高效液相色谱法测定非离子造影剂欧乃帕克

建立了一种快速、灵敏的细径柱高效液相色谱测定非离子造影剂欧乃帕克的方法。用高氯酸去除血浆蛋白,流动相采用简单的7%（体积分数）甲醇水溶液体系。方法的线性范围为0.5~500 mg/L ,相关系数大于 0.999 8 。欧乃帕克的最低检测量为154 pg,保留时间为(3.46 ± 0.02) min,总分析时间少于5 min。日内、日间相对标准偏差(RSD)分别小于4.3%和11.4%。方法的平均回收率大于91.51%。     

Determination of urinary S-sulphocysteine, xanthine and hypoxanthine by liquid chromatography-electrospray tandem mass spectrometry

Molybdenum cofactor and isolated sulphite oxidase deficiencies are two related rare autosomal recessive diseases characterized by severe neurological abnormalities, dislocated lens and mental retardation. Determination of three biochemical markers S-sulphocysteine (SSC), xanthine (XAN) and hypoxanthine (HXAN) in urine is essential for a definitive diagnosis and identification of the exact defect. We developed a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of SSC, XAN and HXAN in urine. The analysis was carried out in the negative-ion selected-reaction monitoring mode. The turnaround time for the assay was 7 min. Linear calibration curves for the three biomarkers were obtained in the range of 12-480 micromol/L. The intra- and inter-day assay variations were <2.5%. Mean recoveries of SSC, XAN and HXAN added to urine at two significantly different concentrations were in the range 94.3-107.3%. At a normal SSC urine excretion value of 3.2 micromol/mmol creatinine, the signal-to-noise ratio was 337:1. This stable isotope dilution LC-MS/MS method is specific, rapid and simple, and provides definitive diagnosis for molybdenum cofactor and isolated sulphite oxidase deficiencies in very small volumes of urine. We have identified seven new cases of isolated sulphite oxidase deficiency from four Saudi families and one Sudanese family.     

Stable isotope dilution analysis of N-acetylaspartic acid in urine by liquid chromatography electrospray ionization tandem mass spectrometry

N-acetylaspartic acid (NAA) is a specific urinary marker for Canavan disease, an autosomal recessive leukodystrophy. We developed a 'dilute and shoot' stable isotope dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of NAA in urine. Deuterated internal standard d(3)-NAA was added to untreated urine and the mixture was injected into the LC-MS/MS system operated in the negative ion mode. Chromatography was carried out on a C(8) minibore column using 50% acetonitrile solution containing 0.05% formic acid at a flow rate of 0.25 mL/min. The retention time was 1.6 min and the turnaround time was 2.2 min. NAA and d(3)-NAA were analyzed in multiple reaction monitoring mode. Calibrators and quality control samples were prepared in pooled control urine. The assay was linear up to 2000 micromol/L with limit of quantification at 1 micromol/L (S/N = 12). Interassay and intraassay coefficients of variation were less than 7% and recovery at three different concentrations was 98.9-102.5%. The LC-MS/MS method for NAA as described involves no extraction and no derivatization, showed no interference and gave excellent recovery with low variability and short analytical time. The method was successfully applied for the retrospective analysis of urine from 21 Canavan disease cases.     

高效毛细管电泳法检测尿液中的假尿嘧啶核苷

假尿嘧啶核苷（ψ）主要来自ｔＲＮＡ的降解,已证实在癌症患者尿液中排泄量异常,可以作为肿瘤诊断的极有用的标志物之一。用高效毛细管电泳法快速测定了人尿中的ψ,用涂层柱（２４ｃｍ×２５μｍｉ．ｄ．）及硼酸盐缓冲液,在线２００ｎｍ检测,可在４ｍｉｎ内使ψ与尿苷等及尿中其它内源物质完全分离。方法的日内、日间变异系数均小于４．０％,用磺基水杨酸作内标,以ψ浓度对相应的峰高或峰面积比定量得标准曲线（ｒ＞０．９９９０）,ψ最低检测限为４μｍｏｌ／Ｌ。     

利用液相色谱–质谱联用技术研究二元表面活性剂体系–啶虫脒微乳剂热贮稳定性

应用液相色谱–质谱联用技术评价复配的阴/非离子二元表面活性剂复合体系–啶虫脒微乳剂的热贮稳定性。将二元表面活性剂复合体系–啶虫脒微乳剂于(54±2)℃下贮藏14天,取出后于室温下干燥24 h,用液相色谱–质谱联用仪测定啶虫脒含量。该法线性拟合方程为y=97 060.72x+542 587.4,线性范围为0.01~0.25 mg/L,相关系数为0.999 8。方法检出限为0.001 mg/L(S/N=3),色谱保留时间和色谱峰面积重复性分别为0.31%,1.44%(n=6),加标回收率为86.23%~107.20%。该法具有简单、准确和灵敏的特点。     

The inclusion of succinylacetone as marker for tyrosinemia type I in expanded newborn screening programs

In expanded newborn screening programs by liquid chromatography/tandem mass spectrometry false negatives for tyrosinemia type I are a significant problem. We describe a method for inclusion of succinylacetone in order to avoid false negatives. We studied spots from 13,000 neonates born in Tuscany (January-May 2007) and ten spots from six patients with tyrosinemia type I. The traditional screening method was modified by adding dioxooctanoid acid (or 13C2-succinylacetone) as an internal standard to the methanolic solution of deuterated acylcarnitines and amino acids. A hydrazine solution was added to the mixture. The times of extraction, butylation and drying were only slightly prolonged. Specific multiple reaction monitoring for derivatized and labelled succinylacetone and dioxooctanoic acid was carried out. The assays were linear up to 100 micromol/L for succinylacetone. Intra- and inter-day imprecision data were in the range of 1.34% to 7.09% and 3.50% to 4.49%. Limits of detection and of quantification were 0.2 micromol/L and 0.4 micromol/L, respectively. Recovery ranged from 97.02% to 100.29%. Succinylacetone levels in samples from unaffected neonates were very close to the detection limit. Of the 46 recalls, eight (17.4%) were for abnormal tyrosine levels and all these cases had succinylacetone levels within the normal range (<2.4 micromol/L). In ten spots from six affected patients succinylacetone values ranged from 3.3 to 35.0 micromol/L. Including succinylacetone in newborn screening programs for amino acids and acylcarnitines avoids false-negative results for tyrosinemia type I. Newborn screening laboratories should consider implementing these modifications.     

Assay of tyrosine hydroxylase based on high-performance liquid chromatography separation and quantification of L-dopa and L-tyrosine

An assay of L-tyrosine (Tyr) hydroxylating activity operating in lincomycin biosynthesis is described. The assay development consisted of HPLC procedure development, assessing the effect of reaction mixture components on non-enzymatic Dopa and Tyr oxidation, and sample stability evaluation. The HPLC procedure with isocratic elution and fluorescence detection was developed and validated. The method showed a wide linear range of Dopa determination of 0.125-25 micromol/L with lower limit of quantification (LLOQ) of 0.125 micromol/L, RSD of 7.2% and accuracy of 101.7%. The studied linear range of Tyr was 15.625 mmol/L to 500 mmol/L with LLOQ of 15.625 mmol/L, RSD of 1.1%, and accuracy of 98.1%. Recoveries for Dopa and Tyr were 100.66 +/- 0.89% and 94.76 +/- 0.94%, respectively. The inter- and intra-day accuracies and precisions were all within 10%. Samples of the reaction mixture were stable for at least 24 h at room temperature (RT) and 28 days at -20 degrees C. The method was tested for the enzyme activity monitoring in purified as well as crude preparations and enabled micro preparation of the enzyme product during confirmation of its identity. The influence of pH and ascorbic acid content in reaction mixture was studied with respect to non-enzymatic Tyr oxidation.     

Rapid column-switching liquid chromatography/mass spectrometric assay for DHEA-sulfate in the plasma of patients with Alzheimer's disease

A simple and highly sensitive method for the quantification of dehydroepiandrosterone-3-sulfate (DHEAS) in human plasma was developed. DHEAS was directly determined in plasma using column-switching liquid chromatography/mass spectrometry (LC-MS). The plasma was filtered with a membrane filter. The filtrate was injected onto a pre-column without further sample preparation such as extraction or derivatization. The pre-column was washed with an aqueous solution to remove interference and the analyte was eluted into a reversed-phase C(18) analytical column for separation and detection using a column-switching valve. The calibration range of DHEAS was 0.01-10 micromol/L, and the linearity of the method was 0.999. The limit of detection (LOD) at a signal-to-noise (S/N) ratio of 3 was 5 nmol/L. The accuracy and precision (%CV) were less than 10% in within-day and day-to-day variations. To explore the relationship between Alzheimer's disease and the DHEAS level in human plasma, the concentrations of DHEAS in female patients with Alzheimer's disease (n = 20) and in normal female subjects (n = 20) were measured. The level of DHEAS was significantly decreased in the plasma of patients with Alzheimer's disease (p < 0.0002) compared with that in normal subjects. From the results, we concluded that our method is sufficiently sensitivity and reliability for the quantification of DHEAS in clinical samples. Plasma DHEAS concentration could be an important marker to understand the pathogenesis of Alzheimer's disease.     

Determination of amino acid enantiomers in human urine and blood serum by gas chromatography-mass spectrometry

Amino acid (AA) enantiomers were determined as N(O)-pentafluoropropionyl-(2)-propyl esters by chiral gas chromatography-mass spectrometry (GC-MS) in 24 h samples of the urine of three healthy volunteers and in their blood sera. In urine the largest amounts were determined for D-Ser (64-199 micromol/day) and D-Ala (24-138 micromol/day). In blood sera, D-Ala (2.3-4.2 micromol/L) and D-Ser (1.0-2.9 micromol/L) were most abundant. Varying amounts of the D-enantiomers of Thr, Pro, Asx, Glx, Phe, Tyr, Orn and Lys were also found, albeit not in all urines and sera. Further, enantiomers were quantified in urine samples of two volunteers fasting for 115 h. Quantities of renally excreted D-AAs decreased in fasting, although amounts of D-Ser (69 and 77 micromol/L urine) as well as other D-AAs were still detectable. Time-dependent analyses of urine showed that D-AAs are continuously excreted. Copyright -Copyright 2001 John Wiley & Sons, Ltd.     

IDose^4迭代重建技术应用于低剂量CT肾动脉血管造影的临床分析

为探讨IDose 4迭代重建技术在低剂量CT肾动脉血管造影中的应用价值,选取205例拟行肾动脉CT血管造影患者,随机将其分为4组,A组(51例)采用管电压100 kV、管电流100 mA,碘海醇370 mg I/mL及IDose 4迭代重建技术,B组(51例)采用管电压100 kV、管电流150 mA,碘海醇270 mg I/mL及IDose 4迭代重建技术,C组(51例)采用管电压80 kV、管电流180 mA,碘海醇270 mg I/mL及IDose 4迭代重建技术,D组(52例)管电压120 kV、管电流120 mA,碘海醇370 mg I/mL及滤波反投影(FBP)重建技术。分析各组图像肾主动脉干CT值、噪声(SD)、信噪比(SNR)和噪声比(CNR),受检者接受CT有效剂量(ED)、容积CT剂量指数(CTDI vol)、剂量长度乘积(DLP)、碘摄入量以及重建图像评分差异和诊断效能。结果显示,A、B、C组重建图像评分优于D组,肾动脉主干CT值、SD、SNR、CNR均高于D组,B、C组肾动脉主干CT值、SD略低于A组。A、B、C组CTDI vol、DLP、ED均低于D组,B组CTDI vol、DLP、ED高于A组。A、B、C、D组诊断肾主动脉狭窄准确率分别为92.68%、86.49%、84.62%、82.86%。证实IDose 4迭代重建技术能降低受试者受辐射和造影剂剂量,提高成像质量,在低剂量CT肾动脉血管造影中具有较高可行性和实用性。     

Modulation of Renal Function in a Metabolic Syndrome Rat Model by Antioxidants in Hibiscus sabdariffa L.

Metabolic syndrome (MS) is the association of three or more pathologies among which obesity, hypertension, insulin resistance, dyslipidemia, and diabetes are included. It causes oxidative stress (OS) and renal dysfunction. Hibiscus sabdariffa L. (HSL) is a source of natural antioxidants that may control the renal damage caused by the MS. The objective of this work was to evaluate the effect of a 2% HSL infusion on renal function in a MS rat model induced by the administration of 30% sucrose in drinking water. 24 male Wistar rats were divided into 3 groups: Control rats, MS rats and MS + HSL rats. MS rats had increased body weight, systolic blood pressure, triglycerides, insulin, HOMA index, and leptin (p ≤ 0.04). Renal function was impaired by an increase in perfusion pressure in the isolated and perfused kidney, albuminuria (p ≤ 0.03), and by a decrease in clearance of creatinine (p ≤ 0.04). The activity of some antioxidant enzymes including the superoxide dismutase isoforms, peroxidases, glutathione peroxidase, glutathione-S-transferase was decreased (p ≤ 0.05). Lipoperoxidation and carbonylation were increased (p ≤ 0.001). The nitrates/nitrites ratio, total antioxidant capacity, glutathione levels and vitamin C were decreased (p ≤ 0.03). The treatment with 2% HSL reversed these alterations. The results suggest that the treatment with 2% HSL infusion protects renal function through its natural antioxidants which favor an improved renal vascular response. The infusion contributes to the increase in the glomerular filtration rate, by promoting an increase in the enzymatic and non-enzymatic antioxidant systems leading to a decrease in OS and reestablishing the normal renal function.     

Direct and simultaneous determination of 5-aminolaevulinic acid and porphobilinogen in urine by hydrophilic interaction liquid chromatography-electrospray ionisation/tandem mass spectrometry

Urinary concentrations of 5-aminolaevulinic acid (ALA) and porphobilinogen (PBG) are elevated in patients with acute hepatic porphyrias, especially during acute attacks. Current assays require lengthy sample pre-treatment and derivatisation steps. We report here a rapid, sensitive and specific hydrophilic interaction liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, for the direct and simultaneous quantitation of ALA and PBG in urine following simple dilution with acetonitrile and centrifugation prior to injection. ALA and PBG were detected using selected reaction monitoring mode, following positive electrospray ionisation. Urine samples (N?=?46) from active and latent mutation-confirmed acute hepatic porphyria patients and normal subjects (N?=?45) were analysed and the results compared with those of a commercially available spectrophotometric method. The validated calibration range was 3-3000?μmol/L for ALA and 2-2000?μmol/L for PBG. For both analytes, imprecision (relative standard deviation) was less than 5% and accuracy (percentage nominal concentrations) was between 88 and 109%. The lower limit of quantitation was 0.1 μmol/L for both analytes. The calculated LC-MS/MS and spectrophotometric results from patient samples compared well [Pearson correlation (r2) of 0.99 and 0.95, for ALA and PBG, respectively]. The method was successfully applied to the measurement of ALA and PBG in urine samples for the screening, biochemical diagnosis and treatment monitoring of patients with acute hepatic porphyrias.     

药物间置换相互作用的毛细管电泳-迎头分析法研究

将18-甲基炔诺酮加到人血清白蛋白-酮洛芬平衡溶液中,室温孵育达平衡后应用毛细管电泳-迎头分析法研究了18-甲基炔诺酮和酮洛芬在人血清白蛋白分子上的置换相互作用。一大体积样品虹吸进样至未涂层毛细管(65 cm×50 μm i.d.,有效长度35 cm),进样时间80 s,毛细管两端高度差11 cm,工作电压10 kV,运行缓冲液为67 mmol/L磷酸盐缓冲液(pH 7.4),游离酮洛芬浓度由前沿峰的高度直接测定。当酮洛芬样品溶液中酮洛芬的总浓度分别为100 μmol/L和200 μmol/L时,随着18-甲基炔诺酮加入量的增加(18-甲基炔诺酮的浓度由0增至200 μmol/L),游离酮洛芬的浓度分别从22.4 增至26.4 μmol/L和从82.1增至106.2 μmol/L。由上面的结果可推论:高浓度的18-甲基炔诺酮可将酮洛芬从它的第二类结合位点上置换出来。     

Development of a capillary electrophoretic method for determination of plasma clearance of iohexol in dogs and cats

Renal function can be monitored by estimation of the glomerular filtration rate (GFR), for example, through measurement of the plasma clearance of a marker that is freely filtrated through the kidney without reabsorption. It has been proposed that iohexol is the most accurate marker for GFR determination in cats and dogs. However, there is a need for a validated capillary electrophoretic method that covers the concentration range for a full curve clearance estimate of iohexol. In the final method, the plasma samples were protein precipitated and the supernatant was analyzed in a background electrolyte containing borate buffer (0.06 m , pH 10.0). The method developed was proved to be linear (concentration range 18– 2900 mg/L) and had a good precision (e.g. 2.3–2.9% at 88 mg/L) and accuracy (e.g. 101–105% at 88 mg/L). Finally, the method was compared with a previously published and validated HPLC‐UV method by parallel analysis of clinical plasma samples from dogs and cats administered Omnipaque®. This comparison showed excellent agreement between the two methods and no proportional or systematic error was observed. The proposed method is simple and has a low cost per sample, which makes it applicable for routine analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Simultaneous determination of allantoin, hypoxanthine, xanthine, and uric acid in serum/plasma by CE

Allantoin (All) is an oxidative end product of purines in mammals. The small amount of All present in human plasma or serum results from free radical action on urate and may provide a stable marker of in vivo free radical activity. Because free radicals have been implicated in the development and progression of atherosclerosis, this study focused on the metabolic compounds of the All pathway. We propose a new fast CE (CE/UV) method for the simultaneous determination of All, uric acid (UA), hypoxanthine (HX), and xanthine (X) in human plasma. These products were quantified in the plasma of patients with chronic renal failure before hemodialysis (n = 6), patients with chronic heart failure (n = 6) and controls (n = 6). The filtered plasma were diluted ten-fold before the direct injection in CE/UV (195 nm), which allows separating the four compounds in less than 13 min. The metabolites were detectable at concentrations of 0.3-0.6 micromol/L. The method was linear over the range 0.5-150 micromol/L for All, HX, and X and 10-1500 micromol/L for UA (r > 0.99). The analytical performance of this method is satisfactory with intra-assay CV < 3.4%, inter-assay CV < 5% (HX and X < 7%), and recovery (93-101%). The proposed CE-UV method appears to be a useful tool for studying physiological and pathological changes of HX, UA, and All levels in plasma samples, the latter being a possible indicator of free radical damage in vivo.     

Implementing tandem mass spectrometry as a routine tool for characterizing the complete purine and pyrimidine metabolic profile in urine samples

Purines and pyrimidines are the basic constituents of DNA and RNA and constitute the basis of at least 50 other important compounds that serve equally vital but separate roles as integral components of intracellular mononucleotide pools. They maintain the supply of these basic components to the different nucleotide pools through an extremely efficient mechanism involving the degradation and recycling of the daily waste products of normal cell turnover. We have developed an LC-MS/MS diagnostic and routine monitoring method for known defects due to both purine and pyrimidine metabolism in a single analysis. Precision tests were made by spiking several urine samples with different creatinine concentrations. For nonspiked low-creatinine urine, intraday precision was in the range of 0.1-9.8% and interday precision was between 1.6 and 14.1%. For nonspiked high-creatinine urine, intraday precision was in the range 0.5-17.2% and interday precision was between 1.5 and 29%. Limit-of-detection (LOD) was in the range 0.1-10 micromol/l and limit-of-quantification (LOQ) in the range of 0.2-15 micromol/l. The current 'dilute and shoot' approach monitors many metabolites, and utilizes a reverse phase chromatographic analysis with a detection requiring 17 min of analysis time. Tandem mass spectrometry and isotope dilution technique enable the accurate quantitation of more than 30 metabolites in one analysis.     

High-throughput capillary electrophoretic method for determination of total aminothiols in plasma and urine.

Increased interest in the analysis of aminothiols in body fluids during the last years results in a request for high-throughput analytical methods for their determination. We report here a novel, high-throughput method for the determination of total concentrations of biogenous aminothiols - homocysteine, cysteine, glutathione, cysteinylglycine, gamma-glutamylcysteine, and of penicilamine, mercaptopropionylglycine, and cysteamine, three compounds used to treat disorders of aminothiol metabolism in plasma and urine. Samples were reduced with tris(carboxyethyl)phosphine and labeled with 5-(bromomethyl)fluorescein. Capillary electrophoretic separations were performed in 60 mmol/L borate - 15 mmol/L sodium dodecyl sulfate - 2-amino-2-methyl-1-propanol, pH 10.0, with laser-induced fluorescence detection. Analysis time was less than 2 min. The assay is linear (r > 0.999) up to 500 micromol/L. Reproducibilities of migration times (coefficient of variation, CV) were < 0.5%. Interassay repeatabilities (CV, n = 10) were 5.08% and 6.09% for 5 micromol/L addition of homocysteine and 0.60% and 3.78% for 100 micromol/L addition of cysteine in plasma and urine, respectively. Recovery values were within 94-106% and sensitivity was better than 0.19 micromol/L for all analyzed compounds. Results agreed well with a standard high-performance liquid chromatography (HPLC) method. The diagnostic usefulness of the method has been proven on 79 samples of cystinuric patients and 12 samples of homocystinuric patients. We report here a novel method for the determination of aminothiols in body fluids by capillary electrophoresis (CE). Determination is fast and sensitive enough for diagnostic purposes.     

Analysis of iodinated X-ray contrast agents in water samples by ion chromatography and inductively-coupled plasma mass spectrometry

In this paper, an analytical method for the determination of six iodinated X-ray contrast agents (amidotrizoic acid, iohexol, iomeprol, iopamidol, iopromide, and ioxitalamic acid), iodide, and iodate in water samples is presented. The method is based on a separation of the analytes by ion chromatography (IC) and a subsequent detection by inductively-coupled plasma mass spectrometry (ICP-MS). The method was optimised with respect to separation conditions (column type and eluent composition) and extensively validated. Without pre-concentration of the samples, limits of detection below 0.2 microg/l could be achieved whereby reproducibility was below 6% for all compounds under investigation.     

高效液相色谱-程序波长紫外检测法同时测定血浆中的色氨酸及其代谢物

建立了一种高效液相色谱-程序波长紫外检测法同时测定血浆中色氨酸(Trp)及其主要代谢产物犬尿氨酸(Kyn)和5-羟色胺(5-HT)。以茶碱为内标(IS),采用BDS-Hypersil-C8柱(150 mm×4.6 mm, 5 μm)分离。流动相为10 mmol/L醋酸钠缓冲液(pH 4.5)-乙腈(94:6, v/v),流速为0.6 mL/min;柱温为25 ℃;紫外检测波长设定: Kyn和IS为360 nm, 5-HT为220 nm, Trp为302 nm。3种物质的平均回收率为87%～113%;线性范围分别为3.97～400 μmol/L(Trp), 0.421～20.2 μmol/L(Kyn), 4.36～980 nmol/L(5-HT);检出限分别为0.134 μmol/L(Trp), 0.0160 μmol/L(Kyn), 2.03 nmol/L(5-HT)。利用该方法对15例抑郁症患者和15例健康志愿者的血浆进行测定,结果表明两组间Trp的代谢存在显著的差异。     

Determination of ascorbic acid in human plasma with a view to stability using HPLC with UV detection

A method for the measurement of ascorbic acid using HPLC with UV detection and investigation into the protein precipitation techniques with regard to stability and recovery are described. The effectiveness of various protein precipitants was tested. Stability of ascorbic acid samples for analysis was investigated over 10 h. Ascorbic acid samples extracted with metaphosphoric acid were stable on a cooled autosampler (4 degrees C) for at least 10 h (with a decline of 1.8% for ascorbic acid solution and 2.8% for plasma). Perchloric acid as protein precipitant for ascorbic acid was unsuitable (with a decline of 36.0% for ascorbic acid solution and 7.3% for plasma). Analytical performance of this method is satisfactory. The intra- and interassay coefficients of variation were 2.1% (n = 10) and 5.8% (n = 12), respectively. The calibration curve was linear with the tested range of 2.0-250.0 micromol/L. The recovery was 96.1% with CV = 4.8% (n = 6) and the LOD was 3 micromol/L. The preliminary reference ranges of ascorbic acid in a group of blood donors are 50.8 +/- 22.4 micromol/L. This assay is a highly sensitive and reproducible HPLC method for the determination of ascorbic acid in human plasma.