Determination of nifedipine in human plasma by flow-injection tandem mass spectrometry

Using the murine teratocarcinoma cell line F9 we investigated the influence of serum stimulation and cisplatin treatment on the p53, CK2, MDM2 levels. Both treatments led to an increase of p53, though with different kinetics; the other proteins investigated were not affected. We present direct evidence by immunoprecipitation for an association of protein kinase CK2 holoenzyme (alpha2beta2), p53, and the ribosomal protein L5. The results suggest complexes between the CK2 holoenzyme and p53 but also p53/CKbeta complexes. Furthermore we provide evidence for the existence of high molecular mass complexes of CK2 in vivo. This is the first evidence that, under physiological conditions, protein kinase CK2 does not exist solely as a heterotetramer, but predominantly in association with other proteins.     

Nipple attachment in rats during the first 24 hours of life.

Investigated nipple attachment in 49 Purdue-Wistar laboratory rat pups during the 1st 24 hrs of life and identified determinants that influenced its expression. In Exp I, nipple attachment was shown to be age dependent: 1-hr-old pups exhibited a low percentage of attachment, and 12- and 24-hr-old pups exhibited high percentages when tested against an anesthetized lactator. Exps II–VI established the time course of nipple attachment as age related. Exps VII–X explored the behavioral mechanism responsible for the increase in attachment from Hr 1 to Hr 12. They showed that nipple experience, independent of milk acquisition, was the responsible factor for the increase. The findings suggest that learning is employed as a behavioral strategy by the newborn, and they reemphasize the value of using species-relevant measures in the assessment of behavioral development. (35 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

辉光放电质谱法测定高温合金中的痕量元素

研究了用辉光放电质谱法(GDMS)同时测定高温合金中的痕量元素.通过对分析元素同位素干扰情况的考察,对辉光放电参数如电流、电压、预溅射时间和积分时间的优化,建立了测定高温合金中B,P,Sc,Cu,Ga,Ge,As,Ag,In,Sn,Sb、Te,Hf,Tl,Pb和Bi共16种痕量元素的方法,该方法具有很好的准确度和精密度.     

Application of tandem mass spectrometry for the analysis of long-chain carboxylic acids

The application of MS-MS for the analysis of long-chain carboxylic acids and their esters has proved enormously successful but expensive. It is discussed mainly on basis of results obtained with different instruments with lesser attention to principles of the method, which have been adequately reviewed elsewhere. The use of electrospray ionization (ESI) has greatly increased the sensitivity of the method and has permitted assay of total lipid extracts. The combination of HPLC with electrospray and single quadrupole mass spectrometry, LC-ESI-CID-MS, rivals the triple quadrupole MS-MS application in many instances at considerably lower cost. However, LC-ESI-MS-MS remains the most desirable system at the present time for lipid ester analyses.     

Determination of calcium binding sites in gas-phase small peptides by tandem mass spectrometry

Low-energy (LE) and high-energy (HE) collisionally activated decompositions (CAD) of calcium/peptide complexes of the form [M - H + Ca]+ and [M + Ca]2+ reflect the site of calcium binding in various gas-phase peptides that are models of the calcium binding site III of rabbit skeletal troponin C. The Ca2+ binding sites involve an aspartic acid, glutamic acid, and asparagine, which are in the metal-binding loops of calcium-binding proteins. Both fast atom bombardment (FAB) and electrospray ionization (ESI) were used to generate the metal/peptide complexes. When submitted to LE CAD, ESI-produced Ca2+/peptide complexes undergo fragmentations that are controlled by Ca2+ binding and provide information on the Ca2+ binding site. The LE CAD spectra are simple, indicating that Ca2+ binding involves specific oxygen ligands including acidic side chains and that only a few low-energy fragmentation channels exist. The HE CAD spectra of FAB-produced Ca2+/peptide complexes are more complex, owing to the introduction of high internal energy into the precursor ion. Interactions of the other alkaline-earth metal ions Mg2+ and Ba2+ with these peptides reveal that the ligand preferences of these metal ions are slightly different than those of Ca2+.     

电感耦合等离子体串联质谱法测定高温合金中痕量砷

高温合金中Co含量较高,采用电感耦合等离子体质谱(ICP-MS)测定高温合金中As时,⁵⁹Co¹⁶O⁺会严重干扰As的分析,这一直是ICP-MS测定高温合金中痕量As的研究难点。在串联四极杆(MS/MS)模式下向碰撞/反应池内通入O₂,设置一级质量过滤器(Q1)m/z=75,⁷⁵As⁺可以与O₂反应生成⁷⁵As¹⁶O⁺,而干扰离子不能与O₂发生反应,将二级质量过滤器(Q2)设置为m/z=91,仅⁷⁵As¹⁶O⁺通过并被检测器检测,从而避免了⁵⁹Co¹⁶O⁺的质谱干扰。据此,建立了电感耦合等离子体串联质谱法(ICP-MS/MS)测定高温合金中痕量As的方法。采用As质量浓度为1.000 ng/mL、Co质量浓度为1.000~1 000 μg/mL的系列标准溶液考察了单四极杆和MS/MS两种模式下Co对As测定的质谱干扰。结果表明,在MS/MS模式下,As的回收率均在100%左右,这说明在MS/MS质量转移模式下,采用O₂为反应气,通过两次质量选择,可以成功消除Co基体带来的严重干扰。对O₂流速进行了优化,选择O₂流速为0.375 mL/min。方法线性范围为1.00~100 ng/mL,线性相关系数为1.000 0,检出限为0.006 7 μg/g,定量限为0.023 μg/g。选择纯钴标准样品为测定对象,按照实验方法对其中As进行测定,并进行加标回收试验,回收率在96%~102%之间。采用所建立的方法对镍基高温合金标准物质和高温合金样品中As进行测定,测定结果分别与认定值或原子荧光光谱法测定值基本一致,实际样品测定结果的相对标准偏差(RSD,n=6)为1.6%～2.8%。     

Automated sample preparation for the determination of budesonide in plasma samples by liquid chromatography and tandem mass spectrometry

An automated bioanalytical method for the determination of the glucocorticosteroid drug budesonide in plasma samples at pM levels was investigated. The method was built using three separate automated analytical steps with manual transfer of samples between them. In the first step, a Tecan RSP150 (Genesis) pipetting robot was used to transfer 1 ml of centrifuged plasma samples and deuterated budesonide internal standard solutions into tubes and to homogenise the resulting admixture. In the second step, a solid-phase extraction was performed using an ASPEC Xli (Gilson) with 100 mg Isolute C18 columns. In order to avoid conventional time-consuming evaporation and reconstitution steps, the solid-phase extraction was coupled on-line to a trace enrichment system for further purification and concentration of the sample extracts. The concentrated samples were eluted in 300 microliters ethanol into injection vials, which were capped and transferred to the autosampler in the detection system. In the third step, the pre-treated samples were chromatographed in a gradient LC system and detected using a tandem MS system (Finnigan TSQ 7000), with an atmospheric pressure chemical ionisation interface. The described Analytical System consisting of one Tecan robot, two ASPEC systems and one LC-MS-MS system may analyse up to about 800 samples a week with less routine work for the analyst. The concentration range studied was 15 to 2500 pM in 1 ml spiked plasma samples and the limit of quantitation for the described method was determined as 15 pM, as defined by accuracy and precision better than 20%.     

辉光放电磁控溅射靶材利用率和刻蚀均匀性方法研究与进展

磁控溅射靶表面磁场的非均匀分布使靶的溅射性能受到了靶材利用率低和刻蚀均匀性差的制约,因此寻求一种较高靶材利用率且在靶材表面的大面积范围内均匀溅射的方法成为研究的热点.简要介绍了辉光放电磁控溅射技术基本原理,综述了近20年来国内外研究机构和学者提高靶材利用率及刻蚀均匀性的主要方法.对已报道的优化磁极结构、辅助磁场、动态磁...     

辉光放电质谱法测定高纯金靶材中42种杂质元素

高纯金靶材广泛应用于电子信息行业,靶材产品对其所含杂质元素含量有着极高要求,杂质元素含量偏高将影响其使用性能.对辉光放电质谱法(GDMS)放电电流、放电气体(氢气)流量和样品预溅射时间进行了优化,以丰度高、无干扰或干扰小为原则来选择待测同位素,同时通过选择分辨率来克服可能存在的质谱干扰,建立了GDMS测定高纯金靶材中A...     

A sequencing strategy for the localization of O-glycosylation sites of MUC1 tandem repeats by PSD-MALDI mass spectrometry

It is demonstrated with glycopeptides of the polymorphic epithelial mucin (MUC1) that post-source decay matrix-assisted laser desorption ionization (PSD-MALDI) is a fast, highly sensitive, and reproducible method for the localization of O-glycosylation sites by reflectron time-of-flight (TOF) mass spectrometry. We have analyzed GalNAc-carrying peptides of up to 25 amino acids, and could distinguish even neighboring glycosylation sites. This method was also able to localize and characterize disaccharides (e.g., the Thomsen-Friedenreich disaccharide) on MUC1 derived peptides. PSD-MALDI-MS fragment ion patterns were recorded in the positive ion mode from the synthetic peptide TAP25 [(T1aAPPAHGVT9S10APDT14RPAPGS20) T1bAPPA], an overlapping sequence of MUC1 tandem repeats, which was glycosylated with GaINAc in vitro. The glycosylation sites found were either Thr9 or Thr1b in the monoglycosylated, Thr9 and Thr1b in the diglycosylated, and Thr9, Thr1b, and Ser20 in the triglycosylated peptide. A single PSD-MALDI-MS spectrum of the underivatized and uncleaved di- or triglycosylated TAP25 peptide was sufficient to identify the glycosylation sites, thereby distinguishing six potential, partly adjacent, glycosylation sites. The monoglycosylated fraction was found to consist of a mixture of two glycosylated species with the same molecular weight. This was shown by the analysis of proteolytic digests. PSD-MALDI-MS of the resulting peptides right out of the digestion probe was sufficient to identify the Gal-NAc-glycosylation sites as either Thr9 or Thr1b, respectively. Beyond the methodical aspects the results revealed that in vitro glycosylation of the TAP25 peptide with a transferase system from human milk differs from that obtained with a breast cancer cell transferase system.     

Structural characterization of the cyanelle peptidoglycan of Cyanophora paradoxa by 252Cf plasma desorption mass spectrometry and fast atom bombardment/tandem mass spectrometry

A strategy for the structural characterization of the four major NaBH4-reduced peptidoglycan monomers derived from muramidase-digested peptidoglycan from the cyanelles of the flagellate Cyanophora paradoxa Korschikoff is described. Initial molecular weight determination of these glycopeptides was performed by positive and negative ion plasma desorption mass spectrometry. Due to the presence of two pairs of disaccharide tripeptide and disaccharide tetrapeptide monomers differing in mass by 112 units, respectively, an as yet unknown peptidoglycan modification either at the carbohydrate or at the peptide moiety was assumed. beta-Elimination of the disaccharide unit from the unreduced peptidoglycan monomers yielded the corresponding (modified) N1-lactyltripeptides and -tetrapeptides, respectively. These peptides, N-terminally blocked with lactic acid, unambiguously showed the modification to be located on the peptide moiety. By positive ion fast atom bombardment/hybrid tandem mass spectrometry of the reduced peptidoglycan monomers as well as of the corresponding deglycosylated monomers (= N1-lactylpeptides) the modification was determined to be linked to the glutamic acid moiety. Based on combined data from plasma desorption mass spectrometry, tandem mass spectrometry, accurate mass measurement and amino acid analysis of the acid hydrolysate after derivatization with o-phthaldialdehyde by high-performance liquid chromatography we could establish the structure of the modification as N-acetylputrescine. Finally, the confirmation of the linkage of the glutamic acid to diaminopimelic acid via the gamma-COOH was based on the presence of a-type peptide backbone fragment ions in the positive ion plasma desorption mass spectra of the modified N1-lactylpeptides.     

Liquid-liquid extraction procedure for trace determination of cyclophosphamide in human urine by high-performance liquid chromatography tandem mass spectrometry

A sensitive, specific and accurate high performance liquid chromatography/ionspray-tandem mass spectrometry procedure (HPLC/MS/MS) has been developed to quantify cyclophosphamide in human urine from hospital personnel involved in drug preparation and administration of antineoplastic alkylating agents. This methodology, which includes liquid-liquid extraction with ethylacetate, requires no derivatization procedures, preventing cyclophosphamide (CP) from possible thermal and chemical decomposition reactions. We detected the excretion of this unmetabolized alkylating drug in 50% of all the study participants. The amount of CP ranged from 0.1 ng microL-1 to 1.9 ng microL-1 urine. This methodology was validated by the use of ifosfamide as internal standard. The assay was linear over the range 0 to 3.2 ng microL-1 urine, with a lower limit of quantification of 0.2 microL-1. The limit of detection was assessed at 0.05 ng microL-1 urine. This method is characterized by a coefficient of variation < 10%. Standard calibration curves, obtained on three different days, had correlation coefficients always greater than 0.998. The intra and interday precision were within 11%, and accuracy was in the range 99-103%. The mean extracted recovery assessed at three different concentrations (0.5, 0.8, 3.2 ng microL-1) was always more than 85%. The extraction efficiency of cyclophosphamide from urine samples was also studied at six different pH values (pH 4, 5, 6, 7, 8, 10). The maximum extraction efficiency was obtained when the pH of urine solutions was adjusted to 7.0     

Identification of drug metabolites in biological matrices by intelligent automated liquid chromatography/tandem mass spectrometry

A rapid and systematic strategy for the identification of drug metabolites in biological matrices based on liquid chromatography-tandem mass spectrometry (LC/MS/MS) techniques was utilized for the identification of drug metabolites of the HIV protease inhibitor Indinavir. This strategy integrates intelligent realtime mass spectrometry with HPLC detection and a predictive strategy for detecting metabolites arising from common biotransformations, to rapidly elucidate structures of drug metabolites. Structures of metabolites generated from in vitro incubation mixtures of Indinavir were characterized from a single chromatographic analysis using the automated LC/MS/MS methodology, thus reducing data acquisition time and improving efficiency.     

辉光放电质谱法测定超高纯铜中痕量杂质的记忆效应研究

随着高纯材料的需求日益增大,分析这种材料的辉光放电质谱技术也在不断的完善,其中一个困扰业界的问题就是记忆效应。本文采用辉光放电质谱法在分析锌样品前后分别两次测定了超高纯铜样品中10种痕量杂质(Mg、Al、Cr、Mn、Fe、Ni、Zn、As、Hg、Pb)。结果表明,两次测定结果基本一致,尤其是两次测定所得痕量锌含量结果一致,说明改变样品基体时,采用只清洗辉光放电池的方法可消除记忆效应。     

Seroepidemiologic study on the feasibility of screening for the antibody to HCV by pooling the serum specimens

The feasibility of screening for the antibody to HCV by pooling 10 serum specimens was studied by utilizing the epidemiologic method of single blind random allocation. We changed only the ratio between the pooled serum specimen volume and the serum specimen diluent volume, with their total volume and single serum specimen volume being as much as the volume required by EIA. Other steps were the same as those of EIA. The result showed that taking single serum specimen as the control group, the sensitivity and specificity of pooled serum specimen were 100% and 97.6%, respectively. The false positive rate was 2.4%, Kappa value was 0.95 (P < 0.05). The analysis of profit found that remarkable social and economic benefit will be obtained when serum pooling method is used. These results demonstrate that pooling 10 serum specimens is feasible to screening the antibody to HCV in blood donors by EIA.     

直流辉光放电质谱法测定高纯氧化镧中25种杂质元素

利用铜粉作为导电介质, 与氧化镧粉末混合均匀, 压片, 采用直流辉光放电质谱法(dc-GDMS)测定了高纯氧化镧粉末中的部分杂质元素含量。考察了辉光放电条件, 如放电电流、放电气体流量、离子源温度以及压片条件, 如两种粉末的混合比例、压片机压力等因素对放电稳定性以及灵敏度的影响, 优化了实验条件;尝试了将镧, 氧和铜的总信号归一化进行计算的方法, 用差减法计算了高纯氧化镧粉末中的杂质元素含量。将铜粉作为试剂空白, 连续测定11次, 统计各待测元素检出限为0.005~0.34 μg/g;对高纯氧化镧粉末样品独立测定6次, 测定结果与电感耦合等离子体质谱法基本吻合, 相对标准偏差在20%以内。     

氧气反应模式-电感耦合等离子体串联质谱法测定土壤中砷和硒

采用电感耦合等离子体质谱法(ICP-MS)测定土壤中的As和Se时会受到严重的氩化物、氯化物、氧化物、双电荷等多原子离子干扰,从而导致测定土壤中As和Se难度加大。采用石墨消解仪以王水消解土壤样品,在串接模式下,分别设置第一级四极杆质量过滤器(Q1)质荷比(m/z)为75和80,在碰撞/反应池中通入氧气,⁷⁵As⁺、⁸⁰Se⁺和氧气反应生成⁷⁵As¹⁶O⁺和⁸⁰Se¹⁶O⁺,而干扰离子不能与氧气反应,分别设置第二级质量过滤器(Q3)m/z为91和96,使得⁷⁵As¹⁶O⁺和⁸⁰Se¹⁶O⁺通过并进入检测器中,从而避免了质谱干扰,据此建立了石墨消解-电感耦合等离子体串联质谱法(ICP-MS/MS)测定土壤中As和Se的方法。对氧气流速进行了优化,选择氧气流速为1.0 mL/min。实验表明:As和Se的线性范围均为0.500～100 μg/L,线性相关系数分别为0.999 93和0.999 98,检出限分别为0.008 mg/kg和0.001 mg/kg,定量限分别为0.032 mg/kg和0.004 mg/kg。采用所建立的实验方法对土壤标准物质和土壤样品中的As和Se分别进行测定,结果表明,对于标准物质,As和Se的测定值均在认定值的范围内,相对标准偏差(RSD,n=6)分别在2.0%~4.4%和2.6%~7.8%之间;对于土壤样品,实验方法对As和Se的测定结果与原子荧光光谱法基本一致,相对标准偏差(n=6)分别在2.3%~4.5%和3.7%~7.9%之间。     

电感耦合等离子体串联质谱法测定 高纯氧化镁粉中金属杂质元素

基于电感耦合等离子体串联质谱(ICP-MS/MS)法建立了准确可靠分析高纯氧化镁粉中金属杂质元素的新方法。ICP-MS/MS通过启用新的质量过滤装置,在氧化镁基质的金属杂质元素测定过程中,能有效减少多原子干扰。采用He为碰撞气,O₂以及NH₃/He混合气为反应气,对比了在单四极杆(SQ)模式和串联四极杆(MS/MS)模式下消除干扰的效果。采用He碰撞模式无法消除一些特殊的质谱干扰,特别是双电荷离子干扰;然而,将分析物转移为氧化物离子或团簇离子,能实现待测元素的无干扰分析,并能获得极低的检出限,通过加标回收实验评估了方法的准确性。结果显示,方法的检出限为0.46~65.9ng/L。各元素的线性相关系数(R²)均不小于0.9998,真实样品的加标回收率为93%~108%,相对标准偏差为1.6%~4.4%。方法完全能用于高纯氧化镁粉中金属杂质元素的实时监控。     

Use of high-precision gas isotope ratio mass spectrometry to determine natural abundance 13C in lutein isolated from C3 and C4 plant sources

Previous studies have shown that cholesterol esterification activity by lecithin:cholesterol acyltransferase (LCAT) is progressively inhibited as up to three acidic acid residues are chemically modified. The purpose of this study was to determine whether three glutamic acid residues in LCAT (154, 155, and 165), that align exactly with three acidic acid residues (270, 271, and 281) in the amphipathic phospholipid binding region of apoE, were necessary for enzymatic activity. Site-directed mutagenesis was used to generate mutant constructs of LCAT in which glutamic acid residues 154, 155, and 165 were replaced with glutamine or lysine. Media harvested from transiently transfected COS cells was used as a source of LCAT for cholesterol esterification and phospholipase A2 (PLA2) assays. Cholesterol esterification for all mutant constructs (11-26 nmol CE/h/microg) was similar to or greater than that of wild type LCAT (16 nmol CE/h/microg), except for a triple mutant, in which glutamic acid residues 154, 155, and 165 were changed to lysines (5 nmol CE/h/microg). PLA2 activity followed a similar trend. There was a significant decrease in the cholesterol esterification to PLA2 activity ratio when residue 165 was mutated from its wild type negative charge (E) to an uncharged (Q) or positive (K) charged residue (10.2 vs. 6.0 vs. 4.3, respectively). We conclude that glutamic acid residues 154, 155, and 165 individually or collectively are not necessary for LCAT activity and that residue 165 may be in a region of LCAT that is involved with cholesterol binding or is sensitive to cholesterol binding at the active site of the enzyme.