高效液相色谱检测胆红素及其临床应用

由于常规的血清胆红素检测方法不能灵敏，准确的测定结合胆红素。笔者参考国外文献采用国产填料和试剂建立了敏感和特异的高效液相色谱胆红素测定法，结果表明本法可对血清胆红素作出较为灵敏和准确的测定，未结合胆红素批内和批间变异系数（Ｖ）值分别为９．８％～１０．９％和７．３％～１２．７％，结合胆红素批内和批弹ＣＶ值分别为２．５％～９．２％和９．７％～１１．０％；未结合和结合胆红素的最低检出浓度分别为０．０５μ     

消毒剂中戊二醛的高效液相色谱检测方法研究

摘要 目的 建立一种准确、简便,适用于消毒剂中戊二醛含量测定的高效液相色谱分析方法。方法 采用C18色谱柱,应用反相液相色谱紫外法检测消毒剂中戊二醛的含量,研究流动相组成、离子强度等试验条件对戊二醛色谱保留性能的影响。结果 以 30〖KG*4〗∶〖KG-*4〗70乙腈+0.02 mol/L磷酸二氢钾水溶液（pH 3.5）为流动相,在柱温30.0 ℃、检测波长235 nm的条件下,戊二醛在C18色谱柱上具有良好的保留行为,标准曲线线性范围为0.005%~0.500%,检出限为0.000 5%,日内和日间相对标准偏差<2.0%,样品加标回收率94.5%~101.7%。结论 该反相液相色谱方法简单可靠,线性范围宽,抗干扰能力强,具有良好的准确度和精密度,适用于消毒剂中戊二醛含量的测定。     

生物检材中利血平的高效液相色谱／质谱和高效液相色谱的检测

目的：建立生物检材中利血平的高效液相色谱／质谱（HPLC／MS）和高效液相色谱（HPLC）的检测。方法：采用C18（200mm×4．6mm,5mL）色谱柱,乙腈：水（40：60）用盐酸调节pH值至3．00±0．05为流动相,检测波长258nm。结果：HPLc／Ms分析利血平的选择离子m／z为608,195。心血中利血平HPLC检测的回归方程、线性检测范围、相关系数、回收率、最低检出浓度,分别为Y=56．576X＋0．3168（μg／mL）,（0．5～48）μg／mL,0．995,（96．50土3．O）％,0．5μg／mL;肝组织中利血平HPI。C检测的回归方程、线性检测范围、相关系数、回收率、最低检出浓度,分别为Y=38．567X＋0．054（μg／g）,（o．5～48）μg／g,0．984,（97．5±2．5）％,0．5μg／g。染毒大鼠心血、心、肝、脾、肺、肾和脑中利血平的含量依次为：（316．39±6．43）,（284．96±3．03）,（353．82±7．73）,（185．74±4．07）,（221．64±1．38）,（215．94±2．87）,（170．15±7．05）μg／g。结论：生物检材中HPLC／MS检测方法选择性好,定性准确,HPLC检测简便,快速,灵敏,定量结果准确,可用于利血平中毒的临床快速检验诊断和利血平中毒死亡案件的法医学鉴定。     

超高效液相色谱快速检测万古霉素浓度方法的建立和性能评价

目的建立一种基于超高效液相色谱(UPLC)技术快速测定血清万古霉素浓度的方法并评价其性能。方法以去甲万古霉素为内标,以0.3 mol/L硫酸锌水溶液为蛋白沉淀剂,对血清样本进行前处理。色谱柱为Phenomenex Kinetex C18柱(100 mm×2.1 mm,2.6μm),流速为0.5 mL/min,检测波长为220 nm,柱温40℃。流动相A为25 mmol/L磷酸二氢钾缓冲液(pH值为2.5),流动相B为乙腈,进行梯度洗脱,并对方法的线性、准确度(加标回收率)、精密度、携带污染率、定量限和检测限等分析性能进行评价。分别采用UPLC和高效液相色谱(HPLC)测定50例患者血清万古霉素浓度,并对测定结果进行方法学比对和评价。结果UPLC测定万古霉素的线性范围为2.0~99.6μg/mL,定量限和检测限分别为1.0和0.1μg/mL。低、中、高浓度样本的平均批内变异系数(CV)分别为3.28%、2.21%、2.59%,批间CV分别为5.73%、2.75%、0.82%。低浓度(2.0μg/mL)、中浓度(19.9μg/mL)、高浓度(79.6μg/mL)加标样本的平均加标回收率分别为104.06%、99.80%、100.19%。中浓度样本的携带污染率为0.57%,高浓度样本的携带污染率为0.19%。UPLC和HPLC测定患者血清万古霉素浓度具有良好的相关性(r=0.9919)。结论建立了基于UPLC技术的万古霉素血药浓度监测方法,该方法的重现性好、快速、简便、灵敏度高、准确度高,适用于临床常规万古霉素药物浓度监测。     

高效液相色谱法检测全血糖化血红蛋白的稳定性分析及其应用价值

目的 初步分析高效液相色谱法(HPLC)检测全血糖化血红蛋白的稳定性并讨论其应用价值.方法 临床随机采集3个HbA1c水平的乙二胺四乙酸(EDTA-K2)抗凝无变异血红蛋白的全血标本90例,应用HPLC对全血标本在不同存放时间(即时、30、50、60、70 d)进行HbA1c检测.结果 全血糖化血红蛋白60 d内检测的结果均在可接受范围内,判定为稳定.结论 应用离子交换高效液相色谱法检测在2～8 ℃条件下保存的EDTA-K2抗凝全血,对糖化血红蛋白稳定性研究有着重要的临床应用价值.     

高效液相色谱快速测定尿假尿苷

介绍一种不用苯硼酸亲和柱的高效液相色谱法快速测定尿假尿甙。本法只需简单的标本预处理，使用ＰＨ３．３磷酸盐缓冲液动相和２６８ｎｍ检测，每个标本测定只需１２分钟，批内和批间变异分别为３．２７％和４．８６％，线性范围超过１７００μｍｏｌ／Ｌ，常见的１０种抗肿瘤药物不干扰测定。     

高效液相色谱荧光检测法测定尿雌三醇

报告用高效液相色谱测定孕妇尿内总雌三醇的方法，尿标本在酸水解后用乙醚提取Ｅ３，蒸于乙醚，残留物用流动相重组，标本成分用Ｃ８柱分离，以２８５ｎｍ波长激发，在６１０ｎｍ测定Ｅ３的自然荧光。本法批内ＣＶ为１．２％－１．５％批间ＣＶ分别为２．３％和４．０％本法在４．３３－１３８．７１μｍｏｌ／Ｌ（１．２５－４０．００ｍｇ／Ｌ）间呈线性，最低检出１．８μｍｏｌ／Ｌ（０．５２ｍｇ／Ｌ，信号／噪声比为３）。     

Simultaneous determination of phenylalanine and tyrosine in peripheral capillary blood by HPLC with ultraviolet detection

Objectives

Phenylalanine (Phe) and tyrosine (Tyr) are the most reliable indicators for the diagnosis of phenylketonuria (PKU). The purpose of this study is to establish a simple and rapid method for the determination of Phe and Tyr in peripheral capillary blood from newborns and children by high performance liquid chromatography with ultraviolet detection (HPLC-UV).

Methods

Peripheral blood specimens were taken from newborns or children by heel stick or skin puncture on the fingers. Blood samples were deproteinized by equal volume of 5% perchloric acid. Amino acid separation was carried out on a Hypersil C8 column. The mobile phase containing 5% acetonitrile (v/v) was run at a flow rate of 1.5 mL/min. Phe and Tyr were determined in the ultraviolet detector at 210 nm.

Results

The determination was within 10.0 min. The linear range was from 6.0 to 1512.0 μmol/L for Phe and 5.5 to 1250.0 μmol/L for Tyr. Although Phe and Tyr levels from peripheral blood samples were relatively lower than those from serum or plasma, the data showed a good agreement between them.

Conclusion

The method we developed is simple, rapid and convenient. It is useful for screening and diagnosis of PKU in newborns and children.     

高效液相色谱——紫外、荧光双检测器同时测定人尿中犬尿氨酸和犬尿喹啉酸

目的建立高效液相色谱（HPLC）同时测定人尿中犬尿氨酸（KYN）和犬尿喹啉酸（KYNA）方法。用KYNA／KYN比值评价人体犬尿氨酸氨基转移酶（KAT）活性。方法采用HPLC测定KYN、KYNA,分析柱为安捷伦HCC18反相色谱柱;流动相为10mmol／L乙酸钠-乙酸缓冲液：7％乙腈=93：7;流速1mL／min;检测器波长：紫外365nm,荧光344nm（激发）／398nm（发射）。结果KYN和KYNA保留时间分别为6．9和11．5min。水溶液标准与标准加入工作曲线斜率差异无统计学意义（KYN：P=0．4068;KYNA：P=0．6462）。KYN和KYNA的平均回收率分别为98．74％±7．53％、95．17％±8．17％;稳定性试验的变异系数（CV）分别为3．35％和0．88％;精密度CV分别为2．73％和2．79％;最低检测限分别为0．4和0．2μmol／L。30份健康成年人尿样的KYNA／KYN比值为2．45±1．54。尿中KYNA与KYN含量呈正相关关系（r2=0．3758,P〈0．001）。结论测定方法灵敏、准确、稳定。KYNA／KYN比值评测KAT活性具有简便、实用等特点,可用于相关疾病的临床辅助诊断和基础研究。     

HPLC法测定血清甲氨蝶呤浓度的方法建立及其与荧光偏振法的比较

目的建立测定血清甲氨蝶呤（MTX）浓度的高效液相色谱（HPLC）法,并与荧光偏振法（FPIA）作比较,评价其临床实用性。方法血清样本经20%高氯酸沉淀蛋白质,吸取上清液进行HPLC分析。以MTX标准品外标法建立标准曲线,检测62例用药后患者血清MTX浓度,同时以FPIA测定。结果 HPLC法在0.049~25 ng/L内线性关系良好,日内及日间相对标准偏差（RSD）均〈6%,最低检测浓度为0.025 ng/L。HPLC法和FPIA具有良好的相关性（r=0.973,P〈0.01）,2种方法测定值的差异无统计学意义（P=0.705）。结论 HPLC法具有便捷、准确、成本低等特点,与FPIA相关性良好,适用于临床MTX的血药浓度监测。     

谷氨酰胺高效液相测定法的研究应用

目的 建立一种准确、简便、快速的高效液相色谱法,快速测定血中谷氨酰胺含量,监测给予经谷氨酰胺强化的安全肠外营养病人血清谷氨酰胺浓度并对照组比较。方法 反相Ｃ１８柱为固定相,异硫氰酸苯酯作柱前衍生剂,５０ｍｍｏｌ／Ｌ醋酸缓液（ｐＨ６．５０）－乙腈（９７．７：２．３）为流动相,进行单泵等梯度洗脱,在λ２５４ｎｍ处检测谷氨酰胺含量。结果 线性范围４０～２５００μｍｏｌ／Ｌ相关系数０．９９８,批内和比间的     

Rapid HPLC measurement of thiamine and its phosphate esters in whole blood

BACKGROUND: Thiamine (vitamin B(1)) deficiency is associated with severe diseases such as beriberi and Wernicke encephalopathy. Although most Americans have sufficient dietary intake, thiamine deficiency is observed in the alcohol-dependent and elderly populations. Measurement of thiamine concentration in whole blood provides an assessment of vitamin B(1) status in at-risk individuals. Method: We used TCA to precipitate proteins in whole blood. Thiamine and its phosphate esters were derivatized using potassium ferricyanide to thiochromes, which were separated by gradient elution on a reversed-phase HPLC column and detected by fluorescence. The method was validated for linearity, limit of quantification, imprecision, accuracy, and interference. Results obtained with this method were compared with those produced by the method currently used in our clinical laboratory. Reference values of thiamine and its phosphate esters were determined in samples obtained from self-reported healthy adults who were not taking vitamin supplements. To shorten analysis time, our method used whole blood rather than washed erythrocytes, did not require lengthy enzymatic dephosphorylation, and had a simple mobile phase. RESULTS: The method was linear to 4000 nmol/L. The lower limit of quantification was 3 nmol/L. The within-run CV was <3.5% and total CV was <9.4%. This method correlated with our current method (r = 0.97). Approximately 90% of the total thiamine content in whole blood was present as thiamine diphosphate (TDP). The means (ranges) for an apparently healthy population were 114 (70-179) nmol/L for TDP and 125 (75-194) nmol/L for total thiamine. Results for separation and measurement of free thiamine and thiamine phosphate esters in whole blood were obtained within 5.5 min. CONCLUSION: We developed an HPLC method that allows separation and measurement of free thiamine and thiamine phosphate esters in whole blood and provides more rapid results than other methods.     

高压液相色谱检测糖尿病患者尿白蛋白的应用

目的探讨高效液相色谱法（HPLC）测定糖尿病（DM）患者尿白蛋白的应用。方法收集85例DM患者及40名健康体检者（正常对照组）尿液,分别用HPLC和免疫比浊法检测尿白蛋白,并将检测结果使用回归和Bland—Ahman进行一致性分析。结果HPLC线性方程为Y=2．9482X＋38．601,r2=0．9944;标准品和样本的保留时间（RT）均为2．42min;尿白蛋白浓度为40．1和152．6mg／L的样本其批内和批间变异系数（CV）分别为4．1％、4．8％和5．6％、6．5％;最低检测限为2mg／L。DM组尿白蛋白HPLC结果为2t．0（3．4～678．9）mg／L,高于免疫比浊法[8．2（2．0—442．2）mg／L]（P〈0．01）,但偏高的倍数不一致（1．3～6．1倍）;但正常对照组2种方法测定结果差异无统计学意义（P〉0．05）。将2种方法的测定结果用回归分析和Bland—Ahman进行一致性分析,显示2种方法的一致性较差。85例DM患者中用HPLC和免疫比浊法检测达到微量蛋白尿诊断标准[尿白蛋白排泄率（AER）〉30mg／24h]的阳性例数分别为48例（56．5％）和27例（31．8％）。结论HPLC能检测尿中的包括免疫性和非免疫性的总的尿白蛋白,与免疫比浊法相比更能早期检出尿白蛋白。     

反相高效液相色谱法测定人血浆中无环鸟苷

目的对流动相的配方进行改良,建立反相HPLC法测定人血浆中阿昔洛韦浓度。方法采用SphericorbC18色谱柱,以2．5％的乙腈水溶液（内含10mmol/L高氯酸,5mmol/L乙酸钠,3．5mmol/L庚烷磺酸钠）为流动相,紫外检测波长254nm。血浆以高氯酸沉淀后高速离心取上清液分析。结果阿昔洛韦峰和其它杂峰得到很好的分离,最低检测限为30ng/ml（进样50μl,信噪比为3：1）。结论本法快速、简便、灵敏度高,且可检测到阿普洛韦代谢物。     

Switching from HPLC/UV to MEIA for whole blood sirolimus quantitation: comparison of methods

Sirolimus is a immunosuppressive agent for renal transplant recipients. Monitoring of whole blood sirolimus concentration is necessary in order to improve clinical outcomes. An increasing number of clinical laboratories (4-14% during 2005) are using microparticle enzyme immunoassay (MEIA) for sirolimus quantitation but previous reports indicated a high variability, with a mean difference of 17% for MEIA method vs. high-performance liquid chromatography/ultraviolet (HPLC/UV). This study was aimed at comparing the reliability of MEIA with the HPLC/UV method. Blood samples from transplant patients were processed using both HPLC/UV and MEIA assays. Comparison and Bland-Altman plots, as well as regression analysis and paired t-test were used to compare results of the assays. Concentrations were stratified into three groups and used to investigate whether any observed difference between methods could be influenced by sirolimus concentration. Regression analysis yielded a coefficient of correlation R of 0.9756, the line of best fit being y=0.9832x+0.1976. The statistical analysis showed no difference between the two sets of experimental data. The average percentage difference between the two methods was found to be -0.2+/-19.2%. On the basis of our present results, the tested MEIA assay is able to quantify sirolimus concentration with a clinically acceptable imprecision, similar to that of HPLC/UV method.     

EMIT法和HPLC法监测苯妥英和苯巴比妥血药浓度比较研究

目的比较酶增强免疫法(EMIT)和高效液相色谱法(HPLC)监测苯妥英(PHT)和苯巴比妥(PB)血浆药物浓度的相关性。方法收集癫痫患者服药后的稳态谷浓度血样,分别用EMIT法和HPLC法进行测定,考察2种测定方法的相关程度。结果以HPLC法测定结果(X)与EMIT法测定结果(Y)所作线性回归方程如下:YPHT=0.505 1.200X(r=0.991);YPB=0.103 1.115X(r=0.970);EMIT法测定血浆中PHT浓度较HPLC法高3.0μg/mL,EMIT法测定血浆中PB浓度较HPLC法高1.7μg/mL,2种方法测定PHT和PB血浆药物浓度结果差异有统计学意义(P<0.05)。结论EMIT法和HPLC法测定2种抗癫痫药血浆浓度结果差异具有统计学意义,在PHT和PB治疗药物监测中应予以关注并作相应调整。     

EMIT法和HPLC法监测苯妥英和苯巴比妥血药浓度比较研究

目的比较酶增强免疫法（EMIT）和高效液相色谱法（HPLC）监测苯妥英（PHT）和苯巴比妥（PB）血浆药物浓度的相关性。方法收集癫痫患者服药后的稳态谷浓度血样,分别用EMIT法和HPLC法进行测定,考察2种测定方法的相关程度。结果以HPLC法测定结果（X）与EMIT法测定结果（Y）所作线性回归方程如下：YPHT=0.505＋1.200X（r=0.991）;YPB=0.103＋1.115X（r=0.970）;EMIT法测定血浆中PHT浓度较HPLC法高3.0μg/mL,EMIT法测定血浆中PB浓度较HPLC法高1.7μg/mL,2种方法测定PHT和PB血浆药物浓度结果差异有统计学意义（P〈0.05）。结论EMIT法和HPLC法测定2种抗癫痫药血浆浓度结果差异具有统计学意义,在PHT和PB治疗药物监测中应予以关注并作相应调整。