导数—同步荧光光谱法测定尿液肾上腺素和去甲肾上腺素

为了解决肾上腺素（Ｅ）、去甲肾上腺素（ＮＥ）氧化产物的荧光光谱严重重叠难题，将同步荧光与导数光谱两种技术结合起来用于尿液Ｅ、ＮＥ测定。结果发现，在激发波长与发射波长相差Δλ＝４０ｎｍ时获得的Ｅ、ＮＥ二阶导数－同步荧光光谱中，４５０ｎｍ处Ｅ的荧光强度的导数信号不受ＮＥ的干扰；４３２ｎｍ处，ＮＥ的荧光强度的导数信号不受Ｅ的干扰，Ｅ、ＮＥ含量在１～２００ｎｇ／ｍｌ范围内成线性关系；最低检测限为０．５ｎｇ／ｍｌ；批内ＣＶ３．２１％～３．９５％，批间ＣＶ４．２６％～５．１０％，Ｅ和ＮＥ的回收率分别为９４．９％～９５．４％、９４．７％～９６．３％；该法操作简单、快速、成本低廉，适合于％批量测定，是测定尿液Ｅ、ＮＥ较为理想的常规方法。     

同步荧光法测定血清维生素E

同步荧光扫描法测定血清维生素Ｅ有效地消除了溶剂粒曼光谱的干扰，提高了测定方法的灵敏度和准确性。维生素Ｅ含量在０．０１－２０．０ｕｇ／ｍｌ范围内与同步荧光峰高度成线性关系，ｒ＝０．９９９；回收率９９．９５％；批内ＣＶ２．２１％－２．４１％；批间ＣＶ２．８２％－２．９６％；该法操作简单、快速。     

偶氮胂Ⅲ比色法测定血清（浆）钙

对偶氮肿Ⅲ（ＡｒｓｅｎａｚｏⅢ）比色法测定血清（浆）钙的方法学作了系统研究。结果表明：反应混合液的吸收光谱呈Ｍ型，两个吸收峰分别为６００ｎｍ和６５０～６６０ｎｍ，但以６６０ｎｍ为适。试剂中ＡｒｓｅｎａｚｏＩＩＩ的最适浓度为１５０μｎｏｌ／Ｌ，试剂空白Ａ（吸光度曾称光密度ＯＤ）受ｐＨ的影响，当ｐＨ为７．０　时Ａ值最低。本法批内（ｎ＝２０）和批间（ｎ＝４０）ＣＶ分别为２．８％～２．９６％、３．１５％～３．３７％，平均回收率为９９．３％，线性高达６．０ｍｍｏｌ／Ｌ，灵敏度为１ｍｍｏｌ／Ｌ＝０．１７Ａ。本法Ｙ与ＥＤＴＡ自动滴定法Ｘ比较：Ｙ＝１．００５９Ｘ－０．０１４１，ｒ＝０．９９８５、Ｐ＜０．００１、ｎ＝２０。Ｍｇ￣（２＋）、Ｃｕ￣（２＋）、Ｆｅ￣（２＋）、Ｚｎ￣（２＋），Ｈｂ、Ｂｉｌ均不干扰测定。     

偶氮氯磷Ⅲ比色法测定血清钙的实验研究

本文对偶氮氯磷Ⅲ（ＣｈｌｏｒｏｐｈｏｓｐｈｏｎａｚｏⅢ）比色法测定血清钙的实验方法作了系统性研究。结果表明：显色剂中偶氮氯磷Ⅲ浓度以１２０μｍｏｌ／Ｌ为宜，反应最适ｐＨ为２．８～３．２，最佳检测波长为６２０ｎｍ，灵敏度为１ｍｍｏｌ／Ｌ＝０．１６４Ａ，线性为４．５ｍｍｏｌ／Ｌ，批内（ｎ＝２０）和批间（ｎ＝４０）ＣＶ分别为２．０８％和２．２６％，平均回收率为１００．２％。本法（Ｙ）与ＡＡＳ法（Ｘ１）及ＡｒｓｅｎａｚｏⅢ法（Ｘ２）结果比较：Ｙ＝０．９８５６Ｘ１＋０．０３７０、ｒ＝０．９９１２，Ｙ＝０．９５４０Ｘ２＋０．１３６５、ｒ＝０．９８８７．溶血、脂血、黄疸不干扰测定，当标本中Ｍｇ２＋、Ｃｕ２＋、Ｆｅ２＋、Ｚｎ２＋分别达５ｍｍｏｌ／Ｌ、２ｍｍｏｌ／Ｌ、３００μｍｏｌ／Ｌ、３００μｍｏｌ／Ｌ时也不影响测定结果。     

增强化学发光免疫法检测乙型肝炎表面抗原的建立和评价

本法采用Ｌｕｍｉｎｏｌ－Ｈ２Ｏ２－ＨＲＰ系统建立了增强化学发光免疫法（ＥＬＥＩＡ）检测乙型肝炎表面抗原（ＨＢｓＡｇ），该发光信号的强度与血清中ＨＢｓＡｇ浓度呈线性关系，回归方程为：Ｙ＝０．４４４１Ｘ＋０．００６７，相关系数ｒ＝０．９９７１；批内变异系数为３．６％～９．３％、批间变异系数为１０．２％～１２．５％；回收率为８２．５％～１１５．０％；最小检测限为２０ｐｇ／ｍｌ；结果表明本法有灵敏性高，准确性和稳定性好的特点。     

血清冲胆汁酸酶法测定及其临床应用

为了建立酶法测定血清叫胆汁酸的方法并评价其临床应用价值，对试剂配方和测定条件进行优化实验，并作临床观察。结果，本法灵敏度：５０μｍｏｌ／Ｌ Ａ５００ｎｍ为０．１，线性为０－２００μｍｏｌ／Ｌ精密度；总ＣＶ〈６．０％，批内ＣＶ〈３．０％。本法和日本一化试剂的比较：Ｙ＝０．９７３Ｘ＋２．７２，ｒ＝０．９９６３。     

自制尿微量白蛋白试剂盒在生化分析仪上的临床应用

采用自制的羊抗人白蛋白血清，以免疫透射比浊法的原理，用双试剂多点定标法在全自动生化分析信我上建立检测尿微量白蛋白的方法学。方法反应稳定；特异性强；重复性好；无明显干扰因素。白蛋白浓度在０－２００－ｍｇ／Ｌ范围内线性良好。批内误差ＣＶ＝１．９９％－２．３７％；批间误差ＣＶ＝２．６１％－３．４６％；平均在收率９８．７％，灵敏度为１２．５ｍｇ／Ｌ。分别检测３８７例健康者，６８例糖尿病患者，３４例肾病患者     

高密度脂蛋白胆固醇直接测定法与硫酸葡聚糖50-镁法比较

目的评价高密度脂蛋白胆固醇（ＨＤＬ－Ｃ）直接测定法在临床应用的可行性。方法将直接测定法与硫酸葡聚糖（ＤＳ５０）－氯化镁沉淀法进行比较，并分析其线性范围、准确度和干扰因素。结果直接法与ＤＳ５０沉淀法相关良好，Ｙ＝０．９２７Ｘ＋０．２１４，ｒ＝０．９６４，ＨＤＬ－Ｃ浓度为２．４５ｍｍｏｌ／Ｌ范围内线性良好，ｒ＝０．９９９，ＨＤＬ－Ｃ浓度两组低、中、高值血清标本（０．８４、１．３１、２．３１；０．９２、１．４２、２．５３ｍｍｏｌ／Ｌ）的批内和批间ＣＶ值分别为２．３８％、１．１５％、２．４７％和３．４８％、０．９９％、２．０６％。浓度为１０．５８ｍｍｏｌ／Ｌ甘油三酯并不影响ＨＤＬ－Ｃ直接测定法。结论ＨＤＬ－Ｃ直接测定法简易快速，结果准确，适于自动化分析。     

高效液相色谱荧光检测法测定尿雌三醇

报告用高效液用色谱（ＨＰＬＣ）测定孕妇尿内总雌三醇（Ｅ＿３）的方法。尿标本在酸水解后用乙醚提取Ｅ＿２，蒸干乙醚，残留物用流动相（甲醇／乙腈／水＝４０／１７／４３）重组。标本成分Ｃ＿８柱分离，以２８５ｎｍ波长激发，在６１０ｎｍ测定Ｅ＿３的自然荧光。本法批内ＣＶ为１２％～１．５％（ｘ＝３２．７～１２８．２ｕｍｏｌ／Ｌ），批间ＣＶ别为２．３％和４０％（分别是３８．４和９８．９ｕｍｏｌ／Ｌ）。本法在４．３３～１３８．７ｕｍｏｌ／Ｌ（１２５～４０．００ｍｇ／Ｌ）间呈线性，最低检出１．９ｕｍｏｌ／Ｌ（０．５２ｍｇ／Ｌ，信号／噪声比为３）。     

血清新喋呤高效液相色谱测定及其初步应用

用反相高效液相色谱法测定肝癌和正常人血清中新喋呤的浓度。测定条件：荧光激发波长为３６０ｎｍ，发射波长为４４０ｎｍ，流动相为甲醇－水。线性范围为１．９７－１９．７６ｎｍｏｌ／Ｌ，ｒ＝０．９９９７，回收率分别为１０８．０％和１０１．８％相应的变异系数分别为６．５７％和２．０９％。     

高效液相色谱-荧光法同时测定血清中的色氨酸和酪氨酸

目的建立一种高效液相色谱（HPLC）-荧光法（FD）同时测定血清色氨酸（Trp）和酪氨酸（Tyr）的方法。方法色谱条件：Megres C18色谱柱（250 mm×4.6 mm,内径为5μm）,流动相为10%乙腈溶液,流速为1.2 mL/min,Tyr和Trp的荧光检测激发波长和发射波长分别为λexTyr=228 nm、λexTrp=285 nm和λemTyr=306 nm、λemTrp=353 nm。血清样本经5%高氯酸溶液去除蛋白质后取上层清液直接进样进行分析测定。且对样本的保存方法进行了探讨。结果 Tyr的保留时间为3.4 min,线性范围为0.275~275μmol/L,最低检出浓度为0.004μmol/L,回收率为90.5%~108.8%。Trp的保留时间为7.6 min,线性范围为0.490~196μmol/L,最低检测浓度为0.005μmol/L,回收率为88.8%~97.2%。Tyr和Trp的日内、日间测定的相对标准偏差均〈5%,苯丙氨酸、5-羟色胺、犬尿喹啉酸、犬尿氨酸和肌酐等物质对该法均无干扰。样本应-20℃冰冻保存。结论该方法简便、快速、敏感、特异,可同时测定血清Tyr和Trp,适合于临床和科研应用。     

高效液相色谱-荧光法测定SLE患者血清色氨酸及其代谢产物

目的建立一种同时测定血清色氨酸(tryptophan,Trp)、犬尿氨酸(kynurenine,Kyn)和犬尿喹啉酸(kynurenic acid,Kyna)的高效液相色谱-荧光检测法(HPLC-FD),并用此法检测SLE患者血清中Trp及其代谢产物Kyn、Kyna的含量。方法血清标本经0.624 mol/L的高氯酸溶液去除蛋白质后取上清液20μl直接进样分析测定。色谱柱为Hypersil C18柱,流动相为0.20mol/L醋酸锌、8.3 mmol/L醋酸和2.5%的乙腈;流速为1.5 ml/min;荧光检测激发波长和发射波长在0~11 min分别为365nm和480 nm,11~15.5 min变换为344 nm和404 nm,15.5~20 min为254 nm和404 nm。采用建立的方法测定体检健康者和SLE患者血清Trp、Kyn和Kyna的含量。结果Trp的线性范围为0.610~196μmol/L,最低检出浓度为0.005μmol/L,平均回收率为103.71%;Kyn线性范围为0.049~98μmol/L,最低检出浓度为0.025μmol/L,平均回收率为97.45%;Kyna线性范围为1.05...     

高效液相色谱法在类风湿关节炎患者血清TRP和KYN检测中的应用与临床价值

目的 探讨高效液相色谱荧光检测法(HPLC-FLD)同时测定血清色氨酸(TRP)和犬尿氨酸(KYN)对诊断类风湿关节炎(RA)的临床意义.方法 血清标本加等量5%(V/V)高氯酸溶液去除蛋白,离心取上清液20 μl,直接进样分析.色谱柱为HpemilC_8柱(300 mm×6.0 mm i.d,10 μm);流动相为0.25 mol/L醋酸锌和50 mmol/L醋酸溶液(含3%乙腈),流速为1.5 ml/min;0～10 min荧光检测器的激发波长和发射波长分别为365 nm和480 nm,10 min后激发波长和发射波长分别变换为254 nm和404 nm.同时,用该方法测定120名健康成人和110例RA患者血清TRP、KYN和TRP/KYN比值(K/T),并评价其诊断RA的敏感度、特异度和方法学效能.结果 血清标本的KYN和TRP保留时间分别为8.1和11.5 min,两者分离良好.KYN线性范围为0.098 ～19.600 μmol/L,最低检测浓度为0.04 μmol/L;回收率为90.8%～96.2%,日内变异系数为3.68%,日间变异率为4.97%.TRP的线性范围为4.9～196.0μmol/L.最低检测限为0.005 μmol/L,回收率为92.6～106.9%,13内变异系数为3.63%,日间变异系数为4.44%.在本试验的色谱条件下测定苯丙氨酸(Phe)、酪氨酸(Tyr)、犬尿喹啉酸(KYNA)、5-羟色胺(5-HT)和Cr均无干扰.RA组患者血清KYN含量和K/T比值[(2.06±0.38) μmol/L和(55.46±5.81)×10~(-3)]与健康对照组[(1.51±0.35)μmol/L和(32.54 ±9.00)×~(-3)]比较,均显著升高(U=3 251.0,t=10 741,P均为0.000),而RA组TRP含量[(38.24±5.27)μmol/L]与健康对照组[(47.52±5.79)μmol/L]比较,则显著降低(t=10.399,P=0.000).K/T比值诊断RA的敏感度、特异度分别为83.6%(92/110)、85.8%(103/120).结论 HPLC-FLD同时测定血清TRP和KYN的方法精密度、回收率、抗干扰能力、线性范围等均符合临床检测要求.K/T比值可作为RA的辅助性诊断指标.     

Spectrofluorimetric determination of bile acid using a europium-doxycycline probe

A new spectrofluorimetric method was developed for the determination of trace amounts of bile acid (BA). Using europium ion (Eu(3+))-doxycycline (DC) as a fluorescent probe, in a buffer solution of pH=7.0, BA can remarkably reduce the fluorescence intensity of the DC-Eu(3+) complex at lambda=612 nm; the reduced fluorescence intensity of the Eu(3+) is proportional to the concentration of BA. Optimum conditions for the determination of BA were also investigated. The linear range and detection limit for the determination of BA were 5.0 x 10(-8) mol/L to 5.5 x 10(-7) mol/L and 1.1 x 10(-8) mol/L, respectively. This method is practical and relatively free of interference from coexisting substances, and can be successfully applied to assess BA in serum samples.     

高效液相色谱法检测人血清游离和总肉毒碱水平

目的 研究人血清中游离和总肉毒碱HPLC测定方法,建立成年体检人群血清中游离和总肉毒碱水平参考值.方法 血清样品经乙腈沉淀蛋白、衍生化反应后,以Lichrospher SiO2为固定相,乙腈-柠檬酸-三乙胺为流动相进行色谱等度分离,260 nm波长下定量检测,并对347名成年体检人群进行血清中游离和总肉毒碱水平测定.结果 在所建立的分析条件下,肉毒碱衍生化产物的色谱保留时间约为10 min,峰形清晰对称,与样品中其余内源性物质分离完全,定量准确.肉毒碱在0～400 μmol/L浓度范围线性良好.血清中游离肉毒碱和总肉毒碱批间(n=7)及平均批内(n=5)测定的相对标准偏差分别为3.04%、3.36%和1.77%、1.97%,测定平均回收率分别为98.2%和96.3%.对347名成年体检人群血清中肉毒碱水平测定结果显示:男182名总肉毒碱(52.2±8.6)μmol/L,游离肉毒碱(42.3±8.3)μmol/L,酯酰肉毒碱(9.9±2.9)μmol/L;女165名,总肉毒碱(48.2±9.9)μmol/L,游离肉毒碱(37.9±8.7)μmol/L,酯酰肉毒碱(10.3±3.5)μmol/L.统计学分析显示,男性血清中游离肉毒碱及总肉毒碱水平与女性组相比明显偏高,差异具有统计学意义(t=4.88、3.98,P<0.01).两组间酯酰肉毒碱浓度相比,差异无统计学意义(t=-1.32,P>0.05).结论 HPLC方法可同时检测血清游离和总肉毒碱含量,且灵敏度、特异性、重复性好,为有关肉毒碱在临床的合理应用及相关疾病的研究建立了有效的参考指标和检测方法.     

Paediatric reference values for total homocysteine,tryptophan, tyrosine and phenylalanine in blood spots

Determining blood concentrations of the amino acids homocysteine, tryptophan, tyrosine and phenylalanine in children is of value in the clinical practice. Over the past decades, the use of blood spot samples to examine amino acid concentrations is increasing rapidly. In children, the use of blood spot samples is especially of relevance, as this method is much less invasive than venous blood sampling. Currently, no paediatric reference values for amino acids in blood spots are available. The aim of the current study was to establish reference values for blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine in school-age children. Dried blood spots were obtained in a community sample of 104 healthy children, aged 6–12?years old (52% males). Blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were determined by positive electrospray liquid chromatography-tandem mass spectrometry. Parents of participants completed questions regarding demographic characteristics. Our sample consisted of healthy children from various ethnic backgrounds, with varying levels of socioeconomic status, in line with the composition of the Dutch society. Blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were similar in males and females, and independent of age. In conclusion, paediatric reference values for blood spot concentrations of total homocysteine, tryptophan, tyrosine and phenylalanine were established, which could be of use in the clinical practice.     

Simultaneous determination of tryptophan, kynurenine and 5-hydroxytryptamine by HPLC: Application in uremic patients undergoing hemodialysis

ObjectivesTo develop a reliable HPLC method for the simultaneous determination of plasma tryptophan, kynurenine and 5-hydroxytryptamine to analyze tryptophan metabolism.Design and methodsSeparation was carried out on a C8 column with the mobile phase composed of acetate buffer (pH 4.5) and acetonitrile using theophylline as internal standard. The eluates were monitored by ultraviolet detection with programmed wavelength.ResultsAnalysis was achieved in less than 8.0 min. The limits of quantification were 3.97 μmol/L, 4.36 nmol/L and 0.421 μmol/L for tryptophan, 5-hydroxytryptamine and kynurenine, respectively. Reproducibility and recovery were satisfactory. Twenty healthy adults and 20 uremic patients undergoing hemodialysis were analyzed using the present method. Tryptophan metabolism was found to be disturbed in uremic patients and was improved obviously after hemodialysis.ConclusionsThe developed HPLC method is simple, reliable and suitable for monitoring tryptophan metabolism in uremic patients undergoing hemodialysis.     

高效液相色谱法测定人血清甜菜碱方法的建立

目的 采用柱前衍生高效液相色谱法-紫外检测法建立一种测定人血清甜菜碱浓度的方法.方法 以对溴苯乙酰溴和18-冠醚-6溶于乙腈制成衍生液,将其与甜菜碱反应形成的衍生物用Supelcosil LC-SCX色谱柱进行分离.流动相为乙腈∶水体积比90∶10,含16 mmol/L的氯化胆碱,等度洗脱,流速为0.8 ml/min,检测波长为259 nm,利用甜菜碱标准品绘制标准曲线,外标法定量检测20名健康大学生志愿者血清甜菜碱.结果 甜菜碱的测定线性范围为6.25～200.00 μmol/L,回归方程Y=1 568.1X-2 747.5,R2=0.999 8.最低检测限为3.0 μmol/L.批内不精密度为1.88%～3.79%(平均3.24%),批间不精密度为3.14%～6.76%(平均4.39%).方法 回收率为95.89%～102.86%(平均99.16%).结论 成功建立一种检测人血清甜菜碱浓度的方法,适用于实验室及临床常规检测.     

5-Br-PADAP直接光度法测定血清锌

目的建立一种快速、简便、灵敏的分光光度法测定血清锌。方法在表面活性剂TritonX-100存在下,用2-(5-溴-2-吡啶偶氮)-5-二乙氨基酚(5-Br-PADAP)作显色剂,不去蛋白直接光度法测定血清锌。结果该方法显色络合物最大吸收波长为558nm,线性范围达51.0μmol/L,表观摩尔吸光数为1.05×105L*mol-1*cm-1。回收率为98.1%～103.2%,批内和批间变异系数(CV)分别为2.1%与3.6%,与原子吸收分光光度法比较相关良好,Y=1.02X-0.41,r=0.9862,P＞0.05。48名健康人血清锌含量为(9.5～24.3)μmol/L(±2s)。结论该法血清用量少,不必去蛋白,具有操作快速、简便、结果灵敏可靠等优点,适合临床应用。     

Fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase

A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.