Measurement of free urinary cortisol and cortisone using liquid chromatography associated with tandem mass spectrometry method

Free urinary cortisol (UFF) measurement is one of the most useful screening tests for Cushing's syndrome. Immunoassays employed today by most clinical laboratories present limitations, specially concerning specificity. These limitations restrain a widespread application of the method, as well as the comparison of results obtained by the use of different methods. We present the development and characterization of a UFF and cortisone method based on liquid chromatography and tandem mass spectrometry (LC-MS/MS). A 200 microL aliquot from a 24 h urine sample is mixed with a solution containing a known quantity of deuterated cortisol and on-line extracted in solid phase (C18). The eluate is transferred to a second C18 column (Phenomenex Luna, 3 micro, 50 x 2 mm) and the isocratic mode elution profile is directly applied to a tandem mass spectrometer model Quattro Micro operating in positive mode atmospheric pressure chemical ionization (APCI). All process is automated and the quantification is performed by isotopic dilution, based on the analyte and the deuterated internal standard peak area ratios. The specificity study showed that all the steroids tested presented cross reactivity of <1% for cortisol and cortisone. Functional sensitivity is <1 microg/L for both steroids, and the interassay CV <8%. Recovery and linearity studies were satisfactory and comparison of results obtained using a RIA for UFF and the present method in 98 routine samples showed a correlation of r= 0.838, with the results obtained with LC-MS/MS significantly lower (medians of 22.0 vs. 49.4 microg/24 h for RIA) (P<0.0001). Reference values for cortisol were defined as values between 11 and 43 microg/24 h, compatible to those recently described for similar methods. The concomitant measurement of UF cortisone allows the study of the activity of the enzyme 11beta-HSD2 and the diagnosis of the apparent mineralocorticoid excess syndrome. The method represents the first steroid assay of a new generation, based on automated preparative methods and tandem mass spectrometry, described in our country.     

The establishment of a method for serum glycated albumin based on liquid chromatography/tandem mass spectrometry

正Objective To establish a candidate reference method for the determination of serum glycated albumin based on isotope dilution liquid chromatography/tandem mass spectrometry(ID-LC/MS).Methods According to the Japan Society of Clinical Chemistry(JSCC)reference method,the serum albumin was firstly purified by SDS-     

基于多维液相色谱质谱组合分析的痢疾杆菌蛋白质基因组学

目的 应用多维液相色谱质谱组合体系为基础的蛋白质组学方法对福氏痢疾杆菌基因组注释进行完善。方法 痢疾杆菌福氏2a型301株(Sf2a301)的全菌蛋白经胰酶消化,二维液相色谱分离后进行MALDI-TOF/TOF和ESI-MS/MS组合鉴定,质谱数据分别应用MASCOT和SEQUEST软件检索基于Sf2a301全基因组构建的6个读码框数据库,完成对原基因组注释的验证和补充。结果 研究表明多维液相色谱质谱组合体系能够增加鉴定蛋白的覆盖率,共鉴定Sf2a301的1 231个蛋白编码基因产物,涵盖了COGs 数据库22个功能分类组中的20个,包含306个功能未知的假定蛋白。发现了9个未注释的基因,得到RT-PCR和Northern blot的进一步验证。新基因大多数是重叠基因,包含3个嵌套基因。结论 多维液相色谱质谱组合体系相对于单一的串联质谱技术能够更加有效验证、补充痢疾杆菌的基因组注释,更新后的基因组注释库为今后开展痢疾杆菌功能研究提供更多的靶点。     

Quantitation of hepcidin from human and mouse serum using liquid chromatography tandem mass spectrometry

The hepatic peptide hormone hepcidin is considered the central regulator of iron metabolism. Characterizing the circulating levels of this peptide is critical to understanding its role in the development of clinically relevant syndromes, such as anemia of inflammation/chronic disease, and may provide insight into potential clinical interventions. While quantitative methods have been published for the determination of urinary hepcidin and serum prohepcidin, no definitive methods have been published for the determination of hepcidin in serum. In this report, we describe a quantitative method for the determination of both human and mouse hepcidin in serum and plasma. The method employs protein precipitation and solid-phase extraction followed by liquid chromatographic separation and tandem mass spectrometry detection. The method has a quantitative range of 0.25 ng/mL to 500 ng/mL serum for mouse hepcidin and 1 ng/mL to 500 ng/mL serum for human hepcidin. The method uses small sample volumes (50 microL for mice and 100 microL for humans) and 96-well formats for rapid sample processing. The method was used to establish baseline serum and plasma concentrations of hepcidin in normal C57Bl/6 mice and healthy human volunteers.     

A proteome analysis of pig pancreatic islets and exocrine tissue by liquid chromatography with tandem mass spectrometry

Liquid chromatography with tandem mass spectrometry (LC-MS/MS) is a proteome analysis method, and the shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolving power. In this study, we analyzed the protein expression in pancreatic tissue by LC-MS/MS. Islets isolated from porcine pancreata (purity ≥95%) and exocrine tissue (purity ≥99%) were used in this study. LC-MS/MS showed that 13 proteins were expressed in pancreatic islets only (Group I), 43 proteins were expressed in both islets and exocrine tissue (Group I&E), and 102 proteins were expressed in exocrine tissue only (Group E). Proteins involved in islet differentiation and cell proliferation were identified in Group I (e.g. CLUS, CMGA, MIF). In addition, various functional proteins (e.g. SCG2, TBA1A) were identified in islet by using the new method of ‘principal component analysis (PCA)’. However, the function of such proteins on islets remains unclear. EPCAM was identified in Group E. Group E was found to include proteins involved in clinical inflammatory diseases such as pancreatitis (e.g. CBPA1, CGL, CYTB, ISK1 and PA21B). Many of these identified proteins were reported less frequently in previous studies, and HS71B, NEC2, PRAF3 and SCG1 were newly detected in Group I while CPNS1, DPEP1, GANAB, GDIB, GGT1, HSPB1, ICTL, VILI, MUTA, NDKB, PTGR1, UCHL3, VAPB and VINC were newly detected in Group E. These results show that comprehensive expression analysis of proteins by LC-MS/MS is useful as a method to investigate new factors constructing cellular component, biological process, and molecular function.     

液相串联质谱法定量检测高密度脂蛋白中脂质类化合物

目的本文旨在建立一种快速提取高密度脂蛋白(HDL)中脂质并结合液相串联质谱定性及快速定量检测的方法。方法采用酸化甲醇沉淀结合超声萃取的方法抽提脂质,高速离心去除蛋白颗粒,取上清液,直接采用液相串联质谱在多反应监测模式下进行定量分析。结果该分离提取脂质的方法简单迅速,采用多反应监测模式可同时定量监测30多种具有标准品的脂质成份,包括鞘氨醇类、鞘磷脂、神经酰胺和氧化型的卵磷脂。结论酸化甲醇超声抽提离心法可简便迅速地获得HDL中的脂质成份,结合液相串联质谱可对HDL中的多种脂质进行定量检测,该方法具有高效、重现性好等优点,适用于大量液体生物样本的分析。     

Diagnosis of glutaric aciduria type 1 by measuring 3-hydroxyglutaric acid in dried urine spots by liquid chromatography tandem mass spectrometry

Accumulation of glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA) in body fluids is the biochemical hallmark of type 1 glutaric aciduria (GA1), a disorder characterized by acute striatal degeneration and a subsequent dystonia. To date, methods for quantification of 3HGA are mainly based on stable isotope dilution gas chromatography mass spectrometry (GC-MS) and require extensive sample preparation. Here we describe a simple liquid chromatography tandem MS (LC-MS/MS) method to quantify this important metabolite in dried urine spots (DUS). This method is based on derivatization with 4-[2-(N,N-dimethylamino)ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DAABD-AE). Derivatization was adopted to improve the chromatographic and mass spectrometric properties of the studied analytes. Derivatization was performed directly on a 3.2-mm disc of DUS as a sample without extraction. Sample mixture was heated at 60°C for 45 min, and 5 μl of the reaction solution was analyzed by LC-MS/MS. Reference ranges obtained were in excellent agreement with the literature. The method was applied retrospectively for the analysis of DUS samples from established low- and high-excreter GA1 patients as well as controls (n = 100). Comparison of results obtained versus those obtained by GC-MS was satisfactory (n = 14). In populations with a high risk of GA1, this approach will be useful as a primary screening method for high- or low-excreter variants. In these populations, however, DUS analysis should not be implemented before completing a parallel comparative study with the standard screening method (i.e., molecular testing). In addition, follow-up DUS GA and 3HGA testing of babies with elevated dried blood spot C5DC acylcarnitines will be useful as a first-tier diagnostic test, thus reducing the number of cases requiring enzymatic and molecular analyses to establish or refute the diagnosis of GA1.     

Ultra-high performance liquid chromatography tandem mass spectrometry method for detection of anti-schizophrenic drugs and the evaluation of their therapeutic reference ranges

正Objective A highly feasible and reliable ultra-high performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS)method was presented for therapeutic drug monitoring of five anti-schizophrenic drugs(aripiprazole,amisulpride,olanzapine,paliperidone and ziprasidone)simultaneously and the reference range of plasma concentration was investigated.Methods The     

A simple and reliable method of detecting variant transthyretins by multidimensional liquid chromatography coupled to electrospray ionization mass spectrometry.

Transthyretin (TTR) variants cause amyloidosis. A method, originally reported by us, of detecting the variants by high performance liquid chromatography/electrospray ionization mass spectrometry (ESIMS) using materials precipitated with anti-TTR antiserum, has been successfully applied by several institutions. The method is simple and reliable, but some variants may not precipitate with the antiserum or may precipitate in different yields compared to normal TTR. Moreover, unidentified minor peaks were observed, which may have been derived from cross reactive materials. We have now devised a new procedure to overcome these problems. An anion exchange and reversed phase liquid chromatography system and ESI mass spectrometer were connected in a tandem fashion using a 6 port valve and a protein trap cartridge. The profile of ion peaks by the method was the same as that by MS with immunoprecipitates. The minor peaks were proved not to be derived from cross reactive materials, and the molecular species of these peaks were characterized. This method is faster than immunoprecipitation method and using no antibody is a great benefit. The method can be applied widely to the study various proteins, when antibodies are not available.     

Late-night salivary cortisol for diagnosis of Cushing's syndrome by liquid chromatography/tandem mass spectrometry assay

Background Late‐night salivary cortisol (LNSC) measurements have been increasingly used by physicians as an initial diagnostic test for evaluation of patients with clinical suspicion of Cushing’s syndrome (CS). Published studies include various numbers of cases, controls and importantly, various assay methods (vast majority various immunoassays), as well as various methods to generate cut‐points. Materials and Methods The retrospective study evaluated the diagnostic utility of LNSC measurements in 249 patients evaluated for possibility of CS because of various clinical conditions using liquid chromatography/tandem mass spectrometry method (LC‐MS/MS). CS was confirmed in 47 patients (18·9%) and excluded in 202 (81·1%) patients at the time of analysis. Results Late‐night salivary cortisol was abnormal or >2·8 nmol/l in 35 of 47 patients with CS; sensitivity of 74·5% and elevated in 20 of 202 patients who were found not to have CS; specificity 90·1%. Using receiver‐operator characteristic statistics for calculation of the most optimal sensitivity and specificity, the cut‐off based on this data was LNSC > 2·1 nmol/l with sensitivity of 83·0% and specificity of 84·2%. Conclusion Analysis of data at one referral institution showed somewhat limited sensitivity of LNSC for diagnosis of CS using current reference ranges.     

Principles and clinical applications of liquid chromatography - tandem mass spectrometry for the determination of adrenal and gonadal steroid hormones

Liquid-chromatography - tandem mass spectrometry (LC-MS/MS) is becoming the method of choice for clinical steroid analysis. In most instances, it has the advantage of higher sensitivity, better reproducibility and greater specificity than commercial immunoassay techniques. The method requires only minimal sample preparation and a small sample volume. Furthermore, it has the potential to analyze multiple steroids simultaneously. Modern instruments guarantee high throughput, allowing an affordable price for the individual assay. All this makes LC-MS/MS an attractive method for use in a clinical setting. Reliable reference ranges for the detected analytes are the pre-requisite for their clinical use. If these are available, LC-MS/MS can find application in congenital disorders of steroid metabolism, such as congenital adrenal hyperplasia, disorders of sex development and disorders of salt homeostasis, as well as in acquired disorders of steroid metabolism, such as primary aldosteronism, Cushing's disease, Addison's disease, and hyperandrogenemia, as well as in psychiatric disease states such as depression or anxiety disorders. The principles of LC-MS/MS for steroid measurement, the pros and cons of LC-MS/MS compared with conventional immunoassays and the possible applications in clinical routine, with a special focus on pediatric endocrinology needs, are discussed here.     

The importance of methodology in serum testosterone measurement: comparison between a direct immunoassay and a method based on high performance liquid chromatography and tandem mass spectrometry (HPLC/MS-MS)

Serum testosterone in its total or free form, is a highly valuable diagnostic test and is available in the great majority of clinical laboratories. This reality was possible due to the development of simple and direct assays, adaptable to large automatic systems. Recent publications have called attention to the limitations of these simplified methodologies, mainly in samples with low concentration, as women and children. In this paper we present results obtained using a reference method based on high performance liquid chromatography and tandem mass spectrometry (HPLC/MS-MS) and its comparison with those obtained with a commercial routine immunoassay (electrochemiluminescent assay, ECLIA). Methods were compared in total testosterone measurement (n = 213), as well as in free testosterone evaluation based on calculation including sex hormone-binding protein (SHBG) levels (n = 135). Values obtained with ECLIA were significantly higher, with more marked dispersion in low concentration. This phenomenon is clearer when presented as a Bland-Altman plot. Difficulties in the implementation of reference methods as the one presented are discussed, as well as the necessity of caution in the interpretation of values obtained with routine assays, a matter of several publications in recent literature.     

Metabolism of an insect diuretic hormone by Malpighian tubules studied by liquid chromatography coupled with electrospray ionization mass spectrometry

The larger of two diuretic hormones of the tobacco hornworm, Manduca sexta, (Mas-DH) is a peptide of 41 residues. It is one of a family of seven currently known insect diuretic hormones that are similar to the corticotropin-releasing factor–urotensin–sauvagine family of peptides. We investigated the possible inactivation of Mas-DH by incubating it in vitro with larval Malpighian tubules (Mt), the target organ of the hormone. The medium was analyzed, and degradation products were identified, using on-line microbore reversed-phase liquid chromatography coupled to electrospray ionization mass spectrometry (RPLC-ESI-MS). This sensitive technique allows identification of metabolites of Mas-DH (present at an initial level of ≈1 μM). An accurate M_r value for a metabolite is usually sufficient for unambiguous identification. Mas-DH is cleaved by Mt proteases initially at L²⁹–R³⁰ and R³⁰–A³¹ under our assay conditions; some Mas-DH is also oxidized, apparently at M² and M¹¹. The proteolysis can be inhibited by 5 mM EDTA, suggesting that divalent metals are needed for peptide cleavage. The oxidation of the hormone can be inhibited by catalase or 1 mM methionine, indicating that H₂O₂ or related reactive oxygen species are responsible for the oxidative degradation observed. RPLC-ESI-MS is shown here to be an elegant and efficient method for studying peptide hormone metabolism resulting from unknown proteases and pathways.     

Drug-drug interactions that reduce the formation of pharmacologically active metabolites: a poorly understood problem in clinical practice

Drug-drug interactions can lead to reduced efficacy of medical treatment. Therapeutic failure may for instance result from combined treatment with an inhibitor of the specific pathway that is responsible for the generation of pharmacologically active drug metabolites. This problem may be overlooked in clinical practice. Several examples of drugs will be discussed -clopidogrel, losartan, tamoxifen and codeine - to illustrate differences in the potential impact on drug treatment in clinical practice. We conclude that the combined use of cytochrome P450-blocking serotonin reuptake inhibitors and tamoxifen or codeine should be avoided, whereas the situation is much more complex regarding the use of proton pump inhibitors together with clopidogrel, and the evidence regarding cytochrome P450 inhibitor-dependent activation of losartan is inconclusive.     

Urine aldosterone radioimmunoassay: validation of a method without chromatography.

A simplified radioimmunoassay of urinary aldosterone is reported. Acid-hydrolyzed urine was extracted with dichloromethane and the extract assayed without further purification, Urinary aldosterone values in patients with Cushing's syndrome, low and normal-renin essential hypertension, congenital adrenal hyperplasia, and primary aldosteronism determined by this method agreed closely (r = 0.95, P less than 0.01) with values obtained using a standardized chromatographic method. This simplified assay represents a significant advance in out capabilitites for evaluating patients for abnormalities in aldosterone physiology.     

Metabolic analysis of the melatonin biosynthesis pathway using chemical labeling coupled with liquid chromatography‐mass spectrometry

Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography‐mass spectrometry (LC‐MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5‐hydroxytryptophan (5HTP), serotonin (5HT), N‐acetylserotonin (NAS), 5‐methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one‐step dispersive solid‐phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS‐Cl), followed by analysis using LC‐MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22‐ to 469‐fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.