柱切换HPLC法分析尿中吗啡和O^6—单乙酰吗啡的研究

目的建立用柱切换HPLC技术分析尿中吗啡和06-单乙酰吗啡的方法.方法尿样用硼砂缓冲液(pH9.2)稀释后进入预处理柱(YWG-C18,33mm×5.0mm,10μm),用H2O洗去杂质,再用CH3OHH2O(6040)将被分析组分洗脱进入分析柱(Lichrospher(R)100CN,125mm×4.0mm,5μm),分析流动相为CH3OH磷酸盐缓冲液(pH6.86)=2278.紫外检测器波长为286nm.结果尿中吗啡和06--单乙酰吗啡的线性范围分别为50～1 600n/ml和100～1 600n/ml.吗啡和O6--单乙酰吗啡的精密度均小于4%.吗啡和O6-单乙酰吗啡的检测限均为40n/ml.结论用CSHPLC测定尿中吗啡和O6-单乙酰吗啡,方法准确、灵敏、快速、简便.     

SPE-LC-MS/MS法测定尿液与血液中海洛因3种代谢物

目的采用SPE-LC-MS/MS方法,同时检测尿液与血液中海洛因主要代谢物3-β-D-葡萄糖醛酸吗啡(M3G)、吗啡和O6-单乙酰吗啡(O6)。方法采用BAKERBONDTMspe Octadecyl(C18)进行提取,应用LC-MS/MS方法检测并通过MRM及内标法进行量化。结果尿液中M3G、吗啡、O6-单乙酰吗啡的最低检测限(LOD)分别为1.24pg、6.71pg、0.47pg;回收率依次为82.25±12.25%、93.75±13.25%、88.70±11.90%。血液中M3G、吗啡、O6-单乙酰吗啡的最低检测限分别为1.50pg、8.21pg、0.52pg。回收率依次为89.85±21.15%、73.70±17.90%、90.10±3.90%。结论本文所建方法同时适用于尿液与血液中海洛因主要代谢物M3G、吗啡、O6-单乙酰吗啡的提取、净化、分析。     

UPLC-MS/MS分析尿液中阿片类化合物

目的 建立尿液中同时分析可待因(codeine,COD)、6-单乙酰吗啡(6-monoacetylmorphine,6-MAM)、吗啡(morphine,MOR)、吗啡-3-葡萄糖醛酸苷(morphine-3-glucuronide,M3G)和吗啡-6-葡萄糖醛酸苷(morphine-6-glucuronide,M6G)的超高效液相色谱-串联质谱(UPLC-MS/MS)方法.方法 以吗啡-d3(MOR-d3)和吗啡-3-葡萄糖醛酸苷-d3(M3G-d3)为内标,尿液用乙腈沉淀蛋白后,过SiroccoTM蛋白沉淀板,UPLC-MS/MS法分离检测.结果 尿液中COD和MAM检出限为0.2 ng/Ml,定量限为0.5 ng/Ml;MOR、M3G和M6G检出限为0.5 ng/Ml,定量限为1 ng/Ml;线性相关系数r≥0.999 7;日内精密度和日间精密度均在10%以内;回收率70.0%～98.3%,基质效应50.5%～99.0%.结论 所建方法简便、快速、准确,可以满足法庭毒物分析的需要.     

氯胺酮、甲基苯丙胺和吗啡金标单抗试剂盒的研制

目的建立同步检测氯胺酮、甲基苯丙胺和吗啡的方法。方法将胶体金标记的抗氯胺酮、抗甲基苯丙胺和抗吗啡单克隆抗体浸涂在玻璃纤维膜上，将氯胺酮、甲基苯丙胺和吗啡的完全抗原以及羊抗鼠多克隆抗体喷涂在硝酸纤维素膜上，分别标定为检测区（T）和质控区（C）。样本中游离的氯胺酮、甲基苯丙胺和吗啡分别与包被的完全抗原免疫竞争结合胶体金标记抗氯胺酮、抗甲基苯丙胺和抗吗啡单克隆抗体。以质控区和检测区是否出现紫红色条带判读结果。结果对66种药品和毒品的特异性测试表明，该试剂盒仅识别氯胺酮及其代谢物、甲基苯丙胺及其衍生物和吗啡类；对人体尿样中的氯胺酮、甲基苯丙胺和吗啡检测阈值分别为1000ng／ml、1000ng／ml和300ng／ml；与GC／MS对照试验结果一致；试剂盒稳定性较好，在常温下可较长时间保存。结论本文研制的试剂盒可用于样本中氯胺酮、甲基苯丙胺和吗啡成分定性的同步检测。     

唾液与血液中海洛因代谢物的检测时限

目的比较唾液与血液中海洛因代谢物吗啡和O6-单乙酰吗啡的检测时限,为实际检测时选择样本采集时间提供依据。方法将实验大耳白兔分为3组,通过耳缘静脉注射浓度为2.6mg/m L的海洛因溶液,给药量分别为0.5m L,1m L,2m L。给药后于0.5~48h期间采集静脉血液;给药后于3~48h采唾液。采用超高效液相色谱串联质谱法,分别检测各时间点,各组不同种类样本中的海洛因代谢产物吗啡和O6-单乙酰吗啡。结果 1O6-单乙酰吗啡在血液中的检测时间为6h(给药量2.6mg和5.2mg)和1.5 h(给药量1.3mg),9h后已检测不到;而唾液样本在24h时虽下降明显,但3种给药剂量下均仍可检出,至48h时均不能检出;2吗啡在血液和唾液中不能检出的时间分别为24h和48h。结论血液中O6-单乙酰吗啡和吗啡的检测时限随用药量的增加而延长,而在唾液的检测时限均明显比在血液中更长久。该结果可作为实际检测时根据样本种类和选择采集时间的依据。     

固体进样杆-GC/MS-SIM技术检验头发中6-单乙酰吗啡

目的建立固体进样杆-GC/MS-SIM技术,检测人头发中毒品海洛因的主要代谢产物6-单乙酰吗啡的方法。方法样品头发洗净剪碎后,经MSTFA衍生化,采用GC/MS-SIM定性定量分析。结果 6-单乙酰吗啡在浓度0.1ng/mg～18.0ng/mg范围内线性相关系数为0.9972,方法检出限为0.1ng/mg。结论本方法头发使用量小,准确度、精密度良好、灵敏度高,是高效、快速、实用的测定头发中6-单乙酰吗啡的方法。     

抗丁丙诺啡单克隆抗体的制备

目的建立抗丁丙诺啡单克隆抗体的杂交瘤细胞株，制备高特异性的丁丙诺啡单克隆抗体，并对其免疫学特性进行鉴定。方法在丁丙诺啡的分子上连接活性羧基基团，通过缩合反应将丁丙诺啡半抗原连接于血蓝蛋白（KLH）和小牛血清白蛋白（BSA），形成完全抗原。以完全抗原免疫Balb／c小鼠，通过细胞融合，筛选等杂交瘤技术，建立稳定的分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株。通过腹腔注射杂交瘤细胞，诱导小鼠产生含有单抗的腹水。用辛酸-硫酸铵加亲和层析法纯化抗丁丙诺啡单克隆抗体。采用酶联免疫反应和胶体金膜层析实验测定丁丙诺啡单抗的特异性以及免疫反应动力学参数。结果共获得3株分泌抗丁丙诺啡单克隆抗体的杂交瘤细胞株，分别命名为7E6，6G4和3C2。7E6，6G4抗体灵敏度为10．0ng／ml，3C2抗体灵敏度为20．0ng／ml。7E6，6CA和3C2抗体的亲和常数分别为3．6×10^-9 mol／L，4．3×10^-9 mol／L和6．3×10^-9 mol／L。特异性测试结果表明7E6和6G4抗体与40种药物、毒品无任何交叉反应，而3C2抗体与吗啡有交叉反应。结论杂交瘤细胞株7E6和6G4产生的抗丁丙诺啡单克隆抗体具有很高的特异性和灵敏度。     

毛发中海洛因及其代谢物的分析综述

对毛发中海洛因及其代谢物—6-单乙酰吗啡和吗啡的提取和检测分析进行了综述。其分析方法大致为6步:收集毛发,清洗毛发,分段,剪(磨)碎毛发,水解,提取净化,检测分析。     

萃取方式对海洛因滥用者毛发中代谢物分析的影晌

目的比较液相萃取和同相萃取对毛发中海洛因毒品代谢物分析的影响。方法对海洛因吸食者毛发和空白添加标准品毛发经甲醇超声后的提取液分别进行液相萃取、同相萃取,然后进行衍生化和GC／MS—SIM检测。结果利用同相萃取法对添加6-单乙酰吗啡的毛发进行萃取和测试,6-单乙酰吗啡的回收率为32．O％,相对标准偏差（RSD）为2．4％;而液相萃取回收率为52．6％,相对标准偏差（RSD）为4．6％。结论固相萃取较之液液萃取,有更好的重复性,更少的杂质干扰和有机溶剂消耗等优势,但甲醇超声液需要挥干后才能进行同相萃取,而且6-单乙酰吗啡的水解率高。     

萃取方式对海洛因滥用者毛发中代谢物分析的影响

目的比较液相萃取和固相萃取对毛发中海洛因毒品代谢物分析的影响。方法对海洛因吸食者毛发和空白添加标准品毛发经甲醇超声后的提取液分别进行液相萃取、固相萃取,然后进行衍生化和GC/MS-SIM检测。结果利用固相萃取法对添加6-单乙酰吗啡的毛发进行萃取和测试,6-单乙酰吗啡的回收率为32.0%,相对标准偏差(RSD)为2.4%;而液相萃取回收率为52.6%,相对标准偏差(RSD)为4.6%。结论固相萃取较之液液萃取,有更好的重复性,更少的杂质干扰和有机溶剂消耗等优势,但甲醇超声液需要挥干后才能进行固相萃取,而且6-单乙酰吗啡的水解率高。     

A screening method for urinary methamphetamine--latex agglutination inhibition reaction test

Methamphetamine in urine samples from abusers was detected by the latex agglutination inhibition reaction test with latex-antibody (Latex-Ab) and latex-methamphetamine (Latex-MA) reagents. Anti-methamphetamine antibody was produced in rabbits by immunization with bovine serum albumin (BSA)-methamphetamine conjugate. Latex particles were coated with antibodies or with rabbit serum albumin (RSA)-methamphetamine conjugate to obtain Latex-Ab and Latex-MA reagents, respectively. The results are read at 4-5 min after mixing the latex reagents. The sensitivity of this method for methamphetamine was 0.4 micrograms/ml urine. Methamphetamine analogs (methylephedrine, amphetamine, phentermine, methoxyphenamine, ephedrine, beta-phenylethylamine, OH-methamphetamine, OH-amphetamine, and OH-ephedrine) all cross-react in varying degrees, while glucosiurea and albuminurea give false positive results in the tests. Though attention must be paid to these effects this simple and rapid test is suitable for the mass screening of urine samples.     

Immunoassay for methamphetamine with a new antibody

p- and o-Aminomethamphetamine were synthesized as haptens to be coupled with carrier protein at the benzene ring of methamphetamine. Immunogens were prepared by the glutaraldehyde method or the MBS (N-(m-maleimidobenzoyloxy)succinimide) type cross-linking reagent method. In particular, immunization with p-aminomethamphetamine-bovine serum albumin (BSA) conjugate prepared by the glutaraldehyde method gave an anti-methamphetamine antiserum having a low cross-reactivity with methylephedrine. With the antiserum, three kinds of immunoassays for methamphetamine were established. An enzyme immunoassay (EIA) and an enzyme-linked immunosorbent assay (ELISA) were developed with alkaline phosphatase (ALP) as a label enzyme. The amount of antibody bound ALP conjugate was determined by its activity in dephosphorylating p-nitrophenyl phosphate in EIA and nicotinamide adenine dinucleotide phosphate (NADP+) in ELISA. The range of methamphetamine measurable by ELISA was 0.025-0.5 ng/well and its sensitivity was superior to that of EIA (0.3-300 ng/tube). A latex agglutination inhibition reaction test (LAIRT) was also developed for the mass screening method of urine samples. The sensitivity of this method for methamphetamine was 0.1 micrograms/ml urine.     

测定单根毛发中吗啡含量的放射免疫方法

目的 建立测定单根毛发中吗啡含量的放免方法。方法 用卵清蛋白-琥珀酰吗啡作免疫原,免疫新西兰白兔获得高品质抗血清;HPLC纯化~(125)Ⅰ-吗啡,建立放射免疫方法,测定正常人和吸毒人员单根毛发。结果 抗体亲和常数为3.25×10~(11)L/M,放化纯度为95％,比放射性112μCi/μg;方法的灵敏度为0.01ng/ml。对5例正常人及5例戒毒所吸毒人员的单根毛发进行了检测。单根毛发长度9～24cm,重量为0.7～2.1mg,5例正常人测值为1.75±0.37ng/mg(x±s);5例吸毒人员测值为471±204ng/mg(x±s)。结论 所建方法可准确定量单根毛发中吗啡的含量。     

Dual-label time-resolved fluoroimmunoassay of psychopharmaceuticals and stimulants in serum

A new method to measure two different drugs simultaneously by time-resolved fluoroimmunoassay (TR-FIA) has been developed. In the TR-FIA reported here, psychopharmaceuticals [chlorpromazine (CPZ) and desipramine (DSP)] and methamphetamine (MA) contained in serum are assayed by a combined use of a new europium (Eu) chelate and a samarium (Sm) chelate, as labels. The drug concentrations were determined by the competition between a labeled antigen with Eu(3+) or biotin and a sample antigen. A microtiter plate coated with a mixture of rabbit IgGs (anti-MA and anti-CPZ or anti-MA and anti-DSP) was used. In the assay of MA and CPZ, Eu(3+) labeled MA-bovine serum albumin conjugate (MA-BSA) and biotinylated CPZ-BSA were added to the well with their non-labeled standard solutions or samples. MA was assayed by measuring the fluorescence intensity of Eu(3+) at 615 nm. After incubation of the Sm(3+) labeled streptavidin, CPZ was assayed by measuring the fluorescence of Sm(3+) at 643 nm. In the assay of MA and DSP, Eu(3+) labeled DSP-BSA and biotinylated MA-BSA were used. In our dual-assay, the minimum detection limits of these drugs were 1ng/ml for MA, 10 ng/ml for CZP and 10 ng/ml for DSP. Since the simultaneous detection of different drugs by TR-FIA is time and sample saving, the method can be employed in rapid and sensitive screening tests.     

抗人同种异型G1m(3)单克隆抗体研制及特异性鉴定

用快速液相色谱仪从G1m（3）阳性人血浆中纯化IgG1蛋白，用其免疫BALB／C／小鼠．建立了一株分泌抗人G1m（3）单克隆抗体的细胞株（D7E8）。经抑制ELISA，直接ELISA及斑点ELISA分析，证明D7E8抗体具有G1m（3）单一特异性。其培养上清液效价为512倍，并初步应用于法医办案。     

Performance of the Emit® II Plus 6-Acetylmorphine Assay on the Viva-E® analyzer

We evaluated the performance of Emit(?) II Plus 6-Acetylmorphine Assay for human urine screening on the Viva-E(?) analyzer. Precision was evaluated using the cutoff and ±25% controls. Recovery and linearity were studied by spiking 6-acetylmorphine (6-AM) into human urine pools. Accuracy was evaluated using urine specimens and the results were compared to those from GC/MS. Cross-reactivity with structurally related drugs was assessed at different cross-reactant concentrations. Interferences were assessed in the presence of 7.5 and 12.5 ng/mL of 6-AM. The qualitative repeatability coefficients of variation (CV's) ranged from 0.3% to 0.4% and the within-lab CV's ranged from 2.0% to 2.2%. In analyte units (ng/mL), the repeatability CV's ranged from 1.3% to 2.2% and the within-lab CV's ranged from 2.6% to 4.3%. The limit of detection of the assay was found to be 2.1 ng/mL. Recovery was within 15% of expected value. Linearity was 2.1-20 ng/mL. Method comparison showed 99% agreement with GC/MS. The assay had minimal cross-reactivity with morphine, morphine-3-glucuronide, morphine-6-glucuronide and other opioids. No interference was observed with endogenous interferences and structurally unrelated drugs. The assay correctly classified CAP survey samples. The Emit(?) II Plus 6-Acetylmorphine Assay will be a suitable screening method for urine specimens in both qualitative and semi-quantitative analyses.     

Postmortem redistribution of morphine and its metabolites

The postmortem redistribution of morphine, morphine-3-glucuronide, morphine-6-glucuronide and total morphine was assessed in 40 heroin-related deaths. In blood taken from subclavian, heart, and femoral regions, concentrations of morphine and its metabolites were similar. While there was a trend for higher concentrations in heart blood, when compared with femoral or subclavian blood, this was not significant. There was also no significant difference in concentrations between admission and autopsy blood in which the postmortem interval was on average 59 h. From our observations, significant postmortem redistribution of morphine and its metabolites seems unlikely.     

单克隆抗体斑点ELISA快速检测血痕ABO血型

本文利用酶标记单克隆抗A、抗B抗体进行直接斑点ELISA检测血痕ABO血型。此法可检测出含量相当于0.005μl的全血,整个实验过程仅需1.5小时。对48份血痕标本均能正确鉴定。与常规方法相比,具有快速、简便、灵敏等特点,有一定的实用价值。