A novel quantification strategy of transferrin and albumin in human serum by species-unspecific isotope dilution laser ablation inductively coupled plasma mass spectrometry (ICP-MS)

Species-specific (SS) isotope dilution analysis with gel electrophoresis (GE)-laser ablation (LA)-ICP-MS is a promising technique for the quantification of particular metal-binding proteins in biological samples. However, unavailable isotopically enriched spike and metal losses in GE separation are main limitations for SS-isotope dilution PAGE-LA-ICP-MS. In this study, we report for the first time the absolute quantification of transferrin (Tf) and albumin (Alb) in human serum by non-denaturing (native) GE combined with species-unspecific isotope dilution mass spectrometry (IDMS). In order to achieve a homogeneous distribution of both protein and isotope-enriched spike (simulated isotope equilibration), immersing the protein strips with ³⁴S spike solution after gel electrophoresis was demonstrated to be an effective way of spike addition. Furthermore, effects of immersion time and ³⁴S spike concentration were investigated to obtain optimal conditions of the post-electrophoresis isotope dilution method. The relative mass of spike and ablated sample (m_sp/m_sam) in IDMS equation was calculated by standard Tf and Alb proteins, which could be applied to the quantification of Tf and Alb in ERM-DA470k/IFCC for method confirmation. The results were in agreement with the certified value with good precision and small uncertainty (1.5–3%). In this method, species-specific spike protein is not necessary and the integrity of the heteroatom-protein could be maintained in sample preparation process. Moreover, the application of species-unspecific isotope dilution GE-LA-ICP-MS has the potential to offer reliable, direct and simultaneous quantification of proteins after conventional 1D and 2D gel electrophoretic separations.     

Phenylalanine in whole blood and plasma--a candidate reference method

A method for the quantification of phenylalanine in whole blood and plasma by isotope dilution gas chromatography-mass spectrometry is presented. The use of an uncommon derivative allows a simple extraction procedure and the most basic mass spectrometer. The relative standard deviation was found to be 1.3% within-batch and 2.1% between-batch under optimum conditions, and the detection limit was found to be 7 pmol injected. The ruggedness of the procedure and sample handling conditions were also examined.     

A Dilute and Shoot Strategy for Determining Alternaria Toxins in Tomato-Based Samples and in Different Flours Using LC-IDMS Separation

Alternaria toxins are emerging mycotoxins whose regulation and standardization are in progress by the European Commission and the European Committee for Standardization. This paper describes a dilute and shoot approach to determine five Alternaria toxins in selected food samples using liquid chromatography–tandem mass spectrometry (LC-MS/MS). The strategy involves sample extraction with acidified aqueous methanol, followed by a solvent change accomplished via sample evaporation and reconstitution. The quantification is based on isotope dilution, applying all corresponding isotopically labeled internal standards to compensate possible matrix effects of the analysis. The main advantages of the present method over other existing methods includes simple and effective sample preparation, as well as detection with high sensitivity. The five-fold sample dilution can decrease matrix effects, which were evaluated with both external and internal standard methods. The results demonstrated a limit of quantification lower than 1.0 µg/kg for all five analytes for the first time. The newly presented method showed acceptable accuracy (52.7–111%) when analyzing naturally contaminated and spiked standard samples at the described levels. The method was validated for tomato-based and flour samples (wheat, rye, and maize). The absolute recovery ranged from 66.7% to 91.6% (RSD < 10%). The developed method could be an alternative approach for those laboratories that exclude sample cleanup and pre-concentration of state-of-the-art instruments with enhanced sensitivity.     

Analysis of corky off-flavour compounds at ultra trace level with multidimensional gas chromatography-electron capture detection

A robust method for routine quality control of corky off-flavour compounds in wine and cork soak matrices has been established. Based on an automated headspace solid phase microextraction (HS-SPME), the method needs only marginal sample preparation and achieves low (sub-ng L⁻¹) trace level detection limits (LODs) for the most relevant off-flavour compounds, such as 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA) and 2,4,6-tribromoanisole (TBA). Particularly for wine matrix, reliable trace level quantification had only been achieved after applying heart-cutting multidimensional gas chromatography (MDGC). Using a halogen-sensitive electron capture detector (ECD) and quantification with a stable isotope dilution assay (SIDA), LODs of 0.1 ng L⁻¹ for TCA, TeCA and TBA could be obtained. Since a SIDA based quantification method is used with a non-mass spectrometric detector, the necessary chromatographic resolution of internal standard and target analyte peaks resulted from the use of highly deuterated [²H₅]-isotopologues.     

Determination of chloride ion in aqueous samples by isotope-dilution Fourier-transform flame infrared emission (ID-FIRE) spectrometry

An isotope dilution method for the determination of chloride ion in aqueous samples is described. The method makes use of the isotopic shift in the rotational lines of the 1–0 band of HCl emitted in the near infrared region of the spectrum by vibrationally excited HCl molecules present in a hydrogen/entrained air flame. Chloride ion in the sample is converted to chlorine gas by electrolysis and swept into a hydrogen/entrained air flame where it is converted into HCl. Because isotope dilution is an absolute method of analysis, matrix effects are minimized, and the chlorine generation step need not be quantitative. With the system described in this paper, samples must contain at least 9 mg of chloride ion per ml, and a 2-ml sample is required. Over the range from 10 to 30 mg Cl⁻ ml⁻¹, the average error was −0.96%, and the average relative standard deviation was 3.3% for seven samples using seven of the more intense lines in the P branch. Compared with standard silver nitrate titrations, the isotope dilution procedure was not affected by such common interferences as bromide ion and iodide ion. The technique was applied to several seawater samples from different regions.     

Application of isotope dilution ICP–MS techniques to quantitative proteomics

ICP–MS techniques based on isotope dilution analysis can be regarded as an emerging tool in quantitative protein analysis. Well-known concepts, for example species-specific and unspecific isotope dilution analysis, which promoted accurate and precise quantification in elemental speciation studies, have nowadays been transferred to the analysis of large biomolecules, e.g. proteins. Besides detection of heteroatom-containing proteins, the artificial introduction of metal-containing labels has attracted much attention and, as a consequence, ICP–MS-based isotope dilution techniques can serve as a valuable quantification tool. In particular, because isotope dilution ICP–MS techniques can enable absolute protein quantification, they can be regarded as an attractive technique in current and prospective proteomics. In this review, recent developments and applications will be highlighted and critically assessed.     

PSAQ™ standards for accurate MS–based quantification of proteins: from the concept to biomedical applications

Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable‐isotope‐labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ?) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here. Copyright © 2012 John Wiley & Sons, Ltd.     

高效液相色谱-非特异性同位素稀释质谱联用技术定量分析全血中总血红蛋白含量

建立了基于同位素稀释(ID)的高效液相色谱(HPLC)-电感耦合等离子体质谱(ICP-MS)联用技术, 通过测量血红蛋白(HGB)中铁(Fe)元素含量实现了全血中总HGB准确定量的策略. 该策略包括2种方法, 第一种方法为在线HPLC-ID-ICP-MS定量技术: 全血中HGB经液相色谱分离后, 通过柱后在线添加浓缩⁵⁴Fe同位素稀释剂的方式, 与液相色谱洗脱液经三通混合后进入ICP-MS在线测量⁵⁴Fe/⁵⁶Fe的同位素比值, 并根据同位素稀释质谱法公式及蛋白中Fe的含量计算HGB的浓度. 针对全血样品中存在的其它主要含铁蛋白[如转铁蛋白(Tf)], 在实现色谱完全分离的基础上, 通过在线优化改变⁵⁴Fe稀释剂流速实现了HGB和Tf的同时定量. 在另一种方法中, 先对全血样品进行消解并通过ID-ICP-MS测定其中Fe的总量, 然后根据全血样品的HPLC-ICP-MS分析结果, 通过计算HGB质谱峰面积占总峰面积的比值从而得到HGB中Fe含量占总Fe含量的比例, 实现了其中HGB的准确定量. 2种定量方法得到的结果分别为(115.3±2.4) mg/g和(115.5±2.1) mg/g, 结果吻合良好, 方法的检出限为1.0×10^?7 mg/g, 方法精密度RSD均<3%. 2种方法均经过HGB标准物质IRMM/IFCC-467进行验证, 测量结果在标准值不确定度范围之内, 可作为临床检验领域HGB分析的参考测量方法.     

Multi-element trace determinations in pure alkaline earth fluoride powders by high-resolution ICP-MS using wet-chemical sample preparation and laser ablation

Four alternative analytical procedures for the determination of ten important trace impurities (Mg, Cr, Fe, Cu, Zn, Sr, Zr, Cd, Ba, and Pb) in pure alkaline earth fluoride powders were applied using high-resolution inductively coupled plasma mass spectrometry (ICP-MS). Two procedures are based on a wet-chemical microwave digestion with boric acid and quantification by the standard addition technique and isotope dilution mass spectrometry (IDMS), respectively. In addition, analyses are also performed by laser ablation as a direct solid sampling technique applying matrix-matched external calibration as well as isotope dilution of the powdered sample. For most elements good agreement between the different methods is found. Detection limits for laser ablation vary between 0.05 ng g^–1 for Zr and 20 ng g^–1 for Mg. They are about one to two orders of magnitude lower than those of the wet-chemical procedures, which is mainly due to the high dilution factor during the sample preparation step. Advantages and restrictions of the different analytical procedures are discussed with respect to their routine applicability. Due to its relatively high accuracy, low detection limits, and time-efficiency LA-ICP-IDMS is the preferred choice if no standard reference materials are available.     

Determination of mannose in honey by isotope dilution gas chromatography mass spectrometryEI北大核心CSCD

建立了一种同位素稀释-气相色谱/质谱(GC-MS)测定蜂蜜中甘露糖含量的方法。选择了一种新型化合物[13C;D]-D-甘露糖作为蜂蜜中甘露糖检测的同位素内标,优化了甘露糖及其内标的气相色谱分离条件和质谱参数。蜂蜜样品经70%乙醇水溶液溶解后加入同位素内标,采用两步衍生法对蜂蜜中糖组分进行衍生化,即先加入2.5%盐酸羟胺-吡啶溶液进行肟化衍生,再加入N,O-双(三甲基硅基)三氟乙酰胺(BSTFA)进行硅烷化衍生,采用GC-MS对甘露糖进行定性分析和定量检测。结果表明,以洋槐蜜、油菜蜜、荆条蜜和椴树蜜为代表性样品基质,甘露糖的检出限均为6 mg/kg,定量限为20 mg/kg,在20,40,200 mg/kg 3个添加水平下方法的平均回收率在66.1%~91.7%之间,相对标准偏差不超过20%。该方法适用于不同类型蜂蜜中甘露糖含量的测定。     

凝胶电泳-激光烧蚀进样电感耦合等离子体质谱与非特异性同位素稀释法联用技术定量分析人血清中蛋白

建立了聚丙烯酰胺凝胶电泳(PAGE)-激光烧蚀进样电感耦合等离子体质谱(LA-ICP-MS)与非特异性同位素稀释法联用技术, 通过测定蛋白条带上硫元素的含量, 实现了人血清中蛋白的定量分析. 血清电泳分离条件为: 分离胶浓度(质量分数)为7.5%, 浓缩胶浓度为4%, 电泳电压100 V. 为优化激光烧蚀实验条件, 以LA-ICP-MS测得不同浓度硫富集凝胶中³²S的信号强度, 在1~400 μg/g范围内呈现良好的线性关系, R²=0.993. 采用外标法对血清中的转铁蛋白和白蛋白进行了相对定量分析. 此外, 重点采用“电泳后同位素稀释”方法实现了蛋白条带上³⁴S稀释剂的加入, 对血清中转铁蛋白和白蛋白进行了绝对定量分析; 采用人血清蛋白标准物质ERM-DA470/IFCC对建立的方法体系进行了验证, 结果表明2种蛋白的定量结果与标准值吻合, 方法的准确性好, 且测量精度优于外标法.     

Quantitation of cyclosporin A in whole blood by liquid chromatography/stable isotope dilution electrospray ionization mass spectrometry.

Therapy with cyclosporin A (CsA) for immunosuppression after organ transplantation requires monitoring of its levels in blood owing to the narrow therapeutic index of the drug and to the high inter-individual variability of the drug absorption and metabolism. We describe the preparation of CsA labelled with stable isotopes ((13)C and (2)H) with an isotopic enrichment of about 99% using labelled glucose and its use as internal standard for quantification of CsA blood levels by isotope dilution/electrospray ionization mass spectrometry. The method was found to be linear in the tested range (1-1000 ng) with and without the matrix. The accuracy of the bracketting calibration curves prepared using 100 ng ml(-1) labelled CsA was within +/-1.7% (bias). The results confirmed the usefulness of the procedure as a reference method for the external quality assessment of the field methods for the evaluation of CsA blood concentration, the imprecision (relative standard deviation) and accuracy (bias) being <2%.     

液相色谱同位素稀释串联质谱法测定人血清中17β-雌二醇含量

研究建立了以人血清中E2-16,16,17-d3为内标测定17β-雌二醇的液相色谱/串联质谱(ID-LC/MS/MS)方法。血清样品经固相萃取装置(SPE)提取雌二醇,乙酸乙酯萃取净化,吹干复溶后用10-乙基吖啶酮-2-磺酰氯(EASC)进行衍生。以Agilent Eclipse XDB-C18色谱分离柱,乙腈、水梯度洗脱,使用电喷雾三重四极杆串联质谱的多重反应监测模式测定,以校准曲线法进行定量。所建立的液相色谱同位素稀释串联质谱法(ID-LC/MS/MS)对于分析血清17β-雌二醇的批内、批间RSD分别为0.29%～0.73%和0.18%～0.28%,回收率为99.6%～100.2%,采用IFCC RELA比对(JCTLM比对)样品进行了方法比较,测定结果与其他实验室相比偏差在0.8%范围内。方法可作为人血清中17β-雌二醇含量测量参考方法。     

Absolute quantification of superoxide dismutase (SOD) using species-specific isotope dilution analysis

Here we report for the first time the use of species-specific isotope dilution mass spectrometry for the absolute quantification of a metalloprotein using non-denaturing gel electrophoresis laser ablation inductively coupled plasma mass spectrometry (GE-LA-ICP-MS). The concept utilises the intrinsic metals of the metalloprotein for labelling of the isotopically labelled spike (⁶⁵Cu, ⁶⁸Zn SOD). The stability of the metal–protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method evaluated. Between 4 and 64 μg, SOD was quantified with a recovery rate between 82% and 110% in a standard. The use of the isotopically enriched SOD was utilised to identify the extent of orthogonal diffusion in 1-D gel electrophoresis. Orthogonal diffusion of natural and isotopically enriched SOD in the gel can interfere with the correct determination of the isotope ratios. The matrix effect of a cytosolic liver extract on the non-covalently bound copper and zinc in SOD was evaluated and no significant metal loss from the SOD spike was observed. This study represents the first step necessary for establishing and evaluating the use of a species-specific isotope dilution approach for the absolute quantification of SOD in real samples based on the combination of gel electrophoresis and LA-ICP-MS.     

Liquid chromatographic-tandem mass spectrometric determination of selected sulphonamides in milk

Liquid chromatography–tandem mass spectrometry is used for the quantitative analysis of selected sulphonamides in milk. Ultrafiltration is the only sample pre-treatment technique which is required. Consequently, sample throughput is much higher than with conventional procedures, and analyte recoveries are high. As for quantification, both external standard and isotope dilution calibration yield satisfactory results. The method is fully validated for five sulphonamides with a maximum residue limit of 100 μg/kg, and which are included in the Dutch control programme on residues. Furthermore, results are presented on the applicability of the method to detect compounds at a much lower concentration level exemplified by a banned sulphonamide, dapsone, which has a provisional action limit of 5 μg/kg. The main conclusion is that the present, novel approach to the trace-level determination of veterinary drugs is simple and straightforward and has a wide-ranging application potential which is briefly exemplified by the analysis of selected benzimidazoles in milk by essentially the same procedure.     

High-throughput biomonitoring of dioxins and polychlorinated biphenyls at the sub-picogram level in human serum

We report on the use of a state-of-the-art method for the measurement of selected polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls in human serum specimens. The sample preparation procedure is based on manual small size solid-phase extraction (SPE) followed by automated clean-up and fractionation using multi-sorbent liquid chromatography columns. SPE cartridges and all clean-up columns are disposable. Samples are processed in batches of 20 units, including one blank control (BC) sample and one quality control (QC) sample. The analytical measurement is performed using gas chromatography coupled to isotope dilution high-resolution mass spectrometry. The sample throughput corresponds to one series of 20 samples per day, from sample reception to data quality cross-check and reporting, once the procedure has been started and series of samples keep being produced. Four analysts are required to ensure proper performances of the procedure. The entire procedure has been validated under International Organization for Standardization (ISO) 17025 criteria and further tested over more than 1500 unknown samples during various epidemiological studies. The method is further discussed in terms of reproducibility, efficiency and long-term stability regarding the 35 target analytes. Data related to quality control and limit of quantification (LOQ) calculations are also presented and discussed.     

同位素稀释气相色谱串联质谱法测定海产品中的苯并（α）芘的残留量

采用同位素稀释法结合固相萃取净化,建立了海产品中苯并（α）芘残留的气相色谱串联质谱（6C—MS／MS）检测方法。样品经乙腈一丙酮（体积比6：4）溶液提取,硅胶固相萃取净化,苯并（μ）芘用GC—MS／MS测定,同位素D12-苯并芘内标法定量。方法的平均回收率为93％-98％,相对标准偏差为6.2％～12．2％（n=6）,定量限为0．2μg／kg。用该方法测定FAPAS烟熏鱼有证标准样品,测定值与标准值一致。     

Headspace solid‐phase microextraction and gas chromatographic analysis of low‐molecular‐weight sulfur volatiles with pulsed flame photometric detection and quantification by a stable isotope dilution assay

Low‐molecular‐weight volatile sulfur compounds such as thiols, sulfides, disulfides as well as thioacetates cause a sulfidic off‐flavor in wines even at low concentration levels. The proposed analytical method for quantification of these compounds in wine is based on headspace solid‐phase microextraction, followed by gas chromatographic analysis with sulfur‐specific detection using a pulsed flame photometric detector. Robust quantification was achieved via a stable isotope dilution assay using commercial and synthesized deuterated isotopic standards. The necessary chromatographic separation of analytes and isotopic standards benefits from the inverse isotope effect realized on an apolar polydimethylsiloxane stationary phase of increased film thickness. Interferences with sulfur‐specific detection in wine caused by sulfur dioxide were minimized by addition of propanal. The method provides adequate validation data, with good repeatability and limits of detection and quantification. It suits the requirements of wine quality management, allowing the control of oenological treatments to counteract an eventual formation of excessively high concentration of such malodorous compounds.     

Isotope abundance ratio measurements using femtosecond laser ablation ionization mass spectrometry

Accurate isotope ratio measurements are of high importance in various scientific fields, ranging from radio isotope geochronology of solids to studies of element isotopes fractionated by living organisms. Instrument limitations, such as unresolved isobaric inferences in the mass spectra, or cosampling of the material of interest together with the matrix material may reduce the quality of isotope measurements. Here, we describe a method for accurate isotope ratio measurements using our laser ablation ionization time‐of‐flight mass spectrometer (LIMS) that is designed for in situ planetary research. The method is based on chemical depth profiling that allows for identifying micrometer scale inclusions embedded in surrounding rocks with different composition inside the bulk of the sample. The data used for precise isotope measurements are improved using a spectrum cleaning procedure that ensures removal of low quality spectra. Furthermore, correlation of isotopes of an element is used to identify and reject the data points that, for example, do not belong to the species of interest. The measurements were conducted using IR femtosecond laser irradiation focused on the sample surface to a spot size of ~12 μm. Material removal was conducted for a predefined number of laser shots, and time‐of‐flight mass spectra were recorded for each of the ablated layers. Measurements were conducted on NIST SRM 986 Ni isotope standard, trevorite mineral, and micrometer‐sized inclusions embedded in aragonite. Our measurements demonstrate that element isotope ratios can be measured with accuracies and precision at the permille level, exemplified by the analysis of B, Mg, and Ni element isotopes. The method applied will be used for in situ investigation of samples on planetary surfaces, for accurate quantification of element fractionation induced by, for example, past or present life or by geochemical processes.