Deproteination of whole blood for LC–MS/MS using paramagnetic micro-particles

Objectives

Liquid chromatography tandem mass spectrometry has become increasingly popular in clinical laboratories over the last decade due to the inherent sensitivity and specificity of the technology. Nevertheless, full automation and hence application in routine laboratories is still hampered by laborious and difficult-to-automate sample pre-treatment protocols. Functionalized paramagnetic micro-particles could simplify sample pre-treatment and ease automation. We evaluated the applicability of a pre-commercial, straightforward paramagnetic micro-particle based kit for the lysis and deproteination of whole blood for the LC–MS/MS analysis of everolimus and compared the performance to our routine protein precipitation method.

Design and methods

Samples were prepared for LC–MS/MS everolimus analysis on an Acquity UPLC chromatographic system coupled to a TQD mass spectrometer (both Waters Ltd.) using a pre-commercial MagSi-TDMprep kit and a routine protein precipitation respectively. Both pre-treatment methods were validated for imprecision, accuracy, linearity, limit of quantification, matrix effect, recovery and process efficiency. A method comparison (n = 63) between both pre-treatment methods was performed.

Results

Imprecision and bias were within pre-defined criteria (15%) for both pre-treatment methods. Both methods were linear from 1.2 to 14.8 μg/L with a limit of quantification of 0.6 μg/L. Process efficiency was on average 65% for protein precipitation pre-treatment and was substantially higher for the MagSi-TDMprep method (85%). A Passing–Bablok regression showed no significant difference between the two pre-treatment methods.

Conclusion

For everolimus in whole blood, the MagSi-TDMprep sample pre-treatment was applicable and comparable to protein precipitation for LC–MS/MS with the possible advantage of easier automation.     

After another decade: LC–MS/MS became routine in clinical diagnostics

Tandem mass spectrometry – especially in combination with liquid chromatography (LC–MS/MS) – is applied in a multitude of important diagnostic niches of laboratory medicine. It is unquestioned in its routine use and is often unreplaceable by alternative technologies. This overview illustrates the development in the past decade (2009–2019) and intends to provide insight into the current standing and future directions of the field. The instrumentation matured significantly, the applications are well understood, and the in vitro diagnostics (IVD) industry is shaping the market by providing assay kits, certified instruments, and the first laboratory automated LC–MS/MS instruments as an analytical core. In many settings the application of LC–MS/MS is still burdensome with locally lab developed test (LDT) designs relying on highly specialized staff. The current routine applications cover a wide range of analytes in therapeutic drug monitoring, endocrinology including newborn screening, and toxicology. The tasks that remain to be mastered are, for example, the quantification of proteins by means of LC–MS/MS and the transition from targeted to untargeted omics approaches relying on pattern recognition/pattern discrimination as a key technology for the establishment of diagnostic decisions.     

Deproteination of serum samples for LC–MS/MS analyses by applying magnetic micro-particles

ObjectivesInvestigation of the practicability and performance of a magnetic micro-particle based method for protein depletion of serum samples, preceding the quantitative analysis of small molecules by LC–MS/MS.Design and methodsA commercially available kit including a protein denaturation reagent and functionalized magnetic particles together with a magnetic separator device was tested by addressing the quantification of amiodarone in serum as an exemplary analyte by LC–MS/MS with on-line SPE. A standard method validation protocol was applied.ResultsThe sample preparation protocol was found to be convenient, straightforward and robust. Validation data characterized the entire analytical method – combining particle-based protein depletion and two-dimensional chromatography – as compatible with the analytical needs regarding selectivity, accuracy (102–106%), linearity (r² ≥ 0.99), reproducibility (CV < 7%), and control of ion suppression.ConclusionsSince this novel approach of sample preparation does neither require centrifugation nor the technically demanding application of positive or negative pressure, as in conventional solid phase extraction protocols, it seems highly attractive for developing fully automatized preparation systems for LC–MS/MS analyzers.     

Retrospective evaluation of therapeutic drug monitoring of clozapine and norclozapine in Belgium using a multidrug UHPLC–MS/MS method

ObjectiveClozapine is an atypical antipsychotic with a narrow therapeutic range and serious toxic side effects. According to AGNP–TDM consensus guidelines, therapeutic drug monitoring (TDM) of clozapine and its metabolite norclozapine is strongly recommended. 330 serum samples, sent to the toxicological laboratory of Ziekenhuis Netwerk Antwerpen for monitoring of clozapine, were tested with a new ultra-high performance liquid chromatography–tandem mass spectrometric method (UHPLC–MS/MS). The aim of this research was to evaluate this method for TDM of clozapine and norclozapine, but also to determine other antipsychotics present in these serum samples.Design and methodsSerum samples were taken just prior to the morning dose of the antipsychotic (trough concentration). All samples were, after a simple liquid–liquid extraction with methyl t-butylether, analyzed using a fully validated UHPLC–MS/MS method which is able to quantitate 16 different antipsychotics and 8 of their major metabolites. Serum concentrations were compared with the therapeutic ranges as defined by the AGNP–TDM guidelines.ResultsFor clozapine, only 22.3% of the serum concentrations were within the therapeutic range of 350–600 ng/mL, while 67.9% of the concentrations were below 350 ng/mL. For norclozapine, 68.2% of the serum samples were within the therapeutic range of 100–600 ng/mL. The mean clozapine:norclozapine ratio was 1.7 (SD 0.8). 218 of the 330 serum samples contained other antipsychotics than clozapine. Only 52.5% of these concentrations were within the proposed range.ConclusionThis retrospective study highlights the importance of TDM for clozapine and other APs, since many patients show suboptimal serum concentrations.     

UPLC–MS and Dereplication-Based Identification of Metabolites in Antifungal Extracts of Fungal Endophytes

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Microbial biopesticides offer an eco-friendly alternative to synthetic chemical pesticides. Bioassay-guided...     

断肠草中毒的GC—MS—MS检测方法

断肠草为马钱科胡蔓藤属植物，该植物产于亚洲和北美，我国主要分布于浙、闽、粤、滇、黔、桂等地的灌木丛中。民间俗名繁多，常称有钩吻、大茶叶、大茶药、胡蔓藤、大炮叶、黄藤根、吻荞、野葛、冶葛、胡蔓草、烂肠草、虎狼草等。     

LHPC—MS／MS法测定人体血浆中丙泊酚浓度的评价

【目的】建立人血浆中丙泊酚浓度的高效液相色谱串联质谱法（HPLC—MS／MS）测定方法,以评价其效果。【方法】采用LC—MS／MS法测定丙泊酚的浓度,色谱柱为Zorbax Eclipse XDB-C18（50mm×4．6mm,5μm）,流动相为甲醇及0．1％氨水溶液;流速0．4mL／min;电喷雾离子化电离源（ESI）,质谱采用MRM模式,以负离子检测,检测离子为丙泊酚m／z177．2→m／z161．4、麝香草酚m／Z147．2→m／z106．7。20名健康志愿者静脉注射丙泊酚2mg／kg后,按预定时间点抽取动脉血进行血浆药物浓度的检测。【结果】丙泊酚血药浓度在线性范围分别为0．012～12．06μg／mI。范围内线性关系良好（r=0．9993）,定量下限为0．012μg／mL;低、中、高3个浓度的日内及日间相对精密度（RSD）〈15％;平均提取回收率86．7％,RSDl．87％。【结论】本方法特异性强、灵敏、高效、精密度及准确度好,适用于人体血浆中丙泊酚的检测。     

国产MALDI-TOF MS系统microTyper MS对革兰阴性菌的鉴定性能评估

目的 评估国产基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)系统microTyper MS鉴定革兰阴性菌的性能.方法 使用国产MALDI-TOF MS系统microTyper MS与进口质谱系统VITEK MS系统同步鉴定该实验室储存的标准菌株及2018年6月至2019年6月临床分离的革兰阴性菌株.鉴定结果不一致时,使用表型试验或16 s rDNA测序进行确认,以此评估microTyper MS对革兰阴性菌的鉴定能力.结果 纳入鉴定的革兰阴性菌株一共45属4509株.VITEK MS系统准确鉴定4419株,准确率为98.0％;microTyper MS准确鉴定4405株,准确率为97.7％.结论 microTyper MS在革兰阴性菌鉴定方面具有与VITEK MS系统相仿的临床细菌鉴定能力.     

MS通道与感觉神经元

各型离子通道是神经系统电兴奋的基础，神经电活动的产生决定于跨膜离子通道的组成和分布特性。离子通道可分为电压门控性（voltage-gated ion channels）、配体门控性（ligand—gated ion channels）和机械敏感性离子通道（mechanosensitive ion channels，MS通道）三种，目前国内外对于电压和配体门控性离子通道的研究较为集中，但有关感觉神经元在生理及病理状态下MS通道功能的研究较少，MS通道在各种躯体感觉，尤其是伤害性信息传人中的作用尚未深入阐明。本文将对MS通道的生理学特性及其在初级感觉神经元各种躯体感觉和伤害性感觉传导中的机制、作用进行综述。     

Tentative Identification of Bioactive Compounds From Aqueous Extract of Punica granatum Peel by HPLC/LC–MS Method

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - The objective of this study was to carry out the phytochemical screening, antibacterial activity in different...     

Vitek MS与Bruker MS对希木龙念珠菌复合体鉴定能力评估

目的评估中国医院侵袭性真菌监测网(CHIF-NET)2010-2015年收集的希木龙念珠菌复合体原始鉴定、Vitek MS和Bruker MS基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)鉴定结果。方法收集CHIF-NET 2010-2015年40株希木龙念珠菌和5株假希木龙念珠菌,所有菌株均经过分子生物学方法进行复核鉴定确认。以复核鉴定结果作为"金标准",评估各医院原始常规方法鉴定结果,并评估Vitek MS和Bruker MS对希木龙念珠菌复合体的鉴定性能。结果与分子生物学复核鉴定结果相比,希木龙念珠菌的原始鉴定准确率为47.5%,分别有15%的菌株被显色培养,ATB Rapid ID32C,API 20C或Vitek 2Compact错误鉴定为奥默毕赤酵母和新型隐球菌;常规鉴定方法无法鉴定假希木龙念珠菌,其中3株被错误鉴定为希木龙念珠菌;与分子生物学鉴定结果相比,Vitek MS IVD2.0和Bruker MS DB 5 989对希木龙念珠菌的鉴定准确率分别为100.0%和57.5%。Vitek MS IVD 2.0数据库中无假希木龙念珠菌的参考谱图,故将5株菌全部错误鉴定为希木龙念珠菌,且可信度90%。Vitek MS RUO数据库中无希木龙念珠菌复合体参考谱图,故无鉴定结果。Bruker MS DB 5989对假希木龙念珠菌的菌种鉴定准确率为20%。Bruker MS自建库后对所有菌株均能正确鉴定到种水平。结论该研究率先在国内评估了Vitek MS与Bruker MS鉴定希木龙念珠菌复合体的性能,对于少见菌种的鉴定可通过增加数据库质谱谱图提高质谱的鉴定能力。     

ID-LC/MS/MS方法测量血清未结合雌三醇的协作研究

摘要：目的用同位素稀释液相色谱串联质谱（ID-LC/MS/MS）候选参考方法进行实验室间血清未结合雌三醇（uE3）协作研究,通过该研究进一步确认方法的性能特征（正确度和精密度）,并将该方法在国内参考实验室推广,推动uE3标准化进程。方法依据ISO/WD 15725-1和GB/T 6379标准,拟定我国血清uE3协作研究方案。协作研究分为两个阶段：初步试验和正式试验,共9家参考实验室参加。按要求收集5个浓度水平样品并进行均匀性评价,分发到各实验室,每个样品重复测量5次,连续测量3天。计算各实验室测量结果的偏移和精密度。测量结果采用格拉布斯（Grubbs）检验和柯克伦（Cochran）检验,识别离群值和离群实验室。根据合格实验室的结果计算靶值,并向离群实验室和偏出允许范围的实验室提供整改建议。结果（1）样品均匀性评价,所有样品测量结果计算F值均小于F0.05（9, 20）临界值,样品中uE3是均匀的。（2）初步试验：Grubbs检验,1个实验室测量结果为离群值;Cochran检验2个实验室检验结果为离群值。剔除离群值后计算靶值,分别为2017E301:（22.08&#177;0.24）nmol/L;2017E302：（33.46&#177;1.67）nmol/L。2个实验室结果超出允许范围。（3）正式试验：Grubbs检验,所有实验室测量结果均未检出离群值。Cochran检验3个实验室数据出现离群值。剔除离群值后计算靶值,分别为2017E303:（10.36&#177;0.35）nmol/L;2017E304:（15.47&#177;0.26）nmol/L;2017E305:（46.97&#177;1.19）nmol/L。;各实验室间测量结果不精密度分别为（1.14%～2.21%,0.79%～1.93%,0.60%～2.09%）,测量结果偏移分别为（-6.18%～4.83%,-2.26%～2.39%,-4.19%～4.07%）。结论通过组织开展协作研究,确认了各参考实验室通过协作研究方案能够快速建立并运行uE3候选参考方法,除个别实验室外各实验室的检测性能满足预定要求（不精密度&lt;3.0%,偏移&lt;7.5%）,进一步确认了该方法的性能和各实验室运行参考方法的能力。通过初步研究和正式研究对研究方案的各实验环节进行了验证和完善;推动了uE3项目参考方法在我国参考实验室的运行,为后续厂商试剂主校准品联合定值或标准物质研制搭建了平台,促进uE3项目的标准化。     

背根神经节上的MS通道

机械敏感性离子通道（mechanosensitive ion channels，MS通道）是一类随细胞膜张力变化而开放概率呈相应变化的通道，可以与细胞骨架相连，感受细胞膜张力及细胞外渗透压的变化。作为一种有生理意义的机械信号转导体，MS通道在生物体的生长、发育、感受内外界环境变化中起重要作用。研究证实，脊椎动物毛细胞、压力感受器、血管内皮细胞、成骨细胞及肾脏、肺等器官的某些细胞中均有这类通道的分布。由于实验条件及研究技术等原因，人们对神经组织上MS通道的研究起步较晚。     

自建MALDI-TOF MS霉菌数据库的研究

目的建立基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)霉菌数据库,提高引起临床相关感染的各种霉菌的鉴定能力。方法收集临床标本来源的霉菌266株,纯培养后进行霉菌18S rDNA扩增测序,获得菌种鉴定结果。选取210株霉菌用于数据库的构建,通过甲酸萃取法提取蛋白,应用MALDI-TOF MS配套的FlexControl软件收集蛋白谱图,采用Biotyper软件构建评价数据库。其余56株霉菌用于数据库鉴定能力的验证,分别用商品化数据库、自建的MALDI-TOF MS数据库和扩展数据库进行菌种鉴定,应用χ2检验比较数据库的鉴定率差异。结果自建本地化MALDI-TOF MS霉菌鉴定数据库包含19个菌属,67个菌种,共计204株霉菌的质谱图。用于数据库验证的56株霉菌中,用商品化数据库鉴定正确18株,鉴定正确率为32.1%,用扩展数据库鉴定正确51株,鉴定正确率为91.1%,差异有统计学意义(P<0.05)。结论自建霉菌数据库提高了霉菌的鉴定能力,从而有助于临床霉菌感染的病原诊断和治疗。