LC-MS/MS法对融合蛋白FP3的氨基酸全序列测定

运用液质联用、两种串联质谱对融合蛋白FP3的氨基酸全序列测定, 确证其一级结构。将样品还原烷基化后, 通过胰蛋白酶酶解蛋白, PNGase F去除多肽混合物中糖肽的糖基化, 将去糖后的总肽用于液质联用系统, 通过液相分离后, 运用Q-TOF和线性离子阱两种串联质谱测定各个肽段的b, y碎片离子, 分析测定融合蛋白FP3的氨基酸全序列。通过LC-ESI-Q-TOF完成了融合蛋白FP3的76%氨基酸序列测定, 通过LC-ESI-Trap完成余下24%氨基酸序列测定。液质联用、串联质谱法测定蛋白质氨基酸序列快速、灵敏、准确, 是对重组蛋白结构分析和确证的重要手段。     

Development of a sensitive bioanalytical method for determination of PNU-83757 in rat,monkey and human plasma: from LC-UV to LC-MS/MS

To support pre-clinical pharmacokinetic/toxicokinetic (PK/TK) evaluation, a sensitive bioanalytical method for determination of N-cyano-N'-(tert-pentyl)-N"-(3-pyridinyl) guanidine (PNU-83757), in rat and monkey plasma was required. Although the UV response of PNU-83757 was quite decent and the extracts using solid phase extraction (SPE) were very selective and concentrated, the best limit of quantitation (LOQ) achieved was 0.4 ng ml(-1) using 0.5 ml plasma for extraction and 2 ng ml(-1) using 0.1 ml plasma for extraction, which was insufficient for PK/TK evaluation at lower doses. When using liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometric detection (LC-APCI-MS/MS, positive ions) and SPE, a LOQ of 0.045 ng ml(-1) for PNU-83757 was reached. Quantitation was accomplished using the precursor --> product ion combinations of m/z 232 --> 162 for PNU-83757 and m/z 236 --> 166 for the internal standard, [2H(4)]PNU-83757, in the multiple reaction monitoring mode. This method has been successfully utilized for PK/TK evaluation in pre-clinical studies and proved to have sufficient sensitivity to determine plasma concentrations for a dose level as low as 1 microg kg(-1) day(-1) in the rat and monkey. Further improvement of this method by using electrospray mass spectrometric detection (LC-ESI-MS/MS, positive ions) and automated membrane SPE, gave an LOQ of 0.008 ng ml(-1), and allowed analysis of large numbers of samples to support clinical PK studies in microg dose levels.     

Quantitative evaluation of protein conformation in pharmaceuticals using cross-linking reactions coupled with LC-MS/MS analysis

The need for a simple and high-throughput method for identifying the tertiary structure of protein pharmaceuticals has increased. In this study, a simple method for mapping the protein fold is proposed for use as a complementary quality test. This method is based on cross-linking a protein using a [bis(sulfosuccinimidyl)suberate (BS(3))], followed by peptide mapping by LC-MS. Consensus interferon (CIFN) was used as the model protein. The tryptic map obtained via liquid chromatography tandem mass spectroscopy (LC-MS/MS) and the mass mapping obtained via matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy were used to identify cross-linked peptides. While LC-MS/MS analyses found that BS(3) formed cross-links in the loop region of the protein, which was regarded as the biologically active site, sodium dodecyl-sulfate polyacrylamide gel electrophoresis demonstrated that cross-linking occurred within a protein molecule, but not between protein molecules. The occurrence of cross-links at the active site depends greatly on the conformation of the protein, which is determined by the denaturing conditions. Quantitative evaluation of the tertiary structure of CIFN was thus possible by monitoring the amounts of cross-linked peptides generated. Assuming that background information is available at the development stage, this method may be applicable to process development as a complementary test for quality control.     

Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC‐MS/MS bioanalysis of human IgG and Fc‐fusion protein drug candidates

The synthesis of a 16‐residue, stable isotopically labeled peptide is described for use as a LC‐MS/MS (Liquid chromatography‐mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc‐fusion protein drug candidates in animal species used in pre‐clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave‐assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable‐labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc‐[¹³C₆, ¹⁵N]‐l ‐leucine onto an 11‐unit segment followed by automated microwave‐assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long‐term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave‐assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side‐chains.     

乙烷硒啉的临床药物动力学和代谢转化特点研究

目的研究肿瘤患者口服乙烷硒啉(1,2-[bis(1,2Benzisoselenazolone-3(2H)-ketone)]ethane,BBSKE)后的药物动力学及体内代谢转化特征。方法3例肿瘤患者单次口服给药剂量为600mg.d-1,采集各个时间点的血浆样品及尿样,用液相色谱-串联四极杆质谱(LC/ESI-MS/MS)联用技术测定血浆样品中BBSKE的含量,用液相色谱-电喷雾离子阱质谱(LC-ESI/MSn)联用技术对尿及血浆中的代谢产物进行分析鉴定。结果得到了BBSKE在血浆中的药时曲线图及主要的药物动力学参数。在尿中共发现了6个BBSKE的氧化、甲基化、葡萄糖醛酸化代谢产物,在血浆中共发现了2个BBSKE的氧化、葡萄糖醛酸化代谢产物。结论BBSKE的血药浓度较低,表观分布容积大。氧化、甲基化、葡萄糖醛酸化反应是BBSKE在人体内的3种重要代谢途径。     

超滤法结合液质联用技术测定抗肿瘤化合物CJ-6在不同种属中的血浆蛋白结合率

目的:通过建立测定血浆中抗肿瘤化合物N-(4-((2-(环丙烷甲酰胺)吡啶-4-基)氧基)-3-氟苯基)-4-甲基-3,5-二氧基-2-苯基-2,3,4,5-四氢-1,2,4-三嗪-6-甲酰胺(CJ-6)的分析方法,测定CJ-6在不同种属(大鼠、猴、人)中的血浆蛋白结合率.方法:采取超滤法测定CJ-6在大鼠、猴、人血浆...     

Utility of high-resolution accurate MS to eliminate interferences in the bioanalysis of ribavirin and its phosphate metabolites

Background: The polar nucleoside drug ribavirin (RBV) combined with IFN-α is a front-line treatment for chronic hepatitis C virus infection. RBV acts as a prodrug and exerts its broad antiviral activity primarily through its active phosphorylated metabolite ribavirin 5′-triphosphate (RTP), and also possibly through ribavirin 5′-monophosphate (RMP). To study RBV transport, diffusion, metabolic clearance and its impact on drug-metabolizing enzymes, a LC-MS method is needed to simultaneously quantify RBV and its phosphorylated metabolites (RTP, ribavirin 5′-diphosphate and RMP). In a recombinant human UGT1A1 assay, the assay buffer components uridine and its phosphorylated derivatives are isobaric with RBV and its phosphorylated metabolites, leading to significant interference when analyzed by LC-MS with the nominal mass resolution mode. Results: Presented here is a LC-MS method employing LC coupled with full-scan high-resolution accurate MS analysis for the simultaneous quantitative determination of RBV, RMP, ribavirin 5′-diphosphate and RTP by differentiating RBV and its phosphorylated metabolites from uridine and its phosphorylated derivatives by accurate mass, thus avoiding interference. Conclusion: The developed LC-high-resolution accurate MS method allows for quantitation of RBV and its phosphorylated metabolites, eliminating the interferences from uridine and its phosphorylated derivatives in recombinant human UGT1A1 assays.     

LC-MS/MS and FT-IR analyses of stones from a patient with Crohn's disease: a case report

This report describes the unusual case of a patient affected by Crohn’s disease suffering from intestinal obstruction with recurrent occlusive symptoms not due to the intestinal disease, but to the presence of two calcified foreign bodies in the pelvis. The stones were surgically removed and analysed by reverse-phase liquid chromatography coupled to UV diode array detection and mass spectrometry (LC-UV-DAD-MS/MS), Chromatoprobe-MS/MS and by Fourier-transform-infrared spectroscopy (FT-IR) techniques. The combined mass spectrometric approaches allowed unequivocally to identify 5-aminosalicylic acid (5-ASA) in stone 1, and to demonstrate that its formation was due to an unmodified 5-ASA tablet, a formulation that must undergo complete dissolution in the small bowel. The second stone was constituted by a solid layer (no solvent-extractable material) identified by FT-IR as a polystyrene fragment. This indicates that accidental ingestion of a plastic material, followed by its calcification, was responsible for its formation.     

Comparative protein profile of human hepatoma HepG2 cells treated with graphene and single-walled carbon nanotubes: an iTRAQ-coupled 2D LC-MS/MS proteome analysis

Graphitic nanomaterials are promising candidates for applications in electronics, energy, materials and biomedical areas. Nevertheless, few detailed studies related to the mechanistic understanding of these nanomaterials with the living systems have been performed to date. In the present study, our group applied the iTRAQ-coupled 2D LC-MS/MS approach to analyze the protein profile change of human hepatoma HepG2 cells treated with graphene and single-walled carbon nanotubes (SWCNTs), with the purpose of characterizing the interactions between living system and these nanomaterials at molecular level. Overall 37 differentially expressed proteins involved in metabolic pathway, redox regulation, cytoskeleton formation and cell growth were identified. Based on the protein profile, we found SWCNTs severely interfered the intracellular metabolic routes, protein synthesis and cytoskeletal systems. Moreover, our data suggested that SWCNTs might induce oxidative stress, thereby activating p53-mediated DNA damage checkpoint signals and leading to apoptosis. However, only moderate variation of protein levels for the cells treated with graphene was observed, which indicated graphene was less toxic and might be promising candidate for biomedical applications. We envision that this systematic characterization of cellular response at protein expression level will be of great importance to evaluate biocompatibility of nanomaterials.     

Bioanalytical method development and validation for a large peptide HIV fusion inhibitor (Enfuvirtide, T-20) and its metabolite in human plasma using LC-MS/MS

A method for measuring a human immunodeficiency virus (HIV) cell membrane fusion inhibitor (T-20/Ro 29-9800) and its metabolite (M-20/Ro 50-6343) in human plasma by liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. The relatively large peptide analytes and their corresponding deuterated (d(10)) peptides used as internal standard were isolated from plasma by protein precipitation with two volumes of acetonitrile to plasma. A large pore size reversed-phase C(18) column was employed to elute the peptides. A triple quadrupole mass spectrometer with electrospray interface operating in positive ion and multiple reaction monitoring modes with transitions m/z 1124-->1343 for both T-20 and M-20 was utilized for peak detection. The advantages of the method were a simple sample preparation, specific and sensitive MS/MS detection, and a wide dynamic range of 10-2000 ng/ml for T-20. The method was validated and used for analyzing samples from clinical studies to provide pharmacokinetic profiles of the HIV fusion inhibitor peptide drug and its metabolite.     

高效液相色谱-质谱联用法测定北沙参中欧前胡素和异欧前胡素的含量

目的建立北沙参中欧前胡素和异欧前胡素含量测定的方法。方法采用高效液相色谱-质谱法:色谱柱:赛默飞世尔Accucore C18(100 mm×2.1 mm,2.6μm);流动相:5 mmol·L-1甲酸溶液-甲醇(80∶20,v/v);流速:0.2 m L·min-1,柱温:20℃。结果欧前胡素和异欧前胡素均在10~5000ng·m L-1与峰面积具有良好的线性关系,平均加样回收率分别为99.3%和99.1%,RSD分别为1.3%和1.1%(n=6)。结论本方法能够快速、准确、方便的检测北沙参中欧前胡素和异欧前胡素的含量,可作为北沙参质量评价的依据。     

LC-MS/MS法测定比格犬血浆中芬太尼浓度及药动学研究

目的 建立比格犬血浆中芬太尼的液相色谱-串联质谱（LC-MS/MS）测定方法,并用于药动学研究。方法 采用固相萃取（SPE）法从血浆中提取芬太尼和内标芬太尼-d5,建立比格犬血浆中芬太尼的LC-MS/MS测定方法,进行特异性、准确度、精密度、基质效应、灵敏度、稀释可靠性、稳定性方法学验证;8只比格犬,分别单次iv给予芬太尼的生理盐水溶液400 mg/只,用LC-MS/MS测定给药后血浆中芬太尼浓度,并用WinNonLin软件计算药动学参数。结果 芬太尼的线性范围为2～1 000 pg/mL,精密度、准确度、基质效应、灵敏度、稀释可靠性、稳定性均符合生物样品分析要求。比格犬体内芬太尼药动学参数：t_1/2为（4.53±0.748）h,AUC_0-t为（19 659±3 889）h·ng/mL,CL为（2 259±284）mL/（h·kg）,符合二室开放模型。结论 建立的LC-MS/MS分析方法准确灵敏,适用于芬太尼的药动学研究。     

A Comparative Analysis of Methods (LC-MS/MS,LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters,Mussels and Pipis

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r² = 0.86) and the PP2A kit (r² = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.     

LC-MS/MS法测定吡非尼酮在大鼠体内的药动学参数

目的建立灵敏的液相色谱-串联质谱(LC-MS/MS)法测定大鼠血浆中吡非尼酮的浓度。方法血浆样品采用乙腈蛋白沉淀方法,色谱柱为Dikma Diamonsil C18;以0.1%甲酸-乙腈(45:55,v/v)为流动相;流速为0.4 m L·min-1;柱温为30℃。结果吡非尼酮血药浓度在5~2000 ng·m L-1内线性关系良好(r=0.9995),最低检测限为5 ng·m L-1;日内、日间RSD均≤10%,高、中、低3种浓度的回收率均在93%左右。6只SD大鼠单剂量口服给予吡非尼酮后药动学参数分别为:Cmax(1354.3±143.5)ng·m L-1;t1/2(2.21±0.24)h;AUC0~t(3474.6±982.5)h·ng·m L-1;AUC0~∞(3687.4±992.4)h·ng·m L-1。结论本方法简便、准确、灵敏、专属性强,同样适用于人血浆中吡非尼酮浓度的测定及其药动学研究,对于评价吡非尼酮疗效和安全性有重要意义。     

Development and validation of a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the determination of sunitinib in rabbit plasma

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the measurement of sunitinib in rabbit plasma. After protein precipitation with acetonitrile, samples were analyzed on a Zorbax Extend-C₁₈ column (150 mm×4.6 mm, 5μm). The mobile phase consisted of a mixture of acetonitrile and deionized water (containing 0.05% formic acid)at a ratio of 27:73 (v/v), and the flow rate was set at 0.8 mL/min.The column temperature was maintained at 30 ^oC. The LC eluate was detected by an electrospray ionization (ESI) source operated in the positive ion mode, and quantification was conducted using MRM of the transitions m/z 399.24→283.01 and m/z 415.19→178.00 for sunitinib and internal standard (IS, diltiazem hydrochloride), respectively. The calibration curve was linear in the range of 2–600 ng/mL. The lower limit of quantification was 2 ng/mL. The method also exhibited satisfactory results in terms of sensitivity, specificity, accuracy (with relative error ranging from –4.0% to 1.1%), precision (with intra- and inter-day relative standard deviations ranging from 2.8% to 9.5%),matrix effect, recovery as well as stability. Taken together, our newly developed method was reliable to monitor sunitinib concentrations in rabbit plasma.     

LC, LC-MS/TOF and MS(n) studies for the identification and characterization of degradation products of nelfinavir mesylate

The objective of the present investigation was to separate, identify and characterize the major degradation products (DPs) of nelfinavir mesylate generated under hydrolytic, oxidative, photolytic and thermal stress conditions as advised in International Conference on Harmonization (ICH) guideline Q1A(R2). The drug was found to degrade under acidic, basic, oxidative and photolytic stress, while it was stable in neutral and thermal stress conditions. A total of three degradation products were formed, which were separated on a C-18 column employing a gradient HPLC method. A complete mass fragmentation pathway of the drug was first established with the help of multi-stage (MS(n)) and MS/TOF accurate mass studies. Then stressed samples were subjected to LC-MS/TOF studies, which provided their fragmentation pattern and accurate masses. The mass spectral data were employed to characterize the DPs and assign structures to them. The total information was also used to establish the degradation pathway of the drug. The degradation products were identified as 3-hydroxy-N-((2R,3R)-3-hydroxy-1-(phenylthio)butan-2-yl)-2-methylbenzamide and (3S,4aS,8aS)-N-tert-butyl-2-((2R,3R)-2-hydroxy-3-(3-hydroxy-2-methylbenzamido)-4-(phenylsulfinyl)butyl)decahydroisoquinoline-3-carboxamide.     

Simultaneous determination of L-arginine, asymmetric dimethylarginine, and symmetric dimethylarginine in the plasma of rodents with LC-MS/MS

Nitric oxide (NO) is synthesized from L-arginine (Arg) by NO synthase (NOS), and asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are endogenous inhibitors of NO formation. Normal distribution values of Arg, ADMA, and SDMA are required to evaluate the effects of cardiovascular drugs on blood vessels, but insufficient normal reference values from rat and mouse plasma exist for new drug development and screening. To determine the means and variations in the basal endogenous materials concentration, Arg, ADMA, and SDMA in blank rat (n = 24) and mouse (n = 37) plasma samples were quantified using LC-MS/MS equipped with an electrospray ionization interface to generate positive mode ions. Accuracy and precision were within 90.42-110.91%, and 0.88-13.84%, respectively, for analyses of Arg, ADMA, and SDMA. The average plasma concentrations of Arg, ADMA, and SDMA were 175.38 +/- 13.87 microM, 0.79 +/- 0.20 microM, and 0.84 +/- 0.20 microM, respectively, in rats and 70.81 +/- 19.38 microM, 0.66 +/- 0.21 microM, and 0.42 +/- 0.10 microM, respectively, in mice. These results will provide a basis on which to evaluate cardiovascular drug effects on ARG, ADMA, and SDMA levels in new drug development.     

LC-MS/MS法和mFPIA法测定全血环孢素A浓度的相关性比较

目的:对高效液相色谱电喷雾串联质谱(LC-MS/MS)法和单克隆荧光偏振免疫(mFPIA)法测定肾移植术后稳定期患者不同时间点的全血环孢素A(CsA)浓度进行相关性比较。方法:分别采用LC-MS/MS法和mFPIA法测定患者全血CsA浓度,对测定结果进行双侧配对t检验比较差异性,用Passing-Bablok回归分析法比较不同采血时间点2种方法测定结果的相关性。结果:2种方法测定值之间具有显著性差异;服药后0、3.0、4.0h时2法测定值的相关性最好,0.5、0.75、12.0h相关性最差。结论:对CsA血药浓度监测结果的分析需考虑监测方法以及不同采血时间点的影响。