串联质谱分析干血滤纸片酰基肉碱方法的建立

目的建立串联质谱方法进行干血滤纸片酰基肉碱(acylcamitine)分析，以便应用于临床相关疾病诊治和新生儿遗传代谢病筛查。方法取含不同浓度的8种酰基肉碱的干血滤纸片(直径3mm)，包括乙酰肉碱(C2)、丙酰肉碱(C3)、丁酰肉碱(C4)、异戊酰肉碱(C5)、己酰肉碱(C6)、辛酰肉碱(C8)、肉豆蔻酰肉碱(C14)和棕榈酰肉碱(C16)。用含已知量相应酰基肉碱内标的甲醇萃取、盐酸正丁醇衍生后，用串联质谱仪检测酰基肉碱及其浓度。分析该方法的精确性、样品回收率及实测值与加入浓度之间的相关性。结果酰基肉碱批内变异系数为6．3％-9．9％，批间变异为10．3％-13．3％。低浓度酰基肉碱回收率为98％-106％，中等浓度酰基肉碱回收率为97％-109％，高浓度酰基肉碱回收率为94％-108％。实测浓度与加入浓度之间的相关系数为0．9986—0．9999。结论串联质谱技术进行血滤纸片酰基肉碱分析，有较高的回收率，能够达到较高的精确性和准确性，为临床进行酰基肉碱分析提供了一项新技术。     

串联质谱检测干血滤纸片氨基酸方法的建立

目的建立可靠的串联质谱技术检测干血滤纸片中氨基酸方法,以便用于氨基酸代谢紊乱疾病的筛查和诊断。方法取含不同浓度氨基酸(苯丙氨酸、酪氨酸、亮氨酸、蛋氨酸、瓜氨酸和缬氨酸)的血滤纸片,用含已知量相应氨基酸内标的甲醇萃取,盐酸正丁醇衍生后,用串联质谱仪检测血片中氨基酸谱及其浓度。分析该方法的精确性、样品回收率及浓度相关性。结果氨基酸批内和批间变异系数分别为6．3％～9．7％、9．9％～14．3％。低浓度回收率为92％～100％,中等浓度回收率为94％～104％,高浓度回收率为98％～。107％。不同浓度的氨基酸实测浓度与加入浓度之间的相关系数为0．9990～0．9999。结论串联质谱技术检测干血滤纸片中氨基酸能够达到较高的精确性、准确性和回收率,为临床进行氨基酸分析提供了一项新技术,可用于新生儿遗传代谢病筛查、高危儿童遗传代谢病的诊断及其他与氨基酸代谢有关的疾病诊治。     

串联质谱检测干血滤纸片氨基酸方法的建立

目的　建立可靠的串联质谱技术检测干血滤纸片中氨基酸方法,以便用于氨基酸代谢紊乱疾病的筛查和诊断。方法　取含不同浓度氨基酸(苯丙氨酸、酪氨酸、亮氨酸、蛋氨酸、瓜氨酸和缬氨酸)的血滤纸片,用含已知量相应氨基酸内标的甲醇萃取,盐酸正丁醇衍生后,用串联质谱仪检测血片中氨基酸谱及其浓度。分析该方法的精确性、样品回收率及浓度相关性。结果　氨基酸批内和批间变异系数分别为6. 3% ~9. 7%、9. 9% ~14. 3%。低浓度回收率为92% ~100%,中等浓度回收率为94% ~104%,高浓度回收率为98% ~107%。不同浓度的氨基酸实测浓度与加入浓度之间的相关系数为0. 999 0~0. 999 9。结论　串联质谱技术检测干血滤纸片中氨基酸能够达到较高的精确性、准确性和回收率,为临床进行氨基酸分析提供了一项新技术,可用于新生儿遗传代谢病筛查、高危儿童遗传代谢病的诊断及其他与氨基酸代谢有关的疾病诊治。     

Quantification of total hexose on dry blood spot by tandem mass spectrometry

Background

Because hypoglycemia and hyperglycemia are harmful and not always associated with overt clinical signs, it is necessary to have methods available to screen for glucose levels to detect hypoglycemia and diabetes as early as possible. A new method for such screening and the clinical determination of blood total hexose on a dry blood spot (DBS) using tandem mass spectrometry (MS/MS) was developed.

Methods

The serum glucose controls and blood were prepared as DBS and then extracted into a methanol solution containing isotope-labeled internal standards. The methanolic extraction was subjected to HPLC, followed by MS/MS in positive ion mode. Multiple-reaction monitoring of m/z 203.1 → 23 was used to detect hexose, and m/z 209.0 → 23 was used for 13C6-D-glucose.

Results

The recoveries of blood glucose by MS/MS were 90%–102% with an R² value of 0.999 after linear regression (p < 0.001). The controls were within an acceptable range, and the coefficients of variation were less than 10%. The blood total hexose in neonates aged 3–7 days (6.41 ± 1.46 mmol/L) was lower than that in neonates aged 8–30 days (6.66 ± 1.38 mmol/L), and it was lower in neonates than in children aged 1–72 months (7.19 ± 1.87 mmol/L).

Conclusion

Quantification of total hexose on a dry blood spot by MS/MS is accurate, reliable and feasible for screening and clinical tests.     

电喷雾串联质谱法检测干血斑中谷氨酰胺、谷氨酸及其临床初步应用

目的?建立一种采用电喷雾串联质谱法（ESI-MS／MS）检测干血斑样品中谷氨酰胺（Gln）和谷氨酸（Glu）含量的方法，评价其用于筛查新生儿鸟氨酸氨甲酰转移酶缺乏症和氨甲酰磷酸合成酶缺乏症的可行性。方法?收集2020年6—9月新生儿筛查足跟血干血斑样品，用非衍生化串联质谱检测试剂盒萃取样品，完成常规串联质谱检测项目后，将残余样品作为Gln 和Glu的检测对象，复溶后选择正离子扫描模式进行分析测定，统计分析ESI-MS／MS 检测Gln 和Glu 的正确度、精密度和稳定性，并评价其临床应用。结果?Gln在125～1 250 μmol／L浓度范围内回收率和正确度均良好，回收率为98.01%～107.24%，偏倚为0.56%～4.8%；Glu在250～1 250 μmol／L浓度范围内回收率良好，回收率为102.06%～113.65%，在250～1 250 μmol／L浓度范围内正确度良好，偏倚为0.09%～7.67%。Gln精密度良好，Glu精密度稍差。稳定性实验证实在样品复溶前后，Gln 差异无统计学意义，但Glu差异有统计学意义。结合Gln和Glu检测结果，6份鸟氨酸氨甲酰转移酶缺乏症确诊患儿干血斑样品检出率为100%，并确定Gln的参考区间为＜106.63 μmol／L，Glu 的参考区间为＜188.24 μmol／L。结论?成功建立了检测Gln 和Glu的ESI-MS／MS法，该方法操作简单、成本低，不影响临床常规检测。     

串联质谱法两种扫描方式检测干血滤纸片上氨基酸含量的精密度和准确度

遗传代谢病是指参与体内代谢的某种酶、运载蛋白、膜受体等编码基因发生突变,使其编码的产物功能不良而引发代谢失常的一组疾病[1].许多种类的遗传代谢病已有特效治疗方法,降低病死率及远期神经系统后遗症发生率依赖于早期快速诊断前提下的及早干预治疗[2].国内外的大型实验室主要通过串联质谱技术来辅助临床医师进行遗传代谢病诊断[3-6].     

串联质谱法两种扫描方式检测干血滤纸片上氨基酸含量的精密度和准确度

遗传代谢病是指参与体内代谢的某种酶、运载蛋白、膜受体等编码基因发生突变,使其编码的产物功能不良而引发代谢失常的一组疾病[1].许多种类的遗传代谢病已有特效治疗方法,降低病死率及远期神经系统后遗症发生率依赖于早期快速诊断前提下的及早干预治疗[2].国内外的大型实验室主要通过串联质谱技术来辅助临床医师进行遗传代谢病诊断[3-6].     

串联质谱法两种扫描方式检测干血滤纸片上氨基酸含量的精密度和准确度

遗传代谢病是指参与体内代谢的某种酶、运载蛋白、膜受体等编码基因发生突变,使其编码的产物功能不良而引发代谢失常的一组疾病[1].许多种类的遗传代谢病已有特效治疗方法,降低病死率及远期神经系统后遗症发生率依赖于早期快速诊断前提下的及早干预治疗[2].国内外的大型实验室主要通过串联质谱技术来辅助临床医师进行遗传代谢病诊断[3-6].     

Accurate measurement of free carnitine in dried blood spots by isotope-dilution electrospray tandem mass spectrometry without butylation

BACKGROUND: To test the feasibility of free carnitine (FC) determination in dried blood spot specimens (DBS) by stable isotope-dilution electrospray-ionisation tandem mass spectrometry (MS/MS). METHODS: The MS/MS method established for newborn screening, measuring acylcarnitines by positive precursor ion scan of m/z 85 in DBS, was adapted by omitting the butylation and heating step during sample preparation. FC measurement in DBS by this non-butylating MS/MS assay was compared with the butylating MS/MS method and the spectrophotometric Cobas method. RESULTS: FC measurement by the non-butylating MS/MS method meets the demands for a bioanalytical microassay with respect to linearity, detection limit (LOD), accuracy, and precision. Formation of FC was 0-1% and 1-4% in liquid samples and in DBS by the non-butylating MS/MS method, while 3-10% and 8-16% by the butylating method, respectively. Acid-catalysed hydrolysis (butanolysis) in liquid samples was higher for short-chain acylcarnitines (acetyl- and propionylcarnitine). Hydrolysis in DBS was more pronounced for long-chain acylcarnitines. FC concentrations in healthy newborns without butylation were 35% lower than those measured by the established newborn screening assay. CONCLUSIONS: The non-butylating MS/MS assay provides a simple and accurate method for FC determination in DBS and represents a trivial but important adaptation of a method already used in many laboratories.     

Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency

BACKGROUND: Recently, several patients with abnormal polyol profiles in body fluids have been reported, but the origins of these polyols are unknown. We hypothesized that they are derived from sugar phosphate intermediates of the pentose phosphate pathway (PPP), and we developed a semiquantitative method for profiling of pentose phosphate pathway intermediates. METHODS: Sugar phosphates in blood spots were simultaneously analyzed by liquid chromatography-tandem mass spectrometry using an ion-pair-loaded C(18) HPLC column. The tandem mass spectrometer was operated in the multiple-reaction monitoring mode. Enzymatically prepared D-[(13)C(6)]glucose 6-phosphate was used as internal standard. The method was used to study sugar phosphates abnormalities in a patient affected with a deficiency of transaldolase (TALDO1; EC 2.2.1.2). RESULTS: In control blood spots, dihydroxyacetone phosphate, pentulose 5-phosphates, pentose 5-phosphates, hexose 6-phosphates, and sedoheptulose 7-phosphate were detected. Detection limits ranged from approximately 100 to approximately 500 nmol/L. Glyceraldehyde 3-phosphate and erythrose 4-phosphate were undetectable. Intra- and interassay imprecision (CVs) were 10-17% and 12-21%, respectively. In blood from the TALDO1-deficient patient, sedoheptulose 7-phosphate was increased. CONCLUSIONS: The new method allows investigation of patients in whom a defect in the PPP is suspected. Measurements of sugar phosphate intermediates of the PPP may provide new insights into metabolic defects underlying the accumulating polyols.     

Newborn screening for hepatorenal tyrosinemia by tandem mass spectrometry: analysis of succinylacetone extracted from dried blood spots

OBJECTIVES: To develop a method for the determination of succinylacetone (SA) in dried blood spots (DBS) using tandem mass spectrometry (MS/MS). METHODS: SA was extracted from DBS with an acetonitrile and water solution (80:20 by volume) containing formic acid and hydrazine hydrate (both at 0.1% by volume), and analyzed by MS/MS with a total run time per sample under 2 min. The reference range for SA in newborns was determined by analyzing a control group of 3199 DBS. SA was also measured in stored newborn specimens from three patients diagnosed clinically with hepatorenal tyrosinemia (HT). RESULTS: The within-run precision was 相似文献   

The analysis of dried blood spot samples using liquid chromatography tandem mass spectrometry

Assays using dried blood samples have been in use for a number of years particularly for the diagnosis of inborn errors of metabolism. This sampling technique has been greatly helped by the sensitivity and specificity offered by liquid chromatography tandem mass spectrometry because of the limited amount of sample available for analysis. There are advantages for patients to take samples at home but these must be weighed against potential problems with the sampling technique. Some assays appear to work very well whilst others suffer from poor recovery because of adsorption of the analyte onto the filter paper. Some newer strategies including impregnation of the filter paper and filter filtration will be discussed. Calibration of assays and the problems with matrix matching of standards are also important issues that need to be addressed before an assay can be used routinely.     

Simultaneous determination of fluoxetine and norfluoxetine in dried blood spots using high-performance liquid chromatography-tandem mass spectrometry

BackgroundTherapeutic drug monitoring (TDM) of the widely prescribed antidepressant fluoxetine (FLU) is recommended in certain situations, such as occurrence of toxicity, inadequate response or suspect of poor adherence. Dried blood spot (DBS) sampling is an increasingly studied alternative for TDM, particularly for outpatients, due to its ease of collection and inherent stability.ObjectivesThe aim of this study was to develop and validate an LC-MS/MS assay for the simultaneous quantification of FLU and norfluoxetine (NFLU) in DBS.Design and methodsThe assay is based on a liquid extraction of single DBS with 8 mm of diameter, using FLU-D6 as the internal standard, followed by reversed phase separation in an Accucore® C18 column (100 × 2.1 mm, 2.6 μm). Mobile phase was composed of water and acetonitrile (gradient from 80:20 to 50:50, v/v), both containing formic acid 0.1%. The assay was validated and applied to 30 patients under FLU pharmacotherapy.ResultsThe assay was linear in the range 10–750 ng mL^− 1. Precision assays presented CV% of 3.13–9.61 and 3.54–7.99 for FLU and NFLU, respectively, and accuracy in the range of 97.98–110.44% and 100.25–105.8%. FLU and NFLU were stable at 25 and 45 °C for 7 days. The assay was evaluated in 30 patients under FLU treatment. Concentrations of both compounds were higher in DBS than in plasma, and the use of the multiplying factors 0.71 and 0.68 for FLU and NFLU, respectively, allowed acceptable estimation of plasma concentrations, with median prediction bias of − 0.55 to 0.55% and mean differences of 0.4 to 2.2 ng mL^− 1.ConclusionsThe presented data support the clinical use of DBS for therapeutic drug monitoring of FLU.     

Newborn screening for Fabry disease by measuring GLA activity using tandem mass spectrometry

BackgroundFabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficiency of α-galactosidase A (GLA). We evaluated a tandem mass spectrometry method to measure GLA activity.MethodsOne 3.2 mm punch from a dried blood spot sample (DBS) was incubated with substrate and internal standard in the reaction buffer for 22 h. The resulting product was quantified against internal standard using MS/MS.ResultsThe median GLA activity of male newborn DBS (N = 5025) was 9.85 ± 6.4 µmol/h/l (CI 95% is 9.67–10.02 µmol/h/l); The median GLA activity of female newborns (N = 4677) was 10.2 ± 6.3 µmol/h/l (CI 95% is 10.02–10.38 µmol/h/l). The difference between the two subgroups is within assay analytical variation. The GLA activities in the DBS samples from 9 juvenile and adult males with previously identified FD were below 1.64 µmol/h/l. The GLA activities from 32 juvenile and adult females with confirmed FD were below 4.73 µmol/h/l. In 5 (16%) females GLA activities were above the 0.5th percentile of lower limit of CI 95% at 3.18 µmol/h/l.ConclusionsThe MS/MS method for Fabry disease newborn screening is robust and can be readily multiplexed with other lysosomal disorders such as Pompe, Gaucher, Niemann–Pick, and Krabbe diseases.