer allows the construction of the mass flow chromatogram. As the only species that is finally measured in the mass spectrometer is CO₂, the peptide content in the standard can be quantified, on the base of its carbon content, using a generic primary standard such as potassium hydrogen phthalate. The approach was validated by the analysis of a reference material (NIST 8327), and applied to the quantification of two commercial synthetic peptide standards. In that case, the results obtained were compared with those obtained using alternative methods, such as amino acid analysis and ICP-MS. The results obtained proved the value of the method for the fast, accurate and precise mass purity assignment of synthetic peptide standards.     

Quantification in capillary electrophoresis-mass spectrometry: long- and short-term variance components and their compensation using internal standards

Different approaches were chosen to examine ionization reproducibility of analytes after separation by capillary electrophoresis-mass spectrometry (CE-MS) in a commercially available sheath-flow electrospray interface. For this task three different standard samples were examined. Sample 1 contained neostigmine bromide (cationic), paracetamol (PCM) (neutral) and nicotinic acid (anionic component). Results were evaluated using internal standard (IS) calculations. Sample 2 represented an isotopically labelled IS of the quantified substance (PCM/D4-PCM), while sample 3 (neostigmine bromide/scopolamine hydrobromide) provided an IS closely migrating to the tested substance. Furthermore, short-time variations inside the interface were examined by multiple injections of the same substance. For sample 1, the relative standard deviations (RSD%s) were between 8 and 25% (n at least 58) for the peak area ratios. Multiple injected samples gave 5.5-19.4% (n = 25) for peak area RSD%. Using a closely migrating IS, sample 3, RSD%s between 6.5 and 10% (n at least 63) were achieved. With isotopically labelled IS, sample 2, an RSD% of 3-4% was achieved for peak area ratios over long periods (n = 25), for shorter periods (n = 9) even 1-2% RSD% was obtained. Keeping the instrument settings constant, the influence on the ionization efficiency and reproducibility was tested, varying the buffer pH, the organic buffer modifier and the sample concentration. Repeatabilities of migration time and peak area were measured and compared. Two 10 mM ammonium acetate buffers with pH 4.0 and 8.5 were investigated. No influence of buffer pH on peak area reproducibility was found. Isopropanol as organic buffer modifier significantly improved the ionisation leading to larger peak areas, but reduced reproducibility. The basic buffer produced slightly better RSD%s for migration times (2.5-4.0%) (n = 180) and faster analysis for the different test analytes of sample 1, while with the acetic buffer, RSD%s from 3.9 to 6.0% were obtained (n at least 163). The positioning of the capillary turned out to be the crucial parameter to ensure reproducible results. Thus, a procedure was established to ensure a defined ion-intensity level after capillary changes. The investigation of the different sample concentrations gave negligible differences in RSD%, showing that the signal-to-noise ratio was not the crucial parameter for reproducibility here, in contrast to CE-UV detection.     

Validation of a simplified field-adapted procedure for routine determinations of methyl mercury at trace levels in natural water samples using species-specific isotope dilution mass spectrometry

A field-adapted procedure based on species-specific isotope dilution (SSID) methodology for trace-level determinations of methyl mercury (CH₃Hg⁺) in mire, fresh and sea water samples was developed, validated and applied in a field study. In the field study, mire water samples were filtered, standardised volumetrically with isotopically enriched CH₃²⁰⁰Hg⁺, and frozen on dry ice. The samples were derivatised in the laboratory without further pre-treatment using sodium tetraethyl borate (NaB(C₂H₅)₄) and the ethylated methyl mercury was purge-trapped on Tenax columns. The analyte was thermo-desorbed onto a GC-ICP-MS system for analysis. Investigations preceding field application of the method showed that when using SSID, for all tested matrices, identical results were obtained between samples that were freeze-preserved or analysed unpreserved. For DOC-rich samples (mire water) additional experiments showed no difference in CH₃Hg⁺ concentration between samples that were derivatised without pre-treatment or after liquid extraction. Extractions of samples for matrix–analyte separation prior to derivatisation are therefore not necessary. No formation of CH₃Hg⁺ was observed during sample storage and treatment when spiking samples with ¹⁹⁸Hg²⁺. Total uncertainty budgets for the field application of the method showed that for analyte concentrations higher than 1.5 pg g^–1 (as Hg) the relative expanded uncertainty (REU) was approximately 5% and dominated by the uncertainty in the isotope standard concentration. Below 0.5 pg g^–1 (as Hg), the REU was >10% and dominated by variations in the field blank. The uncertainty of the method is sufficiently low to accurately determine CH₃Hg⁺ concentrations at trace levels. The detection limit was determined to be 4 fg g^–1 (as Hg) based on replicate analyses of laboratory blanks. The described procedure is reliable, considerably faster and simplified compared to non-SSID methods and thereby very suitable for routine applications of CH₃Hg⁺ speciation analysis in a wide range of water samples.     

同位素内标稀释液相色谱-串联质谱法测定鱼贝类组织中残留的环丙氟哌酸

建立了不同鱼贝类肌肉组织中以氘代同位素为内标测定环丙氟哌酸残留量的液相色谱-串联质谱(LC-MS/MS)方法。样品加入内标环丙氟哌酸-D8和磷酸盐缓冲溶液(pH 7.0)后进行匀质并用乙腈超声提取,经正己烷脱脂后采用Waters Oasis MAX小柱净化,在Cloversil-C18柱上,以乙腈-0.05%三氟醋酸(体积比为25∶75)为流动相,采用多反应监测(MRM)模式,液相色谱-电喷雾质谱法测定。根据环丙氟哌酸和氘代内标物的定量离子质量色谱图的峰面积比值,采用内标法定量。结果表明,环丙氟哌酸和内标的定量离子峰面积比值与环丙氟哌酸浓度在0.1～50.0 μg/kg范围内呈现良好的线性关系,相关系数为0.9991,方法的定量检测限为0.1 μg/kg,回收率为92.5%～98.1%,相对标准偏差(RSD)小于4.3%。将该方法用于市场上10种鱼和贝类样品的检测,结果表明该法具有灵敏、准确的优点,完全满足残留分析的确证检测要求。     

Combining targeted and nontargeted data analysis for liquid chromatography/high‐resolution mass spectrometric analyses

Increasing importation of food and the diversity of potential contaminants have necessitated more analytical testing of these foods. Historically, mass spectrometric methods for testing foods were confined to monitoring selected ions (SIM or MRM), achieving sensitivity by focusing on targeted ion signals. A limiting factor in this approach is that any contaminants not included on the target list are not typically identified and retrospective data mining is limited. A potential solution is to utilize high‐resolution MS to acquire accurate mass full‐scan data. Based on the instrumental resolution, these data can be correlated to the actual mass of a contaminant, which would allow for identification of both target compounds and compounds that are not on a target list (nontargets). The focus of this research was to develop software algorithms to provide rapid and accurate data processing of LC/MS data to identify both targeted and nontargeted analytes. Software from a commercial vendor was developed to process LC/MS data and the results were compared to an alternate, vendor‐supplied solution. The commercial software performed well and demonstrated the potential for a fully automated processing solution.     

实时直接分析-串联质谱法测定葡萄酒中赭曲霉毒素A

建立了葡萄酒中赭曲霉毒素A的实时直接分析-串联质谱(DART-MS/MS)方法。前处理采用乙酸乙酯提取,二甲基十八碳硅烷粉(ODS)粉分散固相萃取净化,采用DART-MS/MS检测,同位素稀释内标法定量。结果表明:在1.0、2.0、10μg/kg 3个加标水平下,回收率为88.7%~105.7%,RSD为8.5%~12.8%,定量限为0.5μg/kg。该方法具有简单、快速、灵敏度高等特点,能满足葡萄酒中赭曲霉毒素A检测的要求。     

Bismuth as a general internal standard for lead in atomic absorption spectrometry

Bismuth was evaluated as internal standard for Pb determination by line source flame atomic absorption spectrometry (LS FAAS), high-resolution continuum source flame atomic absorption spectrometry (HR-CS FAAS) and line source graphite furnace atomic absorption spectrometry (LS GFAAS). Analysis of samples containing different matrices indicated close relationship between Pb and Bi absorbances. Correlation coefficients of calibration curves built up by plotting A^Pb/A^Biversus Pb concentration were higher than 0.9953 (FAAS) and higher than 0.9993 (GFAAS). Recoveries of Pb improved from 52–118% (without IS) to 97–109% (IS, LS FAAS); 74–231% (without IS) to 96–109% (IS, HR-CS FAAS); and 36–125% (without IS) to 96–110% (IS, LS GFAAS). The relative standard deviations (n = 12) were reduced from 0.6–9.2% (without IS) to 0.3–4.3% (IS, LS FAAS); 0.7–7.7% (without IS) to 0.1–4.0% (IS, HR-CS FAAS); and 2.1–13% (without IS) to 0.4–5.9% (IS, LS GFAAS).     

Tandem mass spectrometric sequence characterization of synthetic thymidine-rich oligonucleotides

Tandem mass spectrometry (MS/MS) can provide direct and accurate sequence characterization of synthetic oligonucleotide drugs, including modified oligonucleotides. Multiple factors can affect oligonucleotide MS/MS sequencing, including the intrinsic properties of oligonucleotides (i.e., nucleotide composition and structural modifications) and instrument parameters associated with the ion activation for fragmentation. In this study, MS/MS sequencing of a thymidine (T)-rich and phosphorothioate (PS)-modified DNA oligonucleotide was investigated using two fragmentation techniques: trap-type collision-induced dissociation (“CID”) and beam-type CID also termed as higher-energy collisional dissociation (“HCD”), preceded by a hydrophilic interaction liquid chromatography (HILIC) separation. A low to moderate charge state (−4), which predominated under the optimized HILIC-MS conditions, was selected as the precursor ion for MS/MS analysis. Comparison of the two distinctive ion activation mechanisms on the same precursor demonstrated that HCD was superior to CID in promoting higher sequence coverage and analytical sensitivity in sequence elucidation of T-rich DNA oligonucleotides. Specifically, HCD provided more sequence-defining fragments with higher fragment intensities than CID. Furthermore, the direct comparison between unmodified and PS-modified DNA oligonucleotides demonstrated a loss of MS/MS fragmentation efficiency by PS modification in both CID and HCD approaches, and a resultant reduction in sequence coverage. The deficiency in PS DNA sequence coverage observed with single collision energy HCD, however, was partially recovered by applying HCD with multiple collision energies. Collectively, this work demonstrated that HCD is advantageous to MS/MS sequencing of T-rich PS-modified DNA oligonucleotides.     

同位素稀释液相色谱-串联质谱法测定化妆品中残留的丙烯酰胺

建立了化妆品中丙烯酰胺残留的同位素稀释液相色谱-串联质谱的分析方法。水溶性化妆品样品以水为提取溶剂进行提取,提取液经高速离心处理后,上清液用Oasis HLB固相萃取柱净化;脂溶性化妆品样品以正己烷和水混合溶剂液-液分配萃取。经Waters Atlantis T3色谱柱(150 mm×2.1 mm,3 μm)分离后在多反应监测模式下进行串联质谱定性及定量分析,以13C3-丙烯酰胺为内标定量。方法的定量限为0.1 mg/kg,在0.1～1.0 mg/kg 3个添加水平范围内的平均回收率为87.7%～95.8%,日内测定精密度均小于10%,日间测定精密度均小于12%。该方法能够满足化妆品中丙烯酰胺残留的检测要求。     

A flow injection electrospray ionization tandem mass spectrometric method for the simultaneous measurement of trimethylamine and trimethylamine N-oxide in urine

Key metabolites for the diagnosis of the genetic disorder trimethylaminuria are trimethylamine (TMA) and trimethylamine N-oxide (TMAO). A rapid, automatable flow injection ESI-MS/MS method for their measurement in urine has been developed. The TMA was derivatized with ethyl bromoacetate to form ethyl betaine bromide. The 2 min ESI-MS/MS analysis employed four multiple reaction monitoring (MRM) ion pairs for derivatized TMA (146.1, 118.1), derivatized (2)H(9)-TMA (155.1, 127.1), TMAO (76.1, 58.1) and (2)H(9)-TMAO (85.1, 66.1). In control urine samples (n = 27) referred for suspected metabolic problems TMA was 0.11-1.19 mmol/mol creatinine, TMAO was 13.5-181 mmol/mol creatinine and the TMA/TMAO ratio was 0.0025-0.055. In five patients with diagnosed trimethylaminuria, TMA was 5.3-230 mmol/mol creatinine, TMAO was 0.36-607 mmol/mol creatinine and the TMA/TMAO ratio was 0.20-134.     

Development and validation of stable‐isotope dilution liquid chromatography–tandem mass spectrometric method for determination of salivary progesterone

A method for the quantification of progesterone (PROG) in human saliva using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata™‐X cartridge, and subjected to LC‐ESI‐MS/MS. Quantification was based on selected reaction monitoring, and deuterated PROG was used as the internal standard. This method allowed the reproducible (intra‐ and inter‐assay relative standard deviations, <2.2%) and accurate (analytical recovery, 96.6–99.7%) quantification of the salivary PROG using a 400 μL sample, and the limit of quantification was 12.5 pg/mL. The developed method enabled detection of the variation in the salivary PROG concentrations of healthy volunteers during the menstrual cycle and measurement of the salivary concentrations of pregnant women. The method is expected to be an alternative to the blood PROG monitoring in clinical examinations, because saliva collection is easy, non‐invasive and repeatable. Copyright © 2011 John Wiley & Sons, Ltd.     

Atmospheric pressure chemical ionization and atmospheric pressure photoionization for simultaneous mass spectrometric analysis of microbial respiratory ubiquinones and menaquinones

An atmospheric pressure photoionization (APPI) source and an atmospheric pressure chemical ionization (APCI) source were compared for the selective detection of microbial respiratory ubiquinone and menaquinone isoprenologues using tandem mass spectrometry. Ionization source- and compound mass-dependent parameters were optimized individually for both sources, using the available quinone standards. Detection levels for the two ion sources were determined with ubiquinone-6 (UQ6) and menaquinone-4 (MK4, vitamin K2) standards using flow injection analysis and selected reaction monitoring (SRM). With APPI the calculated lower limit of detection (LLOD) was 1.7 fmol microl(-1) for UQ6 and 2.2 fmol microl(-1) for MK4 at a signal-to-noise ratio of 3. These LLODs were at least three times lower than with APCI. The selectivity of detection afforded by SRM detection reduced complex mixture analysis to 3 min per sample by eliminating the need for chromatographic separations. The detection method was successfully applied to quinone quantification in a variety of environmental samples and cell cultures. Adequate amounts of respiratory quinones can be extracted and quantified from samples containing as low as 2 x 10(7) cells.