LC/MS/MS方法筛查新生儿苯丙酮尿症

苯丙酮尿症 [1～ 4 ] ( PKU )发病原因是患者基因缺陷使肝脏不能合成苯丙氨酸羟化酶而导致体内苯丙氨酸 ( Phe)不能正常代谢为酪氨酸 ( Tyr) ,前者在体内大量堆积并氧化为对人体有害的苯丙酮酸 . PKU是目前筛查范围最广的氨基酸代谢遗传疾病 ,在全世界每年 [5]约有一千万婴儿接受 PKU筛查 ;在我国 ,PKU也是卫生部要求重点筛查的病种 .Chace[5]等在 1 993年报道了 MS/ MS方法筛查新生儿PKU:直接使用 MS/ MS的中性碎片丢失扫描方式检测 Phe和 Tyr,通过氘代内标与待测氨基酸的质谱峰高比来定量 .MS/ MS方法速度快、准确性好、可以…     

GC—CI—MS—SIM方法论断新生儿苯丙酮尿症

苯丙酮尿症（PKU）的发病原因是患者苯丙氨酸羟化酶的缺陷,从而导致体内苯丙氨酸（Phe）不能正常代谢为]酷氨酸（Tyr),前者在体内大量堆积并氧化为有害的七人酮酸,PKU是目前筛查范围最广的氨基酸代谢遗传疾病,全世界每年约有一千万儿接受PKU调查,在中国PKU也是卫生部要求重点筛查的病种^[1],目前常规筛查方法是细菌抑制法（BIA),它的特点是用低、速率慢、空易产生假阳性;Chace等^[2,3]报道了MS－MS）京都是新生儿PKU,曲峻等^[4]发展了Chace的方法,采用HPLC与MS－MS联用方法（LCD－MS－MS）筛查新生儿PKU^[4],利用HPLC富集,MS－MS作为HPLC的检测器定量血样中的苯丙氨酸和酷氨酸,MS－MS和LC－MS－MS方法速度快,准确性好,由于是通过测定Phe与Tyr的比值来筛查PKU,克服了假阳性的产生^[2~4],但是LC－MS－MS和MS－MS价格都很昂贵,不适合国内普及推广,本文将血样的Phe与Tyr进行化学修饰后,采用化学电离源选择离子的方式用气相色谱质谱（Gas chromatography-chemical ionizatino -mass spectrograph-selective ion measurement简称GC－CI－MS－SIM）进行了分析,测定了Phe和Tyr相应衍生物的准分子离子峰面积,用外标法计算出血样中的Phe与Tyr摩尔浓度,与MS－MS、LC－MS－MS一样取得同样满意的效果,由于GC－MS价格较低,适合国内普及推广。     

高效液相色谱-同位素稀释质谱法测定血清中的苯丙氨酸

苯丙酮尿症(PKU)是一种世界性分布的遗传疾病,病人体内缺少苯丙氨酸羟化酶(PAH),导致苯丙氨酸(Phe)不能正常代谢为酪氨酸,引起Phe代谢产物在人体中过量蓄积,造成患者中枢神经系统受到不可逆的损害而影响智力.     

苯丙酮尿症新生儿血斑中辅酶及葡萄糖等物质的分析

苯丙酮尿症(PKU)是新生儿先天性苯丙氨酸羟化酶缺陷所引起的苯丙氨酸代谢障碍疾病.本研究采用超高效液相色谱-质谱联用技术, 测定了5例PKU新生儿出生3天和出生30天后的血斑与20例年龄相仿的正常新生儿血斑中辅酶Q10的绝对含量和辅酶Q9的相对含量,其中,健康新生儿血斑中辅酶Q10的含量为(122.1±24.9) ng/mL,PKU新生儿组的含量为(59.0±12.0) ng/mL.采用气相色谱-质谱联用技术测定了胆固醇和葡萄糖的相对含量.研究结果表明,与对照组相比,PKU新生儿血斑中辅酶Q10、Q9、胆固醇和葡萄糖的含量均显著降低,辅酶Q10的降低与血斑中苯丙氨酸含量升高呈现显著反向相关.本研究结果为PKU患儿的饮食治疗方案提供了依据.     

应用多模型共识偏最小二乘法和FTIR/ATR光谱技术建立苯丙酮尿症的筛查模型

利用多模型共识偏最小二乘法(cPLS)建立新生儿苯丙酮尿症(PKU)的红外光谱筛查模型,比较PLS和cPLS模型的性能。对原始光谱进行一阶微分预处理,分别用PLS和cPLS建立干血片中苯丙氨酸浓度的定量校正模型,各运行40次,以预测均方根误差(RMSEP)、平均相对误差(MRE)和预测准确率(Acc)为指标,考察两种模型对独立测试集的预测效果。PLS模型的RMSEP、MRE、Acc的平均值和标准差分别为103.3、0.32、97.1和30.0、0.07、4.4;而cPLS模型的RMSEP、MRE、Acc的平均值和标准差分别为88.4、0.26、99.3和19.8、0.04、2.4。cPLS较PLS模型预测更准确,稳定性更好,更适于建立PKU的红外光谱筛查模型。     

GC/MS和ESI/MS/MS同位素内标法检测甲基丙二酸血症

以甲基丙二酸血症为对象,分别用GC/MS和ESI/MS/MS方法对该疾病进行了定性和定量检测.通过对样品前处理和分离条件的改善,对疾病的标识化合物之一甲基丙二酸进行了定量测定,其稳定性、精密度和回收率结果很好.同时比较了GC/MS和ESI/MS/MS两种方法的特点,发现两种方法的结合不仅可满足新生儿代谢疾病筛查的要求,同时还可对高危人群进行诊断.     

GC-MS方法诊断新生儿苯丙酮尿症

用甲醇提取血样中的苯丙氨酸(Phe)、酪氨酸(Tyr),提取液离心去蛋白质,用正丁醇将其中的苯丙氨酸和酪氨酸丁酯比,再用三氟醋酸酐酰化后进行GC-MS分析,测定Phe,Tyr特征离子峰面积,用外标法计算出Phe和Tyr的摩尔浓度,根据Phe和Tyr摩尔浓度比值来诊断新生儿苯丙酮尿症(PKU)。     

单柱离子色谱法同时分离分析9种多价金属阳离子的研究

近年来 ,对生物样品中的重金属和过渡金属离子的分析已引起人们的关注 .目前 ,对这些离子的分析方法主要有分光光度法 [1] 、原子吸收法 (AAS) [2 ] 、原子发射法 (AES) [3 ] 和高频电感偶合等离子体发射光谱 -质谱 (ICP- MS) [4 ] 等 .这些方法或操作步骤冗长费时 ,需要多种化     

小儿哮喘患者呼出气冷凝液代谢组学研究

利用气相色谱-质谱联用法(GC-MS/MS)对小儿哮喘患者(Childhood asthma)和健康儿童(Healthy control)的呼出气冷凝液(Exhaled breath condensate,EBC)进行分析,寻找小儿哮喘患者EBC中的潜在标记物,为其发病机制及早期筛查提供科学依据。收集了21例小儿哮喘患者(年龄(8.2±1.6)岁)及17例健康儿童(年龄(8.1±1.3)岁)的EBC样本,采用GC-MS/MS获得化学成分的全扫描数据,通过主成分分析法对两组代谢物进行聚类分析,使用Metaboanalyst 3.0归属涉及的代谢通路。结果表明,小儿哮喘组和正常组EBC代谢图谱能很好地区分,鉴定了8个差异表达物可作为潜在的内源性生物标记物,提示淀粉和蔗糖代谢、赖氨酸降解、氨基糖核苷酸糖代谢、苯丙氨酸代谢可能在小儿哮喘发生发展过程中发挥重要作用。     

镧诱导植物产生抗病性的生化机制

为了进一步探究镧的诱导抗病性机制,通过盆栽试验测定了镧对水稻幼苗体内蛋白质、糖类的代谢,及苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶等防御性酶的活性的影响.结果表明,镧浸种处理后,能参与植物体的物质代谢,提高了植株体内蛋白质和还原糖的含量;提高了水稻幼苗体内与抗菌物质合成有关的苯丙氨酸解氨酶、过氧化物酶和多酚氧化酶等一些关键酶的活性,从而通过改变植物体内的物质代谢,提高防御性酶的活性,增加酚类等抗菌物质的积累,提高植物的抗病性.     

Newborn screening of phenylketonuria using direct analysis in real time (DART) mass spectrometry

Phenylketonuria (PKU) is commonly included in the newborn screening panel of most countries, with various techniques being used for quantification of l-phenylalanine (Phe). To diagnose PKU as early as possible in newborn screening, a rapid and simple method of analysis was developed. Using direct analysis in real time (DART) ionization coupled with triple-quadrupole tandem mass spectrometry (TQ-MS/MS) and with use of a 12 DIP-it tip scanner autosampler in positive ion mode, we analyzed dried blood spot (DBS) samples from PKU newborns. The concentration of Phe was determined using multiple reaction monitoring mode with the nondeuterated internal standard N,N-dimethylphenylalanine. The results of the analysis of DBS samples from newborns indicated that the DART-TQ-MS/MS method is fast, accurate, and reproducible. The results prove that this assay as a newborn screen for PKU can be performed in 18 s per sample for the quantification of Phe in DBS samples. DART-TQ-MS/MS analysis of the Phe concentration in DBS samples allowed us to screen newborns for PKU. This innovative protocol is rapid and can be effectively applied on a routine basis to analyze a large number of samples in PKU newborn screening and PKU patient monitoring.

Fast Diagnosis of Neonatal Phenylketonuria by Gas Chromatography-Mass Spectrometry Following Microwave-Assisted Silylation

Phenylketonuria (PKU) is a fairly common autosomal recessive disease. Phenylalanine (Phe) and tyrosine (Tyr) are the biomarkers of PKU, and it can be diagnosed by the measurement of Phe and Tyr in neonatal blood samples. A fast diagnostic procedure for neonatal PKU has been developed using microwave-assisted silylation followed by gas chromatography-mass spectrometry. Amino acids extracted from neonatal blood samples are rapidly derivatized with N, O-bis(trimethylsilyl)-trifluoroacetamide under microwave irradiation; the derivatives are then analyzed by GC-MS. The silylation conditions have been optimized, and the method validated for linear range, detection limit, precision and recovery. The proposed method is linear from 20 to 800 µM, detection limit is less than 0.35 µM, relative standard deviation (RSD) is less than 9.0%, and recoveries of 87% and 83% were obtained for Phe and Tyr. The proposed method was tested by the determination of Phe and Tyr in blood spots from eight PKU-positive neonates and twelve control neonates. Microwave irradiation considerably accelerates the derivatization reaction of amino acid with BSFTA, and shortens the whole analysis time. Microwave-assisted silylation coupled to GC-MS is a powerful tool for fast screening of neonatal PKU.     

Spectrofluorimetric determination of phenylalanine based on fluorescence enhancement of europium ion immobilized with sol-gel method

The analysis of phenylalanine (Phe) in serum is widely performed for the screening of newborn phenylketonuria (PKU). In this work, a novel spectrofluorimetric method for the determination of Phe was developed based on the fluorescence enhancement of Ruhemann's purple, the reaction product between Phe and ninhydrin, upon coordination with Eu(3+). A filter paper disc containing immobilized reactants (ninhydrin and Eu(3+)) was fabricated by sol-gel method. The experimental parameters affecting the determination of Phe, such as the concentrations of immobilized reagents, the pH value, the reaction time and temperature were optimized. Under optimum conditions, the fluorescence intensity of Phe-ninhydrin-Eu(3+) system was linearly proportional to the concentration of Phe in the range from 5×10(-5) to 2×10(-3) mol L(-1), and the limit of detection was found to be 5.2×10(-6) mol L(-1). The relative standard deviation was 2.6% for ten replicate measurements of 1.5×10(-4) mol L(-1) of Phe. The method has merits of sensitivity, simplicity and low cost, and has been applied to the determination of Phe in artificial serum.     

Determination of phenylalanine and tyrosine by liquid chromatography/mass spectrometry

Phenylketonuria is a common metabolic disorder disease. Those affected appear normal at birth, but without treatment they develop severe psychomotor retardation. Throughout life, they must control their blood levels of phenylalanine (Phe) and consume a diet containing adequate amounts of Phe and tyrosine (Tyr). We have developed a liquid chromatographic/mass spectrometric (LC/MS) method for the quantitative evaluation of Phe and Tyr in food samples. This method takes advantage of the good separation of LC and the selective and reliable quantification provided by MS in the electrospray ionization mode. The LC/MS method is very suitable for the determination of selected amino acids in various matrixes. It is sensitive to levels as low as about 0.30 ppm for Tyr and 0.70 ppm for Phe and robust. Nearly 100 nondietary food samples were analyzed by the developed method.     

Amino acid analysis of dried blood spots for diagnosis of phenylketonuria using capillary electrophoresis-mass spectrometry equipped with a sheathless electrospray ionization interface

We describe a capillary electrophoresis-mass spectrometry (CE-MS) method for newborn screening of a representative amino acid metabolic disease, namely, phenylketonuria (PKU). Underivatized phenylalanine and tyrosine in a dried blood spot (DBS) were simultaneously determined by CE-MS equipped with an ionophore membrane-packed sheathless electrospray ionization interface, which was developed by our group. The method was optimized for rapid determination of the underivatized amino acids, phenylalanine and tyrosine extracted from a DBS. Under the optimized conditions, the limit of detection of phenylalanine and tyrosine (signal-to-noise ratio, 3) was 0.03 and 0.07 mg/L in DBS, respectively, with a CE run time of less than 3 min. For repeated runs of a sample, coefficients of variation (CVs) for migration time were less than 3.7 %, whereas CVs for the area ratio under the curve were 2.1 and 2.9 % for 20 consecutive runs of 49.5 mg/kg Phe and 36.2 mg/kg Tyr, respectively. However, the relative standard deviations of intra- and interday assays for DBS samples were <6.2 and <5.8 %, respectively, which were substantially due to sample extraction from DBS. The analytical method was applied to real clinical samples of Korean neonates, and results were compared with those of conventional methods for PKU diagnosis, which required reference analytical methods such as isotope dilution CE-MS or high-performance liquid chromatography-mass spectrometry for quality assurance of the conventional kit-based assays. The distinct advantages of high sensitivity and extremely low sample volume, as well as a simple, easy, and economic sample pretreatment, were demonstrated for the proposed method.     

Simultaneous determination of creatinine, creatine, and UV-absorbing amino acids using dual-mode gradient low-capacity cation-exchange chromatography

A simple and versatile cation-exchange chromatography technique for the simultaneous determination of urinary creatinine (Cre), creatine (Crn), methionine (Met), tyrosine (Tyr), phenylalanine (Phe), histidine (His), and tryptophan (Trp) was developed. A novel low-capacity cation-exchange column packed with a newly developed sulfoacylated hypercross-linked macroreticular polystyrene-divinylbenzene resin, referred to as TMR-A/75 (capacity: 75 microequiv/column), was successfully used with a binary dual-mode gradient eluting system. Two solvents, (A) 25 mM phosphoric acid-methanol (30:70, v/v) and (B) 25 mM disodium hydrogenphosphate-methanol (30:70, v/v) were pumped through the column by programming solvent delivery ratios as 0 to 5 min: A-B (55:45, pH 3.6); 5-21 min: A-B (49:51, pH 5.3); and 21-35 min: A-B (55:45, pH 3.6). The flow rate was simultaneously time-programmed to be 0.6 mL/min from 0 to 19 min and to be 1.0 mL/min from 19 to 35 min. This eluting system could permit the use of the UV detection at 210 nm. The analytes, Crn, Met, Tyr, His, Cre, Phe, and Trp, were well separated in this order in 27 min with minimum resolution of approximately 2, and the cycle time was about 35 min. Retention time of each analyte was very reproducible with relative standard deviations (RSDs) between 0.05 and 0.38% (n = 5). The peak area responses were also reproducible with RSDs between 0.74 and 2.24% (n = 5). Calibration lines based on area data were linear from 1 to 1000 microM with r2 values of 0.9998 (Crn), 0.9998 (Met), 0.9999 (Tyr), 0.9999 (His), 1.0000 (Cre), 1.0000 (Phe), and 0.9999 (Trp). The method was applicable to the screening and/or chemical diagnosis of inherited metabolic disorders such as phenylketonuria (PKU), tyrosinemia, and Lowe syndrome. The creatinine ratios of diagnostic markers (microM/microM Cre) were easily determined. The Phe/Cre ratios for five urines from patients with PKU ranged from 0.162 to 0.521, and the Tyr/Cre ratio for tyrosinemia was 0.147. The ratios of Tyr/Cre, Phe/Cre, and Trp/Cre for Lowe syndrome were 0.497, 0.321, and 0.495, respectively. In contrast, the creatinine ratios for healthy newborns showed one digit lower than those for patients did. The developed method is very practical and can provide useful information and results for the clinical or biomedical researches with low analytical run costs.     

Determination of phenylalanine in blood by high-performance anion-exchange chromatography–pulsed amperometric detection to diagnose phenylketonuria

We have developed a high-performance anion-exchange chromatography with pulsed amperometric detection method for the detection of phenylalanine (Phe) and diagnosis of phenylketonuria (PKU). Sample pretreatment steps were simplified without derivatization. The analyte was separated within 5 min. The detection limit (S/N = 3) for Phe was 50 pg. Linear dynamic range was 1.23–14.43 mg/dL (r² = 0.9999) for a dried blood spot. The mean recoveries of Phe for intra- and inter-day assays were found to be 96.87–104.16%. This method clearly differentiated PKU-positive groups from normal groups, and proved to be a practical procedure for rapid screening and follow-up monitoring of PKU.