甲卡西酮的LC-MS/MS定性定量分析方法

建立了LC-MS/MS法定性定量分析甲卡西酮。采用三重串联四极杆液质联用仪(LC/QQQ),AgilentZorbax Eclipse Plus C18色谱柱(100 mm×2.1 mm,1.8μm),流动相为0.1%甲酸-乙腈,梯度洗脱,流速为0.3mL/min。质谱应用ESI源、正离子模式、多反应监测(MRM)方式。在0.1～10 000 ng/mL质量浓度范围内线性关系良好,r2=0.999 8,日内与日间保留时间和峰面积的相对标准偏差不大于5.28%,检出限为0.04 ng/mL,回收率为95.6%～100.7%。该方法适用于甲卡西酮的定性、定量分析。     

LC-MS/MS profiling for detection of endogenous steroids and prostaglandins in tissue samples

Roles of steroid hormones, and compounds that can influence their levels in cells, are of increasing interest in e.g. cancer research, partly because resistance to hormone therapies often complicates treatment. To elucidate the processes involved, the hormones and related compounds need to be accurately measured. Reversed-phase liquid chromatography with dynamic multiple reaction monitoring mass spectrometric detection in electrospray mode is capable of providing such measurements. Therefore, LC-MS/MS was developed for sensitive, selective analysis of 11 steroid hormones, cholesterol and two prostaglandins. The effects of the tissue matrix, and solid-phase extraction (SPE) sample clean-up, on the LC-MS/MS signals of the hormones were also investigated. The results show that the developed LC-MS/MS method, following SPE clean-up to reduce matrix interference, can detect selected steroids in extracts of mouse tissues. The method provides linear measurements of the steroids at concentrations up to few ng/μL, and limits of detection in the range 0.03-0.2 pg/μL (for some compounds lower than those of previously reported methods).     

LC-MS/MS fast analysis of androgenic steroids in urine

The liquid chromatography-tandem mass spectrometry method (LC-MS/MS) was developed and validated to detect androgenic steroids: trenbolone, nortestosterone, boldenone, methylboldenone, testosterone, methyltestosterone, 17β-1-testosterone and their metabolites in bovine urine. Sample preparation before LC-MS/MS analysis involved an enzymatic hydrolysis with glucuronidase AS-HP, isolation of free hormones from urine on C(18) SPE column and purification of the extract using liquid-liquid extraction with n-pentane and SPE NH(2) column. For the chromatographic separation of steroids, the Poroshell 120-EC C18 column (150?×?2.1 mm, 2.7 μm) was used. Mass spectrometric measurement was achieved using the API 4000 triple quadrupole (QqQ) instrument with a TurboIon-Spray source operating in positive electrospray ionization mode. The procedure was validated according to the Decision 2002/657/EC. Recovery ranged from 76.5% to 118.9% for all examined compounds. The repeatability was below 20% and reproducibility did not exceed the 25%. The linearity was good for all analytes in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limit (CCα) ranged from 0.10 to 0.17 μg L(-1) for all analytes, whereas the detection capability (CCβ) ranged from 0.17 to 0.29 μg L(-1). The application of an innovative Poroshell column allowed for very good chromatographic separation of steroids with a much shorter time of analysis.     

Multi-mycotoxin analysis of maize silage by LC-MS/MS

This paper describes a method for determination of 27 mycotoxins and other secondary metabolites in maize silage. The method focuses on analytes which are known to be produced by common maize and maize-silage contaminants. A simple pH-buffered sample extraction was developed on the basis of a very fast and simple method for analysis of multiple pesticide residues in food known as QuEChERS. The buffering effectively ensured a stable pH in samples of both well-ensiled maize (pH < 4) and of hot spots with fungal infection (pH > 7). No further clean-up was performed before analysis using liquid chromatography–tandem mass spectrometry. The method was successfully validated for determination of eight analytes qualitatively and 19 quantitatively. Matrix-matched calibration standards were used giving recoveries ranging from 37% to 201% with the majority between 60% and 115%. Repeatability (5–27% RSD_r) and intra-laboratory reproducibility (7–35% RSD_IR) was determined. The limit of detection (LOD) for the quantitatively validated analytes ranged from 1 to 739 μg kg⁻¹. Validation results for citrinin, fumonisin B₁ and fumonisin B₂ were unsatisfying. The method was applied to 20 selected silage samples and alternariol monomethyl ether, andrastin A, alternariol, citreoisocoumarin, deoxynivalenol, enniatin B, fumigaclavine A, gliotoxin, marcfortine A and B, mycophenolic acid, nivalenol, roquefortine A and C and zearalenone were detected.     

Quantitative determination of tiopronin in human plasma by LC-MS/MS without derivatization

Tiopronin (TP) is a synthetic thiol compound without chromophore. By optimizing the chromatographic conditions and sample preparation processes, an improved LC‐MS/MS analytical method without derivatization has been developed and validated to determine TP concentrations in human plasma. After reduction with 1,4‐dithiothreitol, plasma samples were deproteinized with 10% perchloric acid. The post‐treatment samples were analyzed on a C₈ column interfaced with a triple quadrupole tandem mass spectrometer in negative electrospray ionization mode. Methanol–5 mmol/L ammonium acetate (20:80, v/v) was used as the isocratic mobile phase. The assay was linear over the concentration range of 40.0–5000 ng/mL. The intra‐ and inter‐day precisions were within 12.9% in terms of relative standard deviation and the accuracy within 5.6% in terms of relative error. This simple and sensitive LC‐MS/MS method with short analytical time (3.5 min each sample) was successfully applied to the pharmacokinetic study of TP in healthy Chinese male volunteers after an oral dose of 300 mg TP. Copyright © 2011 John Wiley & Sons, Ltd.     

液相色谱-串联质谱外标法定量分析中基质效应的评估

以苯巯基尿酸为目标化合物,考察了色谱分离条件(即强洗脱、弱洗脱及梯度洗脱)对采用外标法定量的液相色谱-串联质谱(LC-MS/MS)分析中基质效应的影响。试验表明:①对无基质标准溶液的分析,以强洗脱条件下的灵敏度最高,且可实现高通量分析;②对同一基质不同浓度的试液,弱洗脱条件的基质效应明显改善,而梯度洗脱的结果最好;③对不同来源的基质和不同浓度的试验,在3种不同的色谱分离条件中以梯度洗脱效果最好,提高了分离度,减少了共流出,有效地降低了基质效应对目标物分析的影响。因此,在LC-MS/MS分析中,色谱分离条件的改善对降低基质效应对测定的影响十分重要。     

LC-MS/MS method for the confirmatory determination of aromatic amines and its application in textile analysis

A confirmation method for the determination of 18 aromatic amines originating from azo dyes after reductive cleavage was developed. The method is based on the use of high-performance liquid chromatography/tandem mass spectrometry with atmospheric-pressure chemical ionization. For the identification of the analytes one precursor ion and two daughter ions (multi-reaction monitoring, MRM) were selected and the LC-MS/MS parameters optimized to obtain high sensitivity and selectivity. The linear ranges varied from 0.1–1 to 30–50 g mL^–1 with correlation coefficients of 0.99 or better. The applicability of the method to determine o-tolidine (3,3-dimethylbenzidine) and 3,3-dimethoxybenzidine in textiles following reductive cleavage of acid red 114, trypan blue, and Chicago sky blue 6B was demonstrated.     

The resistance of metallothionein to proteolytic digestion: an LC-MS/MS analysis

Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.     

血液中卡马西平固相萃取及LC-MS/MS定量方法研究

建立了血液中卡马西平的固相萃取/液相色谱-串联质谱(LC-MS/MS)定量检测方法。血液中的卡马西平用固相萃取柱(Bond Elut Certify)提取,采用Waters AtlantisTMdC18(150 mm×3.9 mm,5μm)色谱柱,电喷雾离子源,正离子检测,多反应监测方式进行定量分析,以SKF-525A为内标。结果表明,该方法对卡马西平的检出限为0.1μg/L,卡马西平的质量浓度在100～6 000μg/L范围内线性关系良好(r=0.997 6),日内、日间精密度RSD(n=6)不高于8.6%,血液中卡马西平的回收率为81%～90%。该方法具有良好的灵敏度、重现性、稳定性和专属性,可用于法庭与临床的毒物分析。     

Quantitative Analysis of Busulfan in Human Plasma by LC-MS–MS

High-performance liquid chromatography with tandem mass spectrometry has been used for rapid, specific, and sensitive analysis of busulfan in human plasma. Busulfan-d₈ was used as internal standard. Analysis was performed on a C₁₈ column (50 mm × 2.1 mm, 3.5-µm particles) with water–methanol 80:20 (v/v) as mobile phase at a flow-rate of 0.30 mL min⁻¹. Detection was by tandem triple–quadrupole mass spectrometry with turbo ion-spray ionization. Linear calibration plots were obtained over the concentration range 1.096–1,096 ng mL⁻¹. The assay is ideally suited to monitoring of busulfan and determination of its pharmacokinetic data.

     

An innovative impurity profiling of Avanafil using LC and LC-MS/MS with in-silico toxicity prediction

Degradation of the drug can lead to the formation of toxic substance hence drug quality and stability is a major concern by pharma regulators. A Selected phosphodiesterase type 5 inhibitor drug Avanafil (AV) structure has amide, arylchloro and hydroxide as functional groups which can easily eliminated during hydrolysis. Henceforth, thoroughly chemical stability of AV was carried out according to ICH guideline Q1A (R2). The drug and marketed tablet formulation undergoes degradation in hydrolytic (acid, base, neutral), thermal, photolytic, oxidative conditions and forms a total new sixteen degradation products (D.P.s) which were identified by LC, characterized by LC-MS/MS and probable degradation mechanism for each stress conditions are proposed. All sixteen D.P.s were identified by optimized LC conditions; C18 column using 10 mM ammonium acetate: ACN (60:40, v/v), pH 4.5 as mobile phase at 0.9 mL min⁻¹ of flow rate, 239 nm wavelength at 20 °C column temperature and the method being LC-MS compatible characterized by LC-MS/MS confirmed by relative retention time (RRT). The structure of D.P.s was confirmed from the fragmentation pattern obtained by LC-MS/MS and further probable degradation mechanism for each stress condition is proposed. The drug and its marketed tablet formulation showed similar degradation peaks which were confirmed using RRT, photodiode array (PDA) and LC-MS. Drug degradation happens due to nucleophilic substitution reaction, amide hydrolysis, electron withdrawing properties of amide, dechlorination and bond cleavage due to energy. The amide group in AV structure can undergo hydrolysis, while due to aryl chloride and hydroxide group in structure it undergoes photodecomposition. A comprehensive stress study reveals that AV is more prone to degrade in light, temperature and moisture; hence AV requires proper storage condition temperature below 25 °C with protection to light and moisture. In silico toxicity prediction of physicochemical properties revealed that all the physicochemical parameters of impurities were within the acceptable limit which indicates that no impurity is at any risk of toxicity. In detail, the LC-MS/MS compatible AV degradation study is fully validated as per ICH Q2 (R1) guideline.     

液相色谱-串联质谱法测定食品中甲基磺草酮

食品用乙腈-水(3+1)溶液进行提取,经凝胶色谱、固相萃取柱净化后,用液相色谱-串联质谱法进行测定和确证,外标法定量。色谱分离用甲醇和甲酸-水(0.1+99.9)溶液以不同体积比混合为流动相梯度洗脱,采用负离子模式电喷雾离子源在多反应监测模式下进行检测。甲基磺草酮的质量浓度在0.01~0.2 mg·L-1范围内与其峰面积呈线性关系。以4种食品样品为基体,加入3种浓度水平的甲基磺草酮标准做回收试验,测得回收率在73.2%~100.6%之间;测定值的相对标准偏差(n=10)在4.1%~11%之间。     

Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.     

Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices

Stability is an important pre-analytical variable for quantitative LC-MS/MS analysis of drug molecules and/or their metabolites in biological matrices. Instability of an analyte in any stage of the bioanalytical process, including sample collection, processing, storage, extraction and LC-MS/MS analysis, can result in under-/over-estimation if an adequate preventive procedure is not in place. In the current review on practical strategies in quantitative LC-MS/MS bioanalysis of unstable small molecules, the common causes of analyte instability were examined. The instability of some analytes is readily predictable because of the presence of certain chemically or biologically labile moieties in the molecules or because the compounds are in an inter-convertible form, e.g. lactone vs hydroxyl carboxylic acid. However, the instability of many other analytes is not readily predictable. Necessary evaluation needs to be conducted to identify the possible instability issues. The current review highlighted some general considerations and specific approaches for developing a robust LC-MS/MS method. In particular, incurred samples should be used as part of routine short-term stability assessment of any unstable analyte during the early stages of method development and validation. This can help unveil any 'hidden' instability issues that, if left unaddressed, could lead to the invalidation of a 'validated' method.     

LC-MS/MS方法快速检测血浆中小檗碱的浓度

小檗碱在生物体内以极低的浓度发挥着良好的疗效,准确测定其血药浓度对其药理学性质的研究具有重要意义。本文建立了高效液相色谱-质谱联用快速测定血浆中小檗碱浓度的方法,采用Waters XTerraTM MSC18色谱柱(2.1×50mm,3.5μm),柱温25℃,以0.2%醋酸水溶液-乙腈为流动相梯度洗脱,流速为0.2mL/min,以苯妥英为内标。质谱采用电喷雾电离源,以选择反应监测模式进行定量分析,血浆样品用乙腈直接沉淀处理。结果表明,小檗碱在0.01～0.5μg/mL浓度范围线性关系良好,最低检测浓度为5ng/mL(S/N=3)。日内、日间RSD均小于15%(n=5)。本文建立的方法操作简单、灵敏度高、分析时间短,适用于小檗碱血药浓度监测及药代动力学研究。     

液相色谱串联质谱测定蔬菜中残留的唑菌胺酯

唑菌胺酯(pyraclostrobin)俗称百泰,德国巴斯夫公司于1993年开发的兼具吡唑结构的甲氧基丙烯酯酯类菌剂~([1]),主要用于防治小麦、水稻、花生、葡萄、蔬菜、香蕉、 柠檬、咖啡、果树、核桃、蔬菜、茶树、烟草和观赏植物、草坪及其他大田作物上由子囊菌纲、担子菌纲、半知菌类和卵菌纲真菌引起的作物病害~([2]).     

非衍生化/液相色谱-串联质谱法测定食品中的二硫代氨基甲酸酯类农药残留

通过优化质谱、液相色谱和萃取缓冲液等条件,建立了一种简单、快速、灵敏测定食品中二硫代氨基甲酸酯的方法。在优化条件下,代森锌(EBDC)、丙森锌(PBDC)的线性范围为2～100μg/L,相关系数均不小于0.997。在花椰菜、萝卜和豌豆中EBDC和PBDC的检出限(S/N>3)为2μg/kg;萝卜和豌豆中EB-DC和PBDC的定量下限(S/N>10)为10μg/kg,而花椰菜中EBDC和PBDC的定量下限(S/N>10)为15μg/kg。在花椰菜、萝卜和豌豆基质中加标5～100μg/kg的EBDC和PBDC时,测定加标回收率为83%～96%,相对标准偏差为4.4%～10.5%,方法可满足定量分析的要求。