3-years experience review of neonatal screening for hemoglobin disorders using tandem mass spectrometry

BackgroundNeonatal screening programs for sickle cell disease are common in North America and in some European countries. Isoelectric Focusing or High Performance Liquid Chromatography is the main technique used for hemoglobin variant detection.MethodsSince tandem mass spectrometry is being used for screening of inherited metabolic disorders and allows protein identification, we had developed an application to identify the most relevant hemoglobin mutations with this technology.ResultsThis approach had been previously validated and has been routinely applied in our laboratory for the last three years. We report here our experience with this new method in the field, applied to our East-Belgian population.ConclusionsTo conclude, mass spectrometry provides an efficient alternative approach for laboratories performing neonatal screening of hemoglobin disorders.     

High-performance liquid chromatography/mass spectrometry: state of the art for the drug analysis laboratory

The combination of HPLC and mass spectrometry has great promise for the toxicology laboratory. In the past five years, significant progress has been made toward producing a reliable interface for these techniques. Thermospray, liquid ion evaporation, ion-spray, and the particle beam separator are all viable "second generation" approaches with significant advantages and disadvantages. I review the operation of these interfaces with an orientation to their use in the drug analysis laboratory. Thermospray, ion-spray, and liquid ion evaporation primarily supply information about molecular mass. The benefits and limitations of using tandem mass spectrometry to obtain additional structural information will also be discussed.     

Tandem mass spectrometry in newborn screening: a primer for neonatal and perinatal nurses

Since 1961, newborn screening for errors of metabolism (EM) has improved the diagnosis, treatment and outcome of newborns with an EM. Recently, advances in laboratory technology with tandem mass spectrometry (MS/MS) has increased the identification of newborns with an EM. With a single dried filter paper blood spot (Guthrie R, Susi A. A simple phenylalanine method for detecting PKU in large populations of newborn infants. Pediatrics. 1963;32:338-343), MS/MS can identify more than 30 disorders of metabolism. This review will explore MS/MS to provide a better understanding of the development and application of this technology to newborn screening for perinatal and neonatal nurses.     

Isolation and structural identification of 9hydroxy-9desmethyl-cyclosporine

A metabolite of cyclosporine has been isolated and its structure identified through use of HPLC and tandem mass spectroscopy. Fast atom bombardment mass spectrometry of an HPLC fraction co-eluting with 1 eta hydroxy-cyclosporine (M17) indicated that the mass of this metabolite was 2 Da greater than that of cyclosporine. Further isolation by HPLC yielded a pure fraction, which we analyzed with tandem mass spectrometry. Linear acyl fragment ions originating from the metabolite under collision-induced dissociation were consistent with the difference in mass being associated with amino acid 9 in the cyclosporine backbone. We propose a nomenclature system for future discussion of cyclosporine metabolites.     

A decade of HPLC-MS/MS in the routine clinical laboratory--goals for further developments

During the past decade, tandem mass spectrometry hyphenated to liquid chromatography separation systems (HPLC-MS/MS) has developed to an important technology in clinical chemistry — not only for research purposes but also for routine use. At present, most important application fields are target analyses in therapeutic drug monitoring (TDM) and metabolic disorders diagnosis. The essential strengths of HPLC-MS/MS include potentially high analytical specificity, wide range of applicability to small and large molecules, capability of multi- and mega-parametric tests, and the opportunity to develop powerful assays with a high degree of flexibility within a short time frame. The technique has overcome important limitations of GC-MS and is characterized by short analytical runtimes, applicability to thermo labile, polar and large molecules, and straightforward sample preparation. However, implementation of HPLC-MS/MS assays still requires substantial expertise and know-how. At the present, its application is limited to a rather small number of clinical routine laboratories. Nonetheless, HPLC-MS/MS has the potential to be further developed to a commonly applied high-throughput technique in clinical chemistry, complementary to present standard techniques as photometry and ligand binding methods. This review intends to characterize working characteristics of present day HPLC-MS/MS instrumentations used in clinical routine laboratories. Limitations of currently available systems and applications will be critically discussed. Required instrument improvements supporting the successful spreading of HPLC-MS/MS in laboratory medicine within the next decade will be outlined.     

基于质谱的蛋白质组学诊断所面临的挑战

过去的几年中,基于质谱技术的蛋白质组学分析方法已经成为寻找新的疾病标志物的重要手段之一.由于它具有高灵敏度、高通量、快速以及能和生物信息学技术结合同时快速分析大量的蛋白质或多肽的优点,使之成为临床研究的有力工具.然而,最近的一些研究显示,因为这项技术的重复性和数据统计分析等方面的不足而受到了争议.本文介绍了采用该方法进行标志物筛查时在实验设计、质谱技术以及数据分析等方面面临的一些挑战,探讨其在血清或血浆分析中的应用.     

Expanded newborn screening of inherited metabolic disorders by tandem mass spectrometry: clinical and laboratory aspects

Newborn screening started in the 1960s for the purpose of identifying phenylketonuric patients to begin early intervention and to prevent mental retardation in these patients. Soon thereafter, screening programs expanded to include additional genetic disorders added individually one at a time. In the 1980s, tandem mass spectrometry (MS/MS) was introduced in clinical laboratories, and in the 1990s, the technique was used for newborn screening. Unlike measuring one analyte at a time, MS/MS allows measurement of >40 analytes, in a few minutes with the use of a single assay. Currently, MS/MS is being used for the identification of several amino acid, organic acid and fatty acid disorders. Several states in the United States and many other countries are using MS/MS in newborn screening. However, there is a significant disparity among different newborn screening programs for disorders being screened by MS/MS and many other challenges are faced by the expanded newborn screening. It is anticipated that in the future the use of MS/MS in newborn screening will expand both at the analyte and geographic levels. Clinicians and laboratory scientists should become familiar with MS/MS, disorders being screened in their patients' population and the future of this emerging technology.     

Recent advances of liquid chromatography–(tandem) mass spectrometry in clinical and forensic toxicology

Liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) has become increasingly important in clinical and forensic toxicology as well as doping control and is now a robust and reliable technique for routine analysis in these fields. In recent years, methods for LC–MS(/MS)-based systematic toxicological analysis using triple quadrupole or ion trap instruments have been considerably improved and a new screening approach based on high-resolution MS analysis using benchtop time-of-flight MS instruments has been developed. Moreover, many applications for so-called multi-target screening and/or quantification of drugs, poisons, and or their metabolites in various biomatrices have been published. The present paper will provide an overview and discuss these recent developments focusing on the literature published after 2006.     

19例cblC型甲基丙二酸血症患儿临床资料及基因型分析

目的 探讨cblC型甲基丙二酸血症患儿的临床特点、实验室检查及基因型的关系,为早期诊断提供思路与方法.方法 收集2015-2020年河北医科大学第二医院小儿内分泌与生长发育科诊治的甲基丙二酸血症患儿19例,对其临床表现、基因结果、串联质谱分析及影像学检查等资料进行回顾性分析.结果 19例中男性10例,女性9例,8例通过...     

Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories

Liquid chromatography tandem mass spectrometry is one of the most specific techniques available in clinical laboratories. In the past, immunoassays were the primary methodology for analysis of steroids in biological samples because they are rapid and easy to perform. However, these methods were shown to suffer from the lack of specificity for measuring many of the diagnostically important steroids. LC-MS/MS overcomes many of the limitations of immunoassays, enhances diagnostic utility of the testing, and expands diagnostic capabilities in endocrinology. In addition to the superior quality of the measurements, LC-MS/MS allows high throughput testing using small sample volume with minimal sample preparation, and frees the laboratory from dependence on suppliers of assay specific reagents. LC-MS/MS is being widely employed for routine measurement of steroids, and the methodology plays an important role in the standardization and harmonization of measurements among clinical laboratories.     

Testosterone measurement by isotope-dilution liquid chromatography-tandem mass spectrometry: validation of a method for routine clinical practice

BACKGROUND: Immunoassay is unsatisfactory for measuring the testosterone concentrations typically found in women. Bench-top tandem mass spectrometers are a viable alternative technology for measurements in the clinical laboratory. METHODS: We used stable-isotope dilution liquid chromatography-tandem mass spectrometry (ID/LC-MS/MS) to measure testosterone in plasma and serum. The sample volume was 50 muL in duplicate; preparation and analysis were carried out in a single tube, and a batch of 192 tubes was analyzed in 17.5 h. RESULTS: Intra- and interassay imprecision was <15% in the range 0.3-49 nmol/L. Recovery of testosterone added to samples at concentrations of 0.625-20 nmol/L was 96% (CV = 12%; n = 26). Six samples were serially diluted with double charcoal-stripped serum to demonstrate linearity. Correlation (r(2)) with isotope-dilution gas chromatography-mass spectrometry for 20 pools of clinical samples (range, 0.5-38.5 nmol/L) was 0.99. Correlations with our extraction RIA were 0.97 for clinical samples from men (range, 8-46.3 nmol/L) and 0.66 for samples from women (range, 0.7-3.0 nmol/L), but were 0.35 for male samples containing <3 nmol/L testosterone and 0.77 for female samples containing >8 nmol/L. Various steroids added to double charcoal-stripped serum showed no interference at the retention time of the testosterone peak. CONCLUSIONS: The ID/LC-MS/MS method has improved accuracy compared with immunoassay. The low sample volume and simplicity, rapidity, and robustness of the method make it suitable for use as a high-throughput assay in routine clinical biochemistry laboratories.     

Procedural overview of molecular genetics technology in the clinical laboratory.

Basic concepts of molecular technology and diagnostic applications in the clinical laboratory are discussed. Advances in molecular biology have led to an increased number of clinical laboratories providing diagnostic support at the genetic level. The clinical applications of such a technology at present is broad, perhaps limited only by the regions mapped on the human genome. As the rapid discovery of new genes ignites a chain reaction of opportunities, the clinical laboratory scientist should be kept informed of the exciting technologic advances in the diagnosis of infectious diseases, genetic-related diseases, and leukemia and lymphomas. Many future diagnostic applications are now being researched.     

AANA Journal course: new technologies in anesthesia: update for nurse anesthetists--mass spectrometry in anesthesia

Despite the development of mass spectrometry in the early 1900's, it has only in this decade been introduced into the clinical anesthesia arena. The mass spectrometer's ability to make simultaneous measurements of specific gases distinguish it from currently available monitoring apparatus. This article will review the basic principle of operation of the mass spectrometer. The cost of implementation of mass spectrometry, clinical applications and potential pitfalls associated with its use will also be discussed.     

Proteomic applications in pathology and laboratory medicine: Present state and future prospects

Clinical mass spectrometry applications have traditionally focused on small molecules, particularly in the areas of therapeutic drug monitoring, toxicology, and measurement of endogenous and exogenous steroids. More recently, the use of matrix assisted laser desorption/ionization time of flight mass spectrometry for the identification of microbial pathogens has been widely implemented. Following this evolution, there has been an expanding role for the measurement of peptides and proteins in pathology and laboratory medicine. This review explores the current state of protein measurement by clinical mass spectrometry and the analytical strategies employed, as well as emerging applications in clinical chemistry, clinical microbiology and anatomical pathology.     

Evaluation of an immunoassay of whole blood sirolimus in pediatric transplant patients in comparison with high-performance liquid chromatography/tandem mass spectrometry.

BACKGROUND: Sirolimus is widely used as an immunosuppressant, along with calcineurin inhibitors. Because of its variable pharmacokinetics and narrow therapeutic range, therapeutic drug monitoring of sirolimus is critical to optimize its therapeutic effect and to minimize toxicity. Although liquid chromatography/tandem mass spectrometry is considered the method of choice, the technical and financial challenges of this method are obstacles to its use. A microparticle enzyme immunoassay on the Abbott IMx has recently been reintroduced to the clinical diagnostic market. METHODS: We evaluated this immunoassay using high-performance liquid chromatography/tandem mass spectrometry as the reference method. Precision and carryover were evaluated using an expanded CLSI EP10-A2 protocol. Linearity was studied by serial dilution of high-level whole blood samples, and clinical utility was demonstrated by correlation with the reference method using 56 de-identified pediatric patient samples. RESULTS: The total imprecision was less than 12% across the concentrations tested. The method was linear from 2.6 to 31 nM. The immunoassay showed a mean positive bias of 11.5% in patient specimens relative to high-performance liquid chromatography/mass spectrometry (p<0.001), with a correlation coefficient (R) of 0.953. CONCLUSION: We conclude that the reintroduced immunoassay is useful for therapeutic drug monitoring of sirolimus.     

High-throughput urine screening for Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis using negative electrospray tandem mass spectrometry

BACKGROUND: Smith-Lemli-Opitz syndrome (SLOS) and cerebrotendinous xanthomatosis (CTX) are disorders affecting cholesterol metabolism. Currently, diagnosis relies on clinical recognition and specific and complex biochemical testing. METHODS: A rapid, high-throughput urine test, suitable for mass screening for these two disorders, was developed using flow injection negative electrospray tandem mass spectrometry with multiple reaction monitoring. Cholestane-pentol glucuronide, a known marker for CTX, was measured and a steroid sulfate with a proposed keto-pregnadien-diol structure was identified and measured for SLOS. Measurement of the two markers was readily incorporated into an existing tandem mass spectrometry method for diagnosing inborn errors of amino and organic acid metabolism. RESULTS: Levels in affected patients were well separated from 1738 controls, ranging from 6.7 to 100 times the 99.7th percentile of controls in SLOS patients (n=3) and 7.3 to 24 times the 99.7th percentile of controls in CTX patients (n=4). CONCLUSIONS: The addition of testing for SLOS and CTX to a routine tandem mass spectrometry urine screening program simplifies the diagnosis of these two disorders and further extends the range of inborn errors of metabolism detected by this technique.     

Proteomics: the next revolution in laboratory medicine?

BACKGROUND: The identification of specific genetic alterations and protein profiles associated with disease offers a unique opportunity to develop proteomics-based assays for early diagnosis. By identifying proteins in serum/plasma, a minimally invasive tool is used to assess the presence of disease and to monitor response to treatment and/or disease progression. The potential clinical applications of this tool are broad-based, including the diagnosis not only of cancer but also cardiovascular and neuromuscular diseases, organ transplantation associated conditions, and infertility. METHODS: A number of competing chromatographic techniques have been proposed for overcoming the complexity and labor-intensive manipulations associated with the traditional technique for proteomic analysis, which is based on two-dimensional gel electrophoretic techniques. However, mass spectrometry has now assumed a central role in most proteomic workflows, and several combinations of ionization sources, analyzers and fragmentations devices have been described and developed. RESULTS: Thanks to proteomic applications in the diagnosis of cancer, several research groups have identified proteomic patterns associated with ovarian, prostatic, colorectal and other cancers. While the sensitivity and specificity of these patterns are highly satisfactory, there are still some open questions concerning the standardization, reproducibility, and inter-laboratory agreement of these data. CONCLUSIONS: Proteomics, and, in particular, serum mass spectroscopic proteomic pattern diagnostics, is a rapid expanding field of research. The plasma proteoma has an important position at the intersection between genes and diseases, and clinical laboratories must adapt to a new era of tests based on proteomics and genomics. In the future, mass spectrometry will become an essential tool in the clinical laboratory.     

Development of a method for rapid quantitation of amino acids by liquid chromatography-tandem mass spectrometry (LC-MSMS) in plasma.

A new analytical method has been developed and is proposed for the rapid determination of eighteen common amino acids, including tryptophan, in plasma and dried blood spots, by liquid chromatography coupled with ionspray tandem mass spectrometry. Potentially the method can include other amino acids and can be used for the diagnosis of metabolic disease. The use of the ionspray tandem mass spectrometry approach permits extremely rapid chromatographic separation of all amino acids requiring less than four minutes for the analysis of each sample, after a simple sample preparation procedure. The chromatographic separation of the analytes was achieved using a CN normal phase column and a water/acetonitrile/trifluoroacetic acid mobile phase at flow rate of 1 ml/min. The mass spectrometer was operated in multiple reaction monitoring mode, where each analyte had its own unique precursor and product ion setting. The quantitative analysis of amino acids was achieved using as internal standards just two representative isotopically labeled amino acids: D4-Ala and D5-Phe. Calibration is made externally by using aqueous solutions with the same labelled amino acids as internal standards. The high specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for the rapid and reliable assay of metabolically significant amino acids. The liquid chromatography-tandem mass spectrometry method is more effective than other published tandem mass spectrometry methods at distinguishing isobaric amino acids like Leu, Ile and HO-Pro and certainly far more rapid than HPLC or ion-exchange chromatographic methods.     

Liquid chromatography-tandem mass spectrometry--application in the clinical laboratory.

This review provides a concise survey of liquid chromatography-tandem mass spectrometry (LC-TMS) as an emerging technology in clinical chemistry. The combination of two mass spectrometers with an interposed collision cell characterizes LC-TMS as an analytical technology on its own and not just as a more specific detector for HPLC compared with conventional techniques. In LC-TMS, liquid chromatography is rather used for sample preparation but not for complete resolution of compounds of interest. The instrument technology of LC-TMS is complex and comparatively expensive; however, in routine use, methods are far more rugged compared to conventional chromatographic techniques and enable high-throughput analyses with very limited manual handling steps. Moreover, compared to both gas chromatography-mass spectrometry (GC-MS) and conventional HPLC techniques, LC-TMS is substantially more versatile with respect to the spectrum of analyzable compounds. For these reasons it is likely that LC-TMS will gain far more widespread use in the clinical laboratory than HPLC and GC-MS ever did. In this article, the key features of LC-TMS are described, method development is explained, typical fields of application are discussed, and personal experiences are related.