高效液相色谱法测定人血清和尿中盐酸青藤碱浓度及药代动力学研究

用RP-HPLC法,以三唑仑为内标,反相C₁₈为分析柱,乙腈—0.01mol·L^-1磷酸二氢钠—四甲基乙二胺(46∶54∶0.22v/v)为流动相,磷酸调至pH6.9,检测波长263nm,测定血清和尿中盐酸青藤碱浓度,线性范围分别为6～480ng·mL^-1和0.06～3μg·mL^-1,平均回收率75.88%和91.35%,日内日间误差小于5%,最低检测浓度血清4ng·mL^-1,尿40ng·mL^-1。8名健康男性志愿者单次口服盐酸青藤碱片80mg,测定血清及尿浓度,该药符合二室开放模型,体内消除符合一级动力学消除过程,主要药代动力学参数:T_1/2α0.791±0.491h,T_1/2β9.397±2.425h,T_{max 1.040±0.274h,Cmax246.604±71.165ng·mL^-1,AUC 2651.158±1039.050ng·h·mL^-1,CL 0.033±0.01ng·mL^-1。}     

LC-MS/MS 法测定人血浆中倍他米松

建立测定人血浆中倍他米松的LC-MS/MS方法。采用Venusil XBP C₈ (200 mm×3.9 mm ID, 5 μm)色谱柱，流动相为甲醇-水(含甲酸铵5 mmol·L^-1)(80∶20)，流速0.4 mL·min^-1；质谱仪离子源为电喷雾离子源(ESI)，正离子模式检测，监测离子为393.3→355.2(倍他米松)和361.3→343.2(泼尼松龙,内标)。血浆样本用乙酸乙酯处理。倍他米松在0.5~80.0 ng·mL^-1线性关系良好(r=0.999 2)， 血浆低、 中、 高3种浓度(1.0， 10.0， 60.0 ng·mL^-1)平均提取回收率为88.24%，定量限为0.5 ng·mL^-1。本方法操作简便、准确、灵敏，适用于复方倍他米松注射液人体药代动力学研究。     

HPLC/MS/MS法测定人血浆中内源性尿嘧啶及二氢尿嘧啶

目的　为评价人体内二氢嘧啶脱氢酶的活性,建立高效液相色谱 串联质谱联用的分析方法,测定该酶的底物以及催化产物在血浆中的基础浓度。方法　血浆样品液 液萃取后,以纯水为流动相,以DiscoveryAmideC₁₆ 色谱柱初步分离,经气动辅助电喷雾离子源负离子化,在三级四极杆质量分析器上以多反应离子监测(MRM)方式检测尿嘧啶(MRM m/z 111.0→41.9)和二氢尿嘧啶(MRM m/z 113.0→42.0 )。结果　尿嘧啶和二氢尿嘧啶的最低定量浓度分别为0.5ng·mL^-1 和5ng·mL^-1 ;线性范围分别为0.5 - 100ng·mL^-1 和5 - 1000ng·mL^-1 ,精密度和准确度符合生物样品分析要求。结论　此法专属、灵敏、准确,适用于测定生物样本中内源性尿嘧啶和二氢尿嘧啶的基础浓度。     

灯盏花素及其β-环糊精包合物在大鼠体内的药代动力学

目的建立测定大鼠血浆中灯盏乙素浓度的反相高效液相色谱法，研究灯盏花素及其β-环糊精包合物(灯盏花素-β-CD)大鼠灌胃后体内药代动力学行为。方法以甲醇-水-醋酸盐缓冲液为流动相，Shim-pack C₁₈为固定相；12只大鼠随机均分为2组，分别灌胃灯盏花素及其包合物后，检测血浆药物浓度。药时数据采用3P97药代计算程序处理。结果线性范围10-400 ng·mL^-1，方法回收率95.32%-98.81%；灯盏花素和包合物的C_max分别为(154±18) ng·mL^-1和(328±31) ng·mL^-1；AUC_0-12h分别为(710±126) ng·h·mL^-1和(1 093±200)ng·h·mL^-1，经t检验两者有极显著性差异(P<0.01)。结论该法准确、灵敏，适用于灯盏乙素血浆浓度的测定；制备的灯盏花素包合物与灯盏花素相比吸收显著增加。     

HPLC-MS/MS法测定人血浆中的左西孟旦及其主要代谢物

建立快速、 灵敏、 易操作的LC-MS/MS法测定人血浆中的左西孟旦及其代谢物OR-1855和OR-1896的浓度。根据待测物的不同性质， 采用两套液相色谱系统和电离方式分别测定人血浆中的左西孟旦和代谢物OR-1855、 OR-1896。测定左西孟旦时， 用瑞舒伐他汀为内标， 血浆样品经甲醇沉淀蛋白， 以甲醇-15 mmol·L^-1醋酸铵-甲酸(55∶45∶0.02， v/v/v)为流动相， Capcell MG III C₁₈柱(35 mm×2.0 mm ID， 3 μm)进行分离， 采用电喷雾电离源，以选择反应监测(SRM)方式进行负离子检测。测定代谢物OR-1855和OR-1896时， 用多索茶碱为内标， 血浆样品经乙酸乙酯萃取， 以甲醇-15 mmol·L^-1醋酸铵-甲酸(65∶35∶0.1， v/v/v)为流动相， Zorbax Extend C₁₈柱(150 mm×4.6 mm ID， 5 μm)进行分离， 采用电喷雾电离源， SRM方式进行正离子检测。测定血浆中左西孟旦方法的线性范围为0.10～50.0 ng·mL^-1， 定量下限可达0.10 ng·mL^-1； 测定血浆中代谢物OR-1855和OR-1896方法的线性范围均为0.20～100 ng·mL^-1， 定量下限均可达0.20 ng·mL^-1。本方法专属性好， 准确、 快速， 适用于左西孟旦注射液的临床药代动力学研究。     

LC-MS/MS同时测定朱砂中掺假染料猩红808和玫瑰红B的含量

目的 建立液相色谱-串联质谱(LC-MS/MS)法同时测定朱砂中的掺假染料猩红808和玫瑰红B的含量。方法 采用Agilent ZORBAX SB-C₁₈(2.1 mm×50 mm,1.8 μm)色谱柱,流动相为乙腈-0.1%甲酸溶液,流速为0.3 mL·min^-1,柱温为30 ℃,离子化方式为ESI,检测模式为MRM,选择离子对：①猩红808 m/z 368.1→275.1,m/z 368.1→219.1,m/z 368.1→77.1;②玫瑰红B m/z 443.2→399.2,m/z 443.2→355.0。结果 该方法猩红808和玫瑰红B的线性范围为10~200 ng·mL^-1和9.69~193.8 ng·mL^-1;检测限分别为2 ng·mL^-1和1.9 ng·mL^-1;回收率(n=6)分别为97.86%和97.34%,RSD为0.70%和0.73%;精密度、稳定性和重复性的RSD均<4%。结论 该方法提取简单,测定方法定量准确,确证性极高、灵敏高,适用于朱砂中红色染料猩红808和玫瑰红B的含量测定,还可为其他红色中药的安全检测提供了借鉴依据。     

全血及血清中羟氯喹HPLC测定方法的建立及其应用

目的：建立高效液相色谱法测定人全血及血清中羟氯喹浓度。方法：200 μL人全血（血清）样品经400 μL的10%甲酸-乙腈（1∶9）沉淀后,取上清液进行色谱分析。采用Zorbax SB-C₁₈为色谱柱,以乙腈-0.2 mol·L^-1磷酸二氢钾（15∶85,v/v,磷酸调pH=3.0）为流动相,检测波长：254 nm,流速：0.8 mL·min^-1,进样量：30 μL。结果：对46例长期（>1年）服用硫酸羟氯喹（200 mg·d^-1）的系统性红斑狼疮患者的血药谷浓度进行检测分析。羟氯喹在10~3000 ng·mL^-1线性关系良好,批内、批间精密度RSD均小于8.6%,全血及血清样品的提取回收率分别为74.5%~78.6%和80.7%~85.4%。患者平均全血/血清谷浓度分别为（707.6±398.4） ng·mL^-1、（282.2±153.8） ng·mL^-1。结论：本法操作简便,灵敏,准确度高,适用于羟氯喹血药浓度检测及药效学研究。     

液相色谱-电喷雾串联质谱法测定人血浆中班布特罗:在药代动力学研究中的应用

目的　建立液相色谱 串联质谱法测定人血浆中班布特罗浓度,研究中国受试者口服该药的动力学特点。方法　血浆样品经液 液萃取后,采用液相色谱电喷雾串联质谱法以选择离子反应监测(SRM)方式进行检测。结果班布特罗的线性范围为0.05 - 4.0ng·mL^-1 ,最低定量浓度为0.05ng·mL^-1 ,该法的日内及日间精密度(RSD)小于8% ,准确度(RE)在±9%范围内。18名中国健康受试者单剂量口服班布特罗10 mg后,主要药动学参数T_max,C_max, T_1/2和AUC_0-t分别为(2.3±1.3)h ,(3.95±2.20 )ng·mL^-1 ,(11.4±6.1)h和(26.85±11.77)ng·h·mL^-1 。结论　该法灵敏度高,操作简便、快速,适用于临床药代动力学研究     

LC-MS/MS法测定人血浆中左舒必利含量的不确定度评定

摘 要 目的：评定LC MS/MS法测定人血浆中左舒必利含量的不确定度。方法: 对测定过程中不确定度的来源进行分析评定,包括称量、纯度、仪器误差、标准溶液的配制、含药血浆样本的配制、标准曲线拟合、重复性、血浆样本的处理等,分析各分量的不确定度与合成不确定度,最终计算扩展不确定度。结果: 人血浆中低(9.0 ng·mL^-1)、中(150.0ng·mL^-1)、高(3 200.0 ng·mL^-1) 3个浓度的扩展不确定度分别为2.46,10.42,173.77 ng·mL^-1(k=2,P=95%)。结论:LC-MS/MS法测定人血浆中左舒必利含量的不确定度主要由血浆样本的处理,仪器误差、标准溶液的配制及标准曲线拟合（低浓度）引入。     

手性和非手性联用色谱法研究尼卡地平对映异构体兔体内过程的差异性

目的研究尼卡地平(NCD)对映体在家兔体内药代动力学和组织分布的差异性。方法生物样品在碱性条件下,正己烷-醋酸乙酯(1:1)提取,用手性和非手性联用色谱法进行分离分析。结果尼卡地平及其对映体分别在反相色谱系统及手性色谱系统中分离良好,浓度为55-550ng·mL^-1线性关系良好。对映体的平均日内、日间RSD分别为5.25%和8.97%,回收率分别为99.99%和97.10%;对映体间主要动力学参数T_max,C_max和AUC,S-NCD为(2.49±0.03)h,(134±2)ng·mL^-1和(1082±32)ng·mL^-1·h,R-NCD为(1.24±0.05)h,(109±2)ng·mL^-1和(778±22)ng·mL^-1·h;在主要脏器和细胞中S-NCD的浓度明显高于R-NCD。药代动力学和靶组织分布均有一定差异性。结论尼卡地平对映体兔体内过程包括代谢动力学和靶细胞浓度分布存在着立体差异性。     

Identification of protein components and quantitative immunoassay for SEC2 in staphylococcin injection

In China, staphylococcin injection has been commonly used in combined cancer therapy to enhance the systemic immune response and reduce the toxicities associated with chemotherapy or radiation therapy in the last decade. It is claimed that the main effective component is staphylococcal enterotoxin C2 (SEC2). However, no standard method based on the concentration of SEC2 has been established for quality control of the injection products. In this study, a sensitive and reliable biotin–streptavidin–ELISA (BS–ELISA) method was established for detection and quantification of SEC2. In addition, 1-D SDS-PAGE coupled with nano-LC–MS/MS was performed to identify the protein components in the injection products from one manufacturing company. The results of the BS–ELISA showed that SEC2 only accounted for less than 0.1% of the total protein in the injection products, and the nano-LC–MS/MS results showed that fifty-five proteins of Staphylococcus aureus were confidently identified in the injection solution. Seventeen out of these proteins, including SEC2, were well-known virulence factors. In addition, eighteen proteins of other Gram-positive bacteria were also confidently identified. Thus, the results indicated that SEC2 is of very low concentration in the injection products and the process of the injection preparation should be improved for health and safety consideration.     

Expression and bioactivity analysis of staphylococcal enterotoxin G and staphylococcal enterotoxin I

Context: The filtrate of Staphylococcus aureus culture, named staphylococcal enterotoxin C injection, has been used for 10 years in China. SEC2 has been claimed to be the only staphylococcal enterotoxins (SEs) without certifiable evidence.

Objectives: To present an efficient procedure for the expression and purification of recombinant proteins SEG and SEI, from S. aureus.

Materials and methods: In present work, we extracted total DNA from S. aureus (FRI 1230) and the recombinant proteins of SEG and SEI were then cloned, expressed and purified using E. coli. Splenic lymphocytes were used as effector cells and K562 and B16 cells were used as target cells to evaluate the inhibitory and stimulatory abilities of purified rSEG and rSEI on in vitro proliferation.

Results: The size of amplified products of SEG and SEI genes were found to be about 400 and 467?bp, respectively. pGEX-SEG and pGEX-SEI were constructed successfully. SEG and SEI were demonstrated to be active stimulators of T-cell proliferation; moreover, they inhibited the proliferation of K562 cells and B16 cells.

Discussion: The current findings suggest that SEC2 might not be the only active component of staphylococcal enterotoxin C injection and may involve other essential proteins like SEG and SEI in its clinical efficacy.

Conclusion: This efficient procedure for the expression and purification of SEG and SEI and may be useful for mass production of therapeutically important proteins. In the future, proteins acting as active stimulators of T-cell proliferation may help in developing effective cancer therapy.     

金黄色葡萄球菌肠毒素SEC2的纯化与活性研究

从金黄色葡萄球菌发酵液中纯化金黄色葡萄球菌肠毒素C2（SEC2）。发酵液经超滤浓缩、硫酸铵沉淀、阴离子交换色谱和分子筛色谱，获得了纯度为90％的SEC2，总收率23％。体外刺激脾淋巴细胞增殖实验以及体外肿瘤细胞增殖抑制试验表明该蛋白具备良好的超抗原活性。     

胸腔内注射金黄色葡萄球菌滤液注射液治疗恶性胸水的疗效

目的 :研究金黄色葡萄球菌滤液注射液 (含金黄色葡萄球菌肠毒素C ,SEC)胸腔内注射治疗恶性胸水的疗效。方法 :经组织学或细胞学检查确诊的恶性胸水病人 12例 ,包括非小细胞肺癌 9例 ,小细胞肺癌 ,子宫肉瘤和黑色素瘤各 1例。行常规胸腔穿刺术并尽量抽尽胸水后注入金黄色葡萄球菌滤液注射液 10mL(含SEC 5 0ng) ,每周 2次 ,治疗时间 2～ 3wk。结果 :完全有效 (CR) 10例 (83% ) ,部分有效 (PR ) 2例 ,总有效率 10 0 %。CR中 1例无病生存达 2 0mo ,另有 2例分别在治疗后 10mo和 6mo死亡 ,但未见胸水复发。与治疗前比较 ,胸腔注射金黄色葡萄球菌滤液注射液 6h后普遍出现周围血液淋巴细胞轻度升高 ,而胸水淋巴细胞比例升高显著。未发现明显不良反应。结论 :胸腔内注射金黄色葡萄球菌滤液注射液治疗恶性胸水的疗效确切 ,使用安全简便     

Superantigen staphylococcal enterotoxin C1 mutant can reduce paraquat pulmonary fibrosis

Abstract

A network of inflammation factors is related to pulmonary fibrosis induced by paraquat (PQ) poisoning. At high doses, the superantigen staphylococcal enterotoxin C1 (SEC1) can induce immunological unresponsiveness and inhibit release of inflammation factors. In this study, site-directed mutagenesis was performed at the H118 and H122 amino acid residues of SEC1 to reduce SEC1 toxicity. The SEC1 mutant showed significantly decreased pyrogenic toxicity, but retained clonal anergy at high dosages in vitro. Pretreatment with the SEC1 mutant prior to PQ poisoning in mice reduced symptom duration and severity, prolonged survival time, and decreased the splenocyte response to ConA induction. The SEC1 mutant also down-regulated several important cytokines related to fibrosis in the plasma after PQ poisoning. SEC1 decreased the expression of genes related to pulmonary fibrosis, including α-SMA, COL1a1, COL3 and TGF-β1, in PQ poisoned mice. Histomorphological observation indicated alleviation of pathological changes in the lungs after SEC1 pretreatment compared to mice in the PQ group. In conclusion, the SEC1 mutant reduced pulmonary interstitial fibrosis induced by PQ poisoning.     

Effects of staphylococcal enterotoxin B on functional and biochemical changes of the lung in rhesus monkeys

Effects of staphylococcal enterotoxin B were studied in anesthetized rhesus monkeys. During the period from 6 to 11 hr following i.v. injection of the toxin (1·0 mg/kg), respiratory quotient increased, while functional residual capacity, CO₂ output, O₂ consumption and expired CO₂ concentration decreased. By 11·5 hr, the surface tension of lung extracts and total lung water content increased, as shown by simultaneous accumulations of extracellular Na⁺ and water. These results provide evidence to support a hypothesis that pulmonary dysfunction and terminal pulmonary edema contribute to death during enterotoxemia by staphylococcal B in rhesus monkeys.     

一种快速分离纯化金葡菌肠毒素C2的方法

Objective To develop a simple and convenient method to purify staphylococcal enterotoxin C₂ (SEC₂) from the supernatant of staphylococcus aureus culture. Methods SEC2was purified by ultrafiltration, anion ion-exchange chromatography, and cation ion-exchange chromatography sequentially. The molecular weight of SEC₂ was detected by the SDS-PAGE and flight time mass-spectrum . The purity of SEC₂ was detected by the SDS-PAGE and high performance liquid chromatography. The purified SEC2 was verified by amino acids sequence analysis. Results The purity of purified SEC₂ was greater than 98% (w)which was consistent with literature, including microheterogeneity of isoelectric point, pI 7.49 and 6.74, the molecular weigh 27.58 KD.The amino acids sequence of purified SEC2 was consistent with what was reported in literatures（ESQPDPTPDELHKSS）. The maximum absorption wavelength of purified SEC2 was 277.5 nm. Conclusions The method is rapid, simple and convenient with high SEC₂ purity, the recovery is more than 50 %(w).This method is suitable to large scale preparation of SEC₂.     

金葡菌肠毒素C2的克隆表达及其生物学活性

目的克隆金葡菌肠毒素C₂全长基因，构建SEC₂的表达载体，实现其可溶性表达，并对纯化的rSEC₂蛋白的生物学活性进行研究。方法通过聚合酶链式反应(polymerase chain reaction，PCR)从金葡菌FRI1230菌株基因中得到肠毒素SEC₂的基因，将其克隆至融合表达载体pGEX-4T-1，转化大肠杆菌进行表达并对融合蛋白进行亲和色谱纯化。通过考察重组SEC₂对淋巴细胞的增殖作用及其对肿瘤细胞杀伤活性的影响，对其超抗原活性和免疫学活性进行研究。结果得到正确的肠毒素SEC₂基因序列并得到高效表达的融合蛋白，MTT法结果表明，重组SEC₂表现出良好的促淋巴细胞增殖活性，且能够增强淋巴细胞对肿瘤细胞的杀伤活性。结论本研究成功克隆了SEC₂基因，表达并纯化出具有抗肿瘤生物学活性的重组SEC₂蛋白，为进一步对其分子抗肿瘤作用机制进行研究以及构建靶向抗肿瘤融合蛋白奠定了基础。     

野生型金黄色葡萄球菌肠毒素C2在E.coli中的表达和纯化研究

目的克隆金黄色葡萄球菌肠毒素C2(SEC2)基因在E.coli重组表达,纯化重组SEC2(rSEC2)并进行生物学活性分析。方法PCR获得正确编码SEC2的基因片断,构建表达质粒pET-28a-sec2在E.coliBL21中表达。利用离子交换和分子筛色谱纯化rSEC2,四甲基偶氮唑盐(MTT)法对rSEC2和天然SEC2(nSEC2)生物学活性进行分析和比较。结果可溶性rSEC2占菌体总蛋白质的40%,两步纯化后蛋白质纯度约达95%,rSEC2和nSEC2均具有显著的超抗原活性。结论成功获得与nSEC2有着类似活性的高纯度rSEC2,为进一步研究该蛋白质的抗肿瘤机理奠定物质基础。     

Quantitative Analysis of Staphylococcal Enterotoxins A and B in Food Matrices Using Ultra High-Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS)

A method that uses mass spectrometry (MS) for identification and quantification of protein toxins, staphylococcal enterotoxins A and B (SEA and SEB), in milk and shrimp is described. The analysis was performed using a tryptic peptide, from each of the toxins, as the target analyte together with the corresponding ¹³C-labeled synthetic internal standard peptide. The performance of the method was evaluated by analyzing spiked samples in the quantification range 2.5–30 ng/g (R² = 0.92–0.99). The limit of quantification (LOQ) in milk and the limit of detection (LOD) in shrimp was 2.5 ng/g, for both SEA and SEB toxins. The in-house reproducibility (RSD) was 8%–30% and 5%–41% at different concentrations for milk and shrimp, respectively. The method was compared to the ELISA method, used at the EU-RL (France), for milk samples spiked with SEA at low levels, in the quantification range of 2.5 to 5 ng/g. The comparison showed good coherence for the two methods: 2.9 (MS)/1.8 (ELISA) and 3.6 (MS)/3.8 (ELISA) ng/g. The major advantage of the developed method is that it allows direct confirmation of the molecular identity and quantitative analysis of SEA and SEB at low nanogram levels using a label and antibody free approach. Therefore, this method is an important step in the development of alternatives to the immune-assay tests currently used for staphylococcal enterotoxin analysis.