高效液相色谱－电化学检测法测定尿液中的多胺

 在已报道的高效液相色谱－电化学检测多胺方法研究的基础上，又进一步探讨了该方法用于尿样本测定的各种条件，并测定了健康志愿者及肿瘤患者尿液中的多胺。结果显示肿瘤患者未水解及水解尿液中的腐胺、尸胺的平均值高于健康志愿者。     

甲亢患者血清和尿液的核磁共振代谢组学研究

应用基于核磁共振(NMR)的代谢组学方法, 研究甲状腺功能亢进(简称甲亢)患者和健康人群的血清和尿液, 分析甲亢疾病的特征代谢物. 实验收集33个甲亢患者和17个健康志愿者的血清样品以及53个甲亢患者和58个健康志愿者的尿液样品, 采用多元统计分析方法研究甲亢组和对照组血清和尿液中的内源性代谢差异. 结果表明, 甲亢组血清中的胆碱、葡萄糖和三甲胺等物质的含量升高, 而VLDL, LDL和胆固醇等脂质以及乳酸、糖蛋白和丙氨酸等代谢物的含量下降; 甲亢组尿液中的葡萄糖、柠檬酸、牛磺酸以及肌氨酸等代谢物的含量升高, 而马尿酸、TMAO、甲酸和琥珀酸等代谢物的含量下降. 结果表明, 甲亢病不仅影响了糖类、脂类和蛋白质三大物质的代谢, 还对能量代谢、肝肠循环和肠道微生物等多个生理系统产生显著影响, 并且可能造成肝脏及肾脏等器官的损伤.     

高效液相色谱法测定尿中多胺

自1971年Russell首先报道肿瘤患者尿中多胺(腐胺、精脒、精胺)排出量比正常人增多,此后许多研究报道已证实多胺与肿瘤的生长及其临床诊断密切相关。所以国内外相继建立了许多测定多胺的方法。 张任恩等利用5-二甲基萘-1-磺酰氯(DNSC1)柱前衍生样品,通过反相液相色谱(RP-HPLC)分析多胺获得满意的结果,而本文则运用HPLC方法测定尿中多胺。     

傅里叶变换红外光谱体表无创探测乳腺肿瘤

利用傅里叶变换红外(FTIR)光谱仪联合衰减全反射(ATR)探头的中红外光导纤维对46名健康志愿者及113名乳腺肿物患者的体表皮肤进行无创红外光谱测定, 并从分子水平比较、分析了正常乳腺和有肿物乳腺体表皮肤的红外光谱特征. 结果表明, 健康志愿者乳房皮肤8个FTIR光谱测定部位的图谱趋于一致; 正常乳腺体表与有肿瘤乳腺体表的吸收峰差异明显, 而1080 cm^-1处核酸相关吸收峰的变化对鉴别肿瘤的良、 恶性有重要意义.     

液相色谱-串联质谱法测定尿液中的内源性类固醇激素

建立了液相色谱-串联质谱(LC-MS/MS)测定尿液中的内源性类固醇激素的方法。尿样经葡萄糖醛酸甙酶酶解后进行液-液提取,以甲醇-0.1%甲酸缓冲液(含0.02 mol/L乙酸铵)(体积比为68:32)为流动相,采用Cosmosil C18色谱柱分离,并以三重四极杆串联质谱多反应监测扫描方式对尿样中的脱氢表雄酮(DHEA)、睾酮、表睾酮、雄酮和苯胆烷醇酮等5种激素进行检测。方法的最低检出限为0.01～10 ng/mL,平均回收率为96.7%～106.5%,日内和日间相对标准偏差(RSD)分别小于7%和11%。应用所建立的方法测定了健康志愿者口服DHEA后尿液中内源性类固醇激素的变化情况,结果表明该方法样品处理简便,色谱分离完全,结果准确可靠,可替代气相色谱-质谱法用于体液中内源性类固醇激素兴奋剂的常规分析。     

超高效液相色谱-串联质谱法测定尿液中118种农药残留

为有效监测人尿液中的农药多残留水平，为健康风险评估提供重要的技术手段，实验利用QuEChERS前处理方法结合超高效液相色谱-三重四极杆质谱技术建立了尿液中118种农药的快速筛查及测定方法。通过对前处理过程、液相色谱分离和质谱条件的系统优化，实现了在2 h内对样品中118种目标分析物的提取及分析测定。具体方法如下：尿液样品中农药目标分析物采用乙腈提取，无水MgSO₄加NaCl作除水盐析剂，再以C₁₈、PSA、无水MgSO₄为净化吸附剂，经QuEChERS法净化，氮吹复溶后，以0.01%甲酸水溶液(含2 mmol/L甲酸铵)及0.01%甲酸甲醇溶液(含2 mmol/L甲酸铵)作为流动相，ZORBAX Eclipse Plus C₁₈柱(100 mm×2.1 mm, 1.8μm)作为分析色谱柱，梯度洗脱分离，超高效液相色谱-三重四极杆质谱正负离子切换动态多反应监测(DMRM)模式检测，外标法定量。结果表明，该方法可以对尿液中的118种农药同时进行快速测定，检出限均可达到0.10μg/L,定量限均可达到0....     

蒜苔中咪鲜胺及其代谢物残留检测方法的研究与比较

建立了水解法与QuEChERS法测定蒜苔中咪鲜胺及其代谢物的残留量,并对两种方法进行了比较。在QuEChERS法中,样品经乙腈提取,QuEChERS净化管净化后用气相色谱法测定咪鲜胺及其代谢产物2,4,6-三氯苯酚的含量,基质匹配标准曲线外标法定量;在水解法中,样品经乙腈提取,吡啶盐酸盐水解后,用硫酸磺化,用气相色谱法测定咪鲜胺的含量。结果表明,水解法和QuEChERS法测定的咪鲜胺标准曲线在0.01～2 mg/L范围内均有良好的线性关系,相关系数(r²)均大于0.999。水解法中咪鲜胺的定量限(LOQ)为0.005 mg/kg; QuEChERS法中咪鲜胺的LOQ为0.039 mg/kg、2,4,6-三氯苯酚的LOQ为0.003 mg/kg。在3个不同浓度添加水平下,方法回收率为81.5%～105.4%,相对标准偏差(RSD)为1.3%～6.8%。在测定阳性样品时,水解法可以较为全面地检测出样品中咪鲜胺及其主要代谢物的总量,QuEChERS法可以检测出咪鲜胺及其主要代谢物2,4,6-三氯苯酚的存在形式和各自的含量,两种方法可以互相补充用于蒜苔中咪鲜胺及其代谢物的...     

大鼠尿液与粪便中氟噻草胺的UPLC-MS/MS法测定

建立了一种测定大鼠尿液和粪便中氟噻草胺含量的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法。粪便与尿液样品采用乙腈提取;液相色谱分离采用Phenomenex反向C18色谱柱(50 mm×4. 6 mm,5μm),以0. 1%甲酸水和乙腈为流动相,流速0. 6 m L/min;质谱检测采用电喷雾离子源,正离子模式和多反应监测(MRM)方式进行扫描。结果表明:氟噻草胺在尿液(0. 10～10. 0 mg/L)和粪便(0. 25～50. 0 mg/L)中线性关系良好,相关系数r 0. 99,尿液和粪便中氟噻草胺的定量下限分别为0. 10 mg/L和0. 25 mg/L;质控样品的日内与日间相对标准偏差不大于9. 9%。样品稳定性为93. 7%～108%,尿中平均提取回收率为97. 0%～98. 8%,基质效应为98. 8%～107%,均符合生物分析方法验证的要求。考察了大鼠单次灌胃给予氟噻草胺400 mg/kg后的排泄动力学,144 h内尿液与粪便的总累积排泄率为12. 62%,其中尿中的累积排泄率为1. 12%,粪便中的累积排泄率为10. 13%,表明氟噻草胺主要经粪便排泄。该法灵敏、专属、准确,可用于大鼠尿液、粪便中氟噻草胺浓度的测定。     

一种癌症早期检测的新方法——尿液的荧光光谱

比较了癌症患者尿液自体荧光光谱与健康人尿液自体荧光光谱的差异，提出了用荧光试剂与尿液中的某些组分反应生成新的发光体，用于癌症患者病情确诊的辅助手段；实验结果表明，直肠癌、鼻咽癌、结节状组织细胞淋巴瘤、肺癌及其它癌症的阳性率分别为71％、71％、67％、50％、61％，该法适于健康普查，将有利于癌症的早期发现，提高癌症的治愈率。     

超高效液相色谱-四极杆-静电场轨道阱质谱联用法用于宫颈癌患者尿液代谢组学研究

利用超高效液相色谱-四极杆-静电场轨道阱质谱联用(UHPLC-Q-Orbitrap MS)方法对宫颈癌(Cervical cancer,CC)患者和健康人(Healthy control,HC)的尿液进行分析,研究宫颈癌患者尿液中的潜在标志物,为其发病机制和诊断提供科学依据。筛选11例宫颈癌患者(Age(45.7±5.6)years)及11例健康人(Age(45.9±3.2)years)尿液样本,采用液相色谱-质谱联用技术对尿液进行测定,通过主成分分析(PCA)和偏最小二乘法-判别分析(OPLS-DA)处理数据,结果表明,两组人群代谢轮廓有显著差别,发现并鉴定了12种潜在的生物标志物,提示特定的肿瘤代谢途径中潜在的代谢标志物可能在宫颈癌发生发展中发挥重要作用。     

Determination of polyamines in human urine by precolumn derivatization with benzoyl chloride and high-performance liquid chromatography coupled with Q-time-of-flight mass spectrometry

A simple and sensitive HPLC/Q-TOF MS method for simultaneous determination of 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine and acetyl-spermine in human urine was developed in electrospray-ionization source by positive ion mode. The samples were firstly pretreated by 10% HClO₄ and then derivatized by benzoyl chloride with 1,6-diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column by a gradient elution with methanol-water, and then sensitively detected with Q-TOF MS. The limits of detection for polyamines ranged from 0.02 to 1.0 ng ml⁻¹ with excellent linearity within the range from 1 to 1000 ng ml⁻¹ except acetyl-spermine from 5 to 1000 ng ml⁻¹. The intra- and inter-day R.S.D. for all polyamines were 2.0-14.7% and 3.9-12.9%, respectively. The method was applied to determine the polyamines in human urine from 10 cancer patients and 15 healthy volunteers. Results showed that the mean levels of polyamines in urine of patients were all higher than those in healthy volunteers. The cluster analysis was used to establish the distinction mode between cancer sufferers and healthy individuals.     

Determination of polyamine metabolome in plasma and urine by ultrahigh performance liquid chromatography–tandem mass spectrometry method: Application to identify potential markers for human hepatic cancer

To evaluate the potential relationship between cancer and polyamine metabolome, a UHPLC–MS/MS method has been developed and validated for simultaneous determination of polyamine precursors, polyamines, polyamine catabolite in human plasma and urine. Polyamine precursors including l-ornithine, lysine, l-arginine and S-adenosyl-l-methionine; polyamines including 1,3-diaminopropane, putrescine, cadaverine, spermidine, spermine, agmatine, N-acetylputrescine, N-acetylspermine and N-acetylspermidine; polyamine catabolite including γ-aminobutyric acid had been determined. The analytes were extracted from plasma and urine samples by protein precipitation procedure, and then separated on a Shim-pack XR-ODS column with 0.05% heptafluorobutyric acid (HFBA) in methanol and 0.05% HFBA in water. The detection was performed on UHPLC–MS/MS system with turbo ion spray source in the positive ion and multiple reaction-monitoring mode. The limits of quantitation for all analytes were within 0.125–31.25 ng mL⁻¹ in plasma and urine. The absolute recoveries of analytes from plasma and urine were all more than 50%. By means of the method developed, the plasma and urine samples from hepatic cancer patients and healthy age-matched volunteers had been successfully determined. Results showed that putrescine and spermidine in hepatic cancerous plasma were significant higher than those in healthy ones, while spermidine, spermine and N-acetylspermidine in hepatic cancerous urine were significant higher than those in healthy ones. The methods demonstrated the changes of polyamine metabolome occurring in plasma and urine from human subjects with hepatic cancer. It could be a powerful manner to indicate and treat hepatic cancer in its earliest indicative stages.     

尿多胺含量与血液系统恶性肿瘤关系的研究——毛细管气相色谱分析尿中多胺

用毛细管色谱柱配以氮磷检测器测定尿中多胺,对几例白血病患者和正常人尿中多胺定性和半定量。结果表明白血病患者尿中多胺总量高于正常人;化疗后较化疗前尿多胺含量明显降低。本法在临床诊断、治疗监视和预后方面均有重要意义。     

Determination of polyamines in urine of normal human and cancer patients by capillary gas chromatography

A capillary gas chromatographic method is described for the determination of polyamines (putrescine, spermidine and spermine) in the urine of normal human and cancer patients. Morning urine after acid hydrolysis is cleaned up on a silica gel column and derivatized with trifluoroacetic-anhydride. Creatinine in human urine is used as internal standard. Recoveries of polyamines are 96.7% putrescine, 102.6% spermidine (Spd), and 98.7% spermine. SD of the method for Spd is 1.949 +/- 0.041 (micrograms/mg creatinine, mean +/- SD, n = 5). The results show that the mean level of polyamines in cancer patients urine is much higher than that in normal human urine. The mean of total polyamines in the normal human and the cancer patients is 2.01 and 44.74, respectively (g/mg creatinine).     

Analysis of polyamines as carbamoyl derivatives in urine and serum by liquid chromatography-tandem mass spectrometry

A quantitative analysis of polyamines in urine and serum by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The polyamines were carbamylated with isobutyl chloroformate, extracted with diethyl ether under pH 9.0, and analyzed by LC-MS/MS with single reaction monitoring mode. The limit of quantification was 1 ng/mL based on a signal-to-noise ratio>3, and the correlation coefficient (r2) for the calibration curves was >0.99 for both urine and serum samples. The present method was applied to urine and serum samples from 30 breast cancer patients and 30 normal female controls. There was no significant difference in the urinary polyamine levels between breast cancer patients and controls. However, 1,3-diaminopropane, putrescine, spermine and N-acetylspermidine levels in serum increased in breast cancer patients. These four serum polyamines may be a good index to study both production and metabolism of polyamines, and a useful tool in assessment of the polyamine status of breast cancer patients.     

人尿中多胺的离子交换富集和气相色谱测定

以离子交换法对尿样中的多胺进行预分离富集，确定了最佳的离子交换条件，同时比较了三种不同酰化剂的酰化效果，确定了最佳酰化条件，并用毛细管气相色谱法测定了尿中腐胺，尸胺，精脒，精胺的含量，结果表明癌症病人尿中多胺含量高于正常人。     

Urine polyamines determination using dansyl chloride derivatization in solid-phase extraction cartridges and HPLC

The derivatization of biogenic amines such as putrescine, cadaverine, spermidine and spermine with dansyl chloride in solid phase extraction cartridges is described. Different types of filling materials were tested in order to have the highest retention of the different analytes. The best results were obtained by using C18 cartridges. The optimal conditions were: amine solution buffered at pH 12, 2 mM dansyl chloride (acetone-bicarbonate solution 20 mM (pH 9-9.5), 2 + 3 v/v) as reagent concentration, room temperature and 30 min reaction time. The developed procedure was applied to the determination of these polyamines in urine samples from healthy controls and cancer patients using HPLC with 1,7-diaminoheptane as internal standard. The concentrations ranged from 0.5 to 5 micrograms mL-1 and the detection limits were 10 ng mL-1 for all polyamines. By concentrating the urine extracts, the detection limits were improved down to 2 ng mL-1. The accuracy and the precision of the method were tested. The proposed dansylation method is advantageous with respect to solution dansylation. It improves the total analysis time, avoids high temperatures that can affect the thermal stability of the derivatives and could make possible the automation of the procedure.     

Determination of polyamines in human plasma by high‐performance liquid chromatography coupled with Q‐TOF mass spectrometry

A high‐performance liquid chromatography coupled with Q‐time of flight mass spectrometry (HPLC/Q‐TOF MS) method was developed and validated for the determination of 1, 3‐diaminopropane, putrescine, cadaverine, spermidine and spermine in human plasma. The plasma samples were first pretreated by 10% HClO₄ and then derived by benzoyl chloride with 1, 6‐diaminohexane as internal standard. The derived polyamines were separated on a C₁₈ column using a gradient program. The detection was performed on a Q‐TOF MS by positive ionization mode. Calibration curve for each polyamine was obtained in the concentration range of 0.4 ~ 200.0 ng ? ml^?1, with limit of detection of 0.02 ~ 0.1 ng ? ml^?1. The intra‐ and inter‐day RSD for all polyamines were 2.5–14.0% and 2.9 ~ 13.4%, respectively. The method was applied to determine the polyamines in human plasma from cancer patients and healthy volunteers. Results showed that the mean levels of polyamines in the plasma of cancer patients were higher than that of healthy volunteers, which suggested that the plasma polyamines could be employed as cancer diagnostic indicators in clinical testing. Copyright © 2012 John Wiley & Sons, Ltd.     

Mass fragmentographic identification of polyamine metabolites in the urine of normal persons and cancer patients, and its relevance to the use of polyamines as tumour markers

The mass fragmentographic identification of N-(2-carboxyethyl)-4-amino-n-butyric acid, N-(3-aminopropyl)-N1-(2-carboxyethyl)-1,4-diaminobutane, N,N1-bis(2-carboxyethyl)-1,4-diaminobutane, and delta-aminovaleric acid in acid-hydrolysed urines of a normal person and two cancer patients is described. A previous study, in which the metabolic fate of intraperitoneally injected polyamines in rats was investigated, revealed that these compounds should be considered as non-alpha-amino acid metabolites of the naturally occurring polyamines. Quantification of polyamines and their non-alpha-amino acid metabolites by gas chromatography with nitrogen--phosphorus detection showed that, relative to the parent polyamines, humans normally excrete higher quantities of polyamine catabolites in urine than rats, suggesting that humans catabolize polyamines more efficiently. As illustrated by the follow-up of the concentrations of polyamines and their catabolites in the urine of a patient with high-grade non-Hodgkin lymphoma during chemotherapy, the catabolic pressure on polyamines may be considerably increased during neoplastic diseases, since an even higher proportion of oxidized polyamine metabolites was observed. It is therefore suggested that the additional measurement of the circulating concentrations of polyamine-degrading enzymes is of importance for the correct interpretation of polyamine (metabolite) determinations for oncological purposes.     

Reversed-phase liquid chromatographic separation and simultaneous fluorimetric detection of polyamines and their monoacetyl derivatives in human and animal urine, serum and tissue samples: an improved, rapid and sensitive method for routine application

A highly sensitive and precise method for the determination of the polyamines putrescine, cadaverine, spermidine and spermine and all their monoacetyl derivatives in a single analysis in human and animal urine, serum and tissue samples is described. For polyamine separation, an ion-pairing reversed-phase high-performance liquid chromatographic (HPLC) method is used, followed by post-column derivatization with o-phthalaldehyde and consecutive fluorescence detection. Urine and serum samples are purified with a Bond Elut silica cartridge. The detection limit for polyamines is 0.5-1.0 pmol and excellent linearity is achieved in the range from 3 pmol up to more than 10 nmol. The influence of some modifications of different analytical steps such as the temperature of the HPLC column and the derivatization reaction coil and the o-phthalaldehyde flow-rate is described. Quality control data and measurements of the reproducibility of the method are presented. In order to establish a rapid analytical method for easy routine use, all steps for preparation and quantitative analysis are minimized. This method was applied to the determination of total polyamines in human urine and serum hydrolysate and of free and acetylated polyamines in human urine and pancreatic tissue of the rat. Values for normal polyamine concentrations in the urine and serum of fifteen male and fifteen female healthy volunteers and in the pancreas of ten normal rats are presented.