NAD(P)‐dependent steroid dehydrogenase‐like protein and neutral cholesterol ester hydrolase 1 serve as novel markers for early detection of gastric cancer identified using quantitative proteomics

BackgroundGastric cancer (GC) is the third most common cause of cancer deaths worldwide. In the present study, we aimed to identify novel GC biomarkers by integrating isobaric tags of relative and absolute quantitation (iTRAQ) for aberrantly expressed proteins in GC patients.MethodsUsing stable isotope tags, we labeled an initial discovery group comprising four paired gastric cancer and adjacent gastric tissue samples, and subjected them to LC‐ESI‐MS/MS. We used a validation set comprising 129 paired gastric cancer and adjacent gastric tissues from patients and benign healthy controls to validate the candidate targets.ResultsWe identified two proteins, NAD(P)‐dependent steroid dehydrogenase‐like (NSDHL) and neutral cholesterol ester hydrolase 1 (NCEH1), that were significantly overexpressed in GC tissues. The sensitivity and specificity of NSDHL were 80.6% and 74.4%, respectively, in GC compared with a sensitivity of 25.6% in adjacent tissues and 24% in benign healthy controls. The area under the ROC curve (AUC) for NSDHL was 0.810 for GC detection. Overexpression of NSDHL in GC was significantly correlated with local tumor invasion. The sensitivity and specificity of NCEH1 were 77.5% and 73.6%, respectively, in GC compared with a sensitivity of 26.4% in adjacent tissues and 20% in benign controls. The AUC for NSDHL was 0.792. Overexpression of NCEH1 was significantly associated with tumor histological classification and local invasion. Moreover, a combined analysis of NSDHL and NCEH1 achieved a sensitivity and specificity of 85.7% and 83%, respectively, and the AUC was 0.872. The combined analysis of NSDHL and NCEH1 was significantly correlated with histological grade and TNM Ⅱ‐Ⅳ staging.ConclusionsiTRAQ‐labeled quantitative proteomics represents a powerful method to identify novel cancer biomarkers. The present study identified NSDHL and NCEH1 as useful biomarkers for screening, diagnosis, and prognosis of patients with gastric cancer.     

Explore potential plasma biomarkers of acute respiratory distress syndrome (ARDS) using GC–MS metabolomics analysis

ObjectivesThe aim of this study was to analyse the metabolomics of patients with acute respiratory distress syndrome (ARDS) for the identification of metabolic markers with potential diagnostic and prognostic value.MethodsThe enrolled subjects included adult patients with ARDS that met the Berlin definition and healthy controls matched based on age, gender, and body mass index (BMI). Plasma samples were collected from 37 patients with ARDS and 28 healthy controls. The plasma metabolites were detected with gas chromatography–mass spectrometry (GC–MS), and the relevant metabolic pathways were predicted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.ResultsA total of 222 metabolites were identified in our study, of which 128 were significantly altered in patients with ARDS compared with healthy controls. Phenylalanine, aspartic acid, and carbamic acid levels were significantly different between all groups of patients with ARDS classified from mild to severe. Furthermore, four metabolites, ornithine, caprylic acid, azetidine, and iminodiacetic acid, could serve as biomarkers to potentially predict the severity of ARDS. We discovered 92 pathways that were significantly different between ARDS and control groups, including 57 pathways linked to metabolism.ConclusionsPlasma metabolomics may improve our understanding of ARDS biology. Specific products related to hypoxia may serve as early biomarkers for ARDS prediction, while the metabolites with significant correlations with partial pressure of arterial oxygen (PaO₂)/percentage of inspired oxygen (FiO₂) may play a role in determining ARDS severity. This study suggests that metabolomic analysis in patients at risk of ARDS or those with early ARDS may provide new insight into disease pathogenesis or prognosis.     

Performance evaluation of three liquid chromatography mass spectrometry methods for broad spectrum drug screening

BackgroundLiquid chromatography-mass spectrometry (LC-MS) and tandem LC-MS (LC-MS/MS) are increasingly used in toxicology laboratories as a complementary method to gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ultraviolet detection (LC-UV) for comprehensive drug screening (CDS). This study was designed to characterize the sensitivity and specificity of three LC-MS(/MS) vendor-supplied methods for targeted CDS and identify the current limitations associated with the use of these technologies.MethodsFive methods for broad spectrum CDS, including LC-UV (REMEDi), full scan GC-MS, LC-MS (ZQ?-Mass Detector with MassLynx?-software), LC-QTRAP-MS/MS (3200-QTRAP® with Cliquid®-software) and LC-LIT-MS/MS (LXQ? Linear Ion Trap with ToxID?-software) were evaluated based on their ability to detect drugs in 48 patient urine samples.ResultsThe tandem MS methods identified 15% more drugs than the single stage MS or LC-UV methods. Use of two broad spectrum screening methods identified more drugs than any single system alone. False negatives and false positives generated by the LC-MS(/MS) software programs were identified upon manual review of the raw data.ConclusionsThe LC-MS/MS methods detected a broader menu of drugs; however, it is essential to establish manual data review criteria for all LC-MS(/MS) drug screening methods. Use of an EI-GC-MS and ESI-LC-MS/MS combination for targeted CDS may be optimal due to the complementary nature of the chromatographic and ionization techniques.     

Plasma metabolic profiling of Alzheimer's disease by liquid chromatography/mass spectrometry

ObjectivesThe identification of Alzheimer's disease (AD) biomarkers may allow for a less invasive and more accurate diagnosis as well as serve as a predictor of future disease progression and treatment response. The aim of this study was to map potential biomarkers in plasma for AD.Design and methodsPlasma metabolic perturbations between AD and healthy old person were investigated using ultra performance liquid chromatography/mass spectrometry (UPLC/MS) and metabonomics approach. The principal component analysis (PCA) of UPLC/MS spectra showed that metabolic changes between two groups.ResultsThe PCA of UPLC/MS spectra showed that metabolic changes observed between AD and control were clear. Nine potential biomarkers in correlation with the extent of AD were found.ConclusionsBased on PCA, several potential biomarkers (LPCs, sphingosine and tryptophan) were found and further identified by the following LC/MS/MS analysis. All of them could be the potential early markers of AD.     

MALDI‐TOF‐MS analysis in low molecular weight serum peptidome biomarkers for NSCLC

ObjectsLung cancer is one of the leading causes of death from cancer in the world. Screening new serum biomarkers is important for the early detection of lung cancer. The purpose of this study was to investigate the serum peptide model between non‐small cell lung cancer (NSCLC) patients and healthy controls, as well as between paired pre‐ and postoperative NSCLC patients, and to find the low molecular weight (LMW) potential tumor markers for NSCLC.Methods56 serum samples from NSCLC patients, 56 controls, and 20 matched pre‐ and postoperative patients were analyzed using magnetic‐bead (MB)‐based purification technique combined with MALDI‐TOF‐MS. To distinguish NSCLC from cancer‐free controls, three models were established. Finally, comparing the three groups of serum protein fingerprints, nano‐liquid chromatography–electrospray ionization tandem mass spectrometry was used to further identify the differential peptides.ResultsAmong the three models constructed, the GA model had the best diagnostic efficacy. Five differential peaks were screened by combining the case group, healthy controls, and postoperative group analysis, which were up‐regulated in the case group and showed a tendency to return to healthy control values after surgery. The protein matching the mass spectrometry peak m/z 2953.73 was identified as fibrinogen α chain.ConclusionThis study shows that the application of MALDI‐TOF‐MS is a promising approach for the identification of potential serum biomarkers for NSCLC, which is potentially valuable for establishing a new diagnostic method for lung cancer. In addition, we found that fibrinogen α chain may be an auxiliary diagnostic indicator for NSCLC.     

Comparison of tear proteins between healthy and early diabetic retinopathy patients

ObjectivesTo identify potential prognostic or diagnostic marker tear proteins for early diabetic retinopathy (DR) and to investigate the pathogenesis of this disease using proteomics techniques.Design and methodsThe tear proteins expressed in patients suffering from diabetes mellitus without the retinopathy symptoms, nonproliferative diabetic retinopathy and healthy volunteers were analyzed by 2-DE. The differentially expressed proteins in patients were identified by ESI-Q-TOF and confirmed by Western blotting.ResultsProteins which were differentially expressed with statistical significance (P < 0.05) in two diabetic groups as compared to those in healthy group were selected and identified by ESI-Q-TOF MS/MS. Among these proteins, three proteins (LCN-1, HSP27 and B2M) were found to exhibit a progressive reduction in two disease groups. The expression levels of which might be useful as diagnostic biomarkers of DR were verified by Western blottingConclusionsProteomic analysis using tear is a novel approach that can provide insight into possible biomarker and the pathogenesis of early DR.     

Identification of possible biomarkers for breast cancer from free fatty acid profiles determined by GC-MS and multivariate statistical analysis

ObjectivesThe aim was to investigate the free fatty acid (FFA) metabolic profiles and to identify biomarkers that can be used to distinguish patients with breast cancer (BC) from benign (BE) patients or healthy controls.Design and methodsA total of 114 subjects were divided into the following three groups: BC patients, BE patients and controls. The FFA profiles in three groups were studied by gas chromatography–mass spectrometry coupled with multivariate statistical analysis.ResultsThree saturated fatty acids (C14:0, C16:0 and C18:0) and three unsaturated fatty acids (C18:2, C18:3 and C20:5) in BC were significantly different than controls. Palmitic acid, stearic acid, linoleic acid and total FFA were identified as potential biomarkers distinguished BC from the other groups.ConclusionThe alterations of FFA could reflect underlying metabolic changes in BC patients, and this study has demonstrated that FFA biomarkers might be helpful for prevention and characterization of BC patients.     

Effects of pretreatment protocols on human amniotic fluid protein profiling with SELDI-TOF MS using protein chips and magnetic beads

BackgroundThere is increasing interest in the use of human amniotic fluid (AF) proteomics with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for diagnosing pregnancy-associated abnormalities. A critical parameter of diagnostic biomarkers is the accuracy and reproducibility of protein patterns. We evaluated the effects of common pretreatment protocols on protein patterns generated using SELDI mass spectrometry with two different protein capture strategies (including functional protein chips and functionalized magnetic beads prior to MS analysis) in AF.MethodVarious extrinsic factors involved in processing and storing amniotic fluid, including matrix composition, sample storage time, temperature and freeze–thaw cycles, were analyzed regarding their impact on AF protein patterns using SELDI mass spectrometry with 2 different protein capture strategies.ResultsThree extrinsic factors (sample storage for 3 days at either room temperature or 4 °C or freeze–thawing the sample 5 times) significantly decreased the number or intensities of protein peaks detected in AF. Matrix dilutions also changed the spectra of AF, with more peaks and higher intensities observed with 50% α-cyano-4-hydroxycinnamic acid (CHCA). Moreover, protein chips captured more proteins or peptides than magnetic beads on SELDI-TOF MS profiling of AF.ConclusionsThese results suggest that extrinsic factors must be taken into account for valid data interpretation to ensure good reproducibility of AF profiling by SELDI mass spectrometry.     

串联质谱和高效液相色谱-串联质谱二次筛查联合应用于新生儿MMA筛查

目的探讨串联质谱和高效液相色谱-串联质谱二次筛查联合应用在甲基丙二酸血症(MMA)中的筛查价值。方法收集新生儿串联质谱初筛结果中C3、C3/C2、C3/C0单一或多个指标异常的新生儿干血滤纸片标本,用高效液相色谱-串联质谱的方法定量检测原始血片中甲基丙二酸、甲基枸橼酸和高半胱氨酸,对二次筛查后疑似阳性的新生儿进行召回复查,并进行尿气相色谱/质谱检测。临床诊断患儿进一步予以基因检测进行确诊。结果共收集423例C3、C3/C2、C3/C0单一或多个指标异常的新生儿筛查标本,初筛阳性率约为1%,行联合筛查检测结果发现8例标本中甲基丙二酸和同型半胱氨酸表达水平明显升高,召回复查尿气相色谱质谱提示甲基丙二酸轻度升高。结论串联质谱和高效液相色谱-串联质谱联合应用可以提高新生儿MMA筛查的阳性预测值、降低假阳性率,在新生儿遗传代谢病筛查中具有重要价值。     

Urine toxicology screening by liquid chromatography time-of-flight mass spectrometry in a quaternary hospital setting

ObjectiveValidation of a non-targeted method for urine drug screening (UDS) by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF), and comparison to an established GC–MS method in a hospital setting.Methods217 UDS specimens sent to a quaternary hospital pathology department, were analysed by a CEDIA® immunoassay screen (six drug panels; amphetamines, barbiturates, benzodiazepines, cocaine metabolites, cannabinoids and opiates) on an Abbott Architect instrument. Specimens were subsequently analysed by an established non-targeted qualitative GC–MS method and results compared with a general unknown screening method by LC-QTOF that was under evaluation as a replacement method.Results42 selected drugs were evaluated; limits of identification ranged from 2 to 100 µg/L and most drugs (n = 39) were stabile for 24 h after preparation. Matrix effects greater than 25% were observed in seven of the selected drugs. 87% of the specimens tested positive to 1 or more drug panels in a CEDIA® screen. A total of 537 positive drug findings were identified by GC–MS compared to 1,267 positive findings by LC-QTOF. On average, each GC–MS screen identified 2.5 ± 1.8 drugs and the LC-QTOF screen identified 5.8 ± 3.2 drugs. No drugs were identified in 11.3% of the GC–MS screens, whereas drugs were detected in 99% of these by the LC-QTOF. In almost all instances, the LC-QTOF screen could provide mass spectrometric confirmatory results of positive immunoassay screens and was able to identify a wider range of additional drugs and drug metabolites.ConclusionsThe described general unknown screening (non-targeted, qualitative) LC-QTOF method can detect a larger range of drugs encountered in a hospital setting. The method has been shown to be suitable for comprehensive toxicology screening in a clinical toxicology laboratory.     

Role of liquid chromatography–high-resolution mass spectrometry (LC-HR/MS) in clinical toxicology

Background. Gas chromatography (GC) and liquid chromatography (LC) coupled with mass spectrometry (MS) are widely used to confirm drug screening results and for urine screening in presumed intoxicated patients. These techniques are better suited to targeted analysis than to general unknown screening and, due to the complexity of testing, results are seldom available rapidly enough to contribute to the immediate care of the patient. High resolution (HR)/MS with time-of-flight (TOF) or orbitrap instruments offer potential advantages in clinical toxicology. Comparison of GC-MS, LC-MS/MS and LC-HR/MS. For unknown analyses, GC-MS and LC-MS/MS require comparison of full-scan spectra against preestablished libraries. Operation in full-scan mode greatly reduces sensitivity and some drugs present in low but significant concentrations may be missed. Selected ion monitoring (SIM) in GC/MS and selected reaction monitoring (SRM) in LC-MS/MS, where only targeted ions are monitored, increase sensitivity but require prior knowledge of what compound is to be measured. LC-HR/MS offers mass assignment with an accuracy of 0.001 atomic mass units (amu) compared with 1 amu in conventional MS. Tentative identification is thus directed to a very limited set of compounds (or even one unique compound) based on the exact molecular formula rather than a fragmentation pattern, since HR/MS can discriminate between compounds with the same nominal molecular mass. LC-MS/MS has clear advantages over GC/MS in ease and speed of sample preparation and the opportunities for its automation. LC-HR/MS is more suitable to clinical toxicology because the drugs present in a sample are rarely known a priori, and tentative identifications of unknowns can be made without the availability of a reference standard or a library spectrum. Blood can be used in preference to urine which is more relevant to the patient's current clinical situation. Methods. A literature search was conducted using PUBMED for clinical toxicology, adulterants in illicit drugs and herbal supplements, and case reports using LC-TOF/MS and LC-HR/MS. Only 42 papers in English were identified in these searches. LC-HR/MS in clinical toxicology. LC-HR/MS has been used to detect designer drugs, doping agents, (neurosteroids) and adulterants such as levamisole, a veterinary antihelmitic found in street cocaine, and pharmaceuticals in herbal medications marketed to contain only natural ingredients. LC-HR/MS has proved useful for cases where existing tests were unable to identify the cause of the intoxication. One patient suffered a drug-induced seizure which was originally thought to be caused by an herbal medication, but diphenhydramine was determined to be the culprit. In another, 5-oxoproline was identified as the cause of metabolic acidosis seen in chronic acetaminophen (paracetamol) use. LC-HR/MS has successfully identified medications that were mislabeled or misrepresented street drugs. In one case, medications sold as diazepam were determined to be glyburide instead. The identification of novel designer amines, stimulants found in “bath salts”, and synthetic cannabinoids are well suited to LC-HR/MS. Dozens or even hundreds of possible compounds cannot realistically be tested on an individual basis by targeted LC-MS/MS or GC/MS analysis. Conclusions. LC-HR/MS offers unique opportunities for time-sensitive clinical analysis of blood samples from intoxicated patients and for comprehensive screening in a wide range of situations and materials. While the identification is not as definitive as that obtained by conventional fragmentation MS, the presumptive identification can be confirmed later with standards and spectral library matches. Optimum utilization of the presumptive diagnosis requires close collaboration between the laboratory analysts and their clinical counterparts.     

Identification and confirmation of haptoglobin as a potential serum biomarker in hypertrophic cardiomyopathy using proteomic approaches

Abstract

Introduction. Aiming at identifying biomarkers for hypertrophic cardiomyopathy (HCM), the serum proteome was explored through a two-dimensional gel-based proteomic approach (2D-DIGE) coupled with mass spectrometry and database interrogation.

Methods. Serum samples from 20 male HCM patients and their sex- and age-matched controls were cleaned from interfering components. Patients and controls were pooled in five matched groups with the same age, and proteins extracts from each pool were labelled with cyanine dyes. Then, gel images were analysed using a fluorescence scanner and proteins were identified. Tryptic peptides were analysed by capillary reversed-phase liquid chromatography coupled online with tandem mass spectrometry (MS/MS).

Results. Four different proteins were observed to be differentially expressed between HCM patients and their matched controls. Of them, decreases in haptoglobin levels were confirmed to be associated with HCM in an independent set of 181 consecutive HCM patients from our monographic clinic and 114 controls with similar age and sex using a nephelometer-based technique. Moreover, a significant negative correlation was observed between haptoglobin and subaortic gradient, thus highlighting the role of haptoglobin in HCM.

Conclusion. All these observations point out the utility of the 2D-DIGE proteomic strategy for the identification of serum proteins indicative of the presence of cardiac injury.     

基于串联质谱标签技术筛选免疫介导性脱髓鞘疾病诊断与鉴别诊断的生物标志物

目的:采用串联质谱标签（TMT）联合液相色谱串联质谱（LC-MS/MS）筛选免疫介导性脱髓鞘疾病诊断与鉴别诊断的潜在生物标志物。方法:选择首都医科大学附属北京天坛医院2020年1月至2021年1月收治的20例脱髓鞘疾病患者（脱髓鞘组）,包括10例吉兰-巴雷综合征（GBS）患者（GBS亚组）与10例多发性硬化（MS）患者...     

Identification of tubulin beta chain,thymosin beta-4-like protein 3, and cytochrome b–c1 complex subunit 1 as serological diagnostic biomarkers of gastric cancer

Objective

Despite major advances in its diagnosis and treatment, gastric cancer (GC) remains a major life-threatening disease. Treatment of the disease is further aggravated by the lack of diagnostic biomarkers that can aid in the early detection of GC and promote its favorable prognosis. The present work aims to identify novel diagnostic biomarkers for GC.

Design and methods

The present work is a case–control study that focuses on proteomic analysis of serum from healthy volunteers and GC patients using ClinProt profiling technology based on mass spectrometry. A pattern of proteins/peptides with the ability to differentiate the studied populations was identified. Deregulated proteins/peptides differentially expressed in the serum of patients compared with healthy volunteers were identified by mass spectroscopy.

Results

A pattern of proteins/peptides consisting of four protein/peptide peaks at m/z 1467, 1867, 2701, and 2094 was identified. These protein/peptide peaks were able to differentiate the studied populations with close to 100% sensitivity and specificity. Three of the deregulated proteins/peptides at m/z 1867, 2701, and 2094 were identified by mass spectroscopy (LTQ Orbitrap XL) as tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b–c₁ complex subunit 1, respectively.

Conclusions

The pattern of proteins/peptides identified in the present work shows great potential for GC diagnosis. Deregulated proteins of tubulin beta chain, thymosin beta-4-like protein 3, and cytochrome b–c₁ complex subunit 1 may be involved in the pathogenesis of GC and serve as potential serological diagnostic biomarkers.     

Preliminary exploration on the serum biomarkers of bloodstream infection with carbapenem‐resistant Klebsiella pneumoniae based on mass spectrometry

BackgroundCarbapenem‐resistant K. pneumoniae (CRKP) bloodstream infections (BSI) must be rapidly identified to improve patient survival rates. This study investigated a new mass spectrometry‐based method for improving the identification of CRKP BSI and explored potential biomarkers that could differentiate CRKP BSI from sensitive.MethodsMouse models of BSI were first established. MALDI‐TOF MS was then used to profile serum peptides in CRKP BSI versus normal samples before applying BioExplorer software to establish a diagnostic model to distinguish CRKP from normal. The diagnostic value of the model was then tested against 32 clinical CRKP BSI and 27 healthy serum samples. Finally, the identities of the polypeptides used to establish the diagnostic model were determined by secondary mass spectrometry.Results107 peptide peaks were shared between the CRKP and normal groups, with 18 peaks found to be differentially expressed. Five highly expressed peptides in the CRKP group (m/z 1349.8, 2091.3, 2908.2, 4102.1, and 8129.5) were chosen to establish a diagnostic model. The accuracy, specificity and sensitivity of the model were determined as 79.66%, 81.48%, and 78.12%, respectively. Secondary mass spectrometry identified the Fibrinogen alpha chain (FGA), Inter‐alpha‐trypsin inhibitor heavy chain H4 (ITIH4) and Serum amyloid A‐2 protein (SAA2) as the source of the 5 serum peptides.ConclusionsWe successfully established a serum peptide‐based diagnostic model that distinguished clinical CRKP BSI samples from normal healthy controls. The application of MALDI‐TOF MS to measure serum peptides, therefore, represents a promising approach for early BSI diagnosis of BSI, especially for multidrug‐resistant bacteria where identification is urgent.     

The screening of inborn errors of metabolism in sick Chinese infants by tandem mass spectrometry and gas chromatography/mass spectrometry

BackgroundAnalyses of amino acid/acylcarnitines in dried blood spots (DBS) and organic acids in urine are the primary tests for inborn errors of metabolism (IEMs). Automated tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) can rapidly and simultaneously detect numerous metabolic compounds with high precision and sensitivity.MethodsThree thousand four hundred and twenty-nine DBSs and 2781 urine samples were collected from our hospital patients with suspected IEMs, and analyzed for amino acid/acylcarnitines and organic acids by MS/MS and GC/MS, respectively. The results were used in a coincidental survey to determine the efficacy of these methods for the diagnosis of IEMs.ResultsNineteen different types of IEMs were detected in 121 affected cases (1.95% of 6210 samples). There were 66.12% amino acid disorders, 29.75% organic acid disorders and 4.13% with fatty acid oxidation disorders. Conclusions: the sick infants tested in this study had high prevalence rates of neonatal intrahepatic cholestasis, methylmalonic acidemia, hyperphenylalaninemia, tyrosinemia type I, and urea cycle disorders.ConclusionThe combined use of MS/MS and GC/MS is an appropriate tool for screening of IEMs in sick infants.     

Database-dependent metabolite profiling focused on steroid and fatty acid derivatives using high-temperature gas chromatography–mass spectrometry

BackgroundA database-dependent gas chromatography–mass spectrometry (GC–MS) based approach was developed for non-targeted metabolite profiling, focusing on 232 steroids, 24 fatty acids, 10 eicosanoids, 10 cannabinoids and 22 steroid-fatty acid esters in biological specimens.MethodsThis method, used to search for potent biomarkers in lipid metabolism, included MS based analysis combined with high-temperature gas chromatographic (HTGC) separation of biological metabolites, statistical clustering and an in-house database (DB) searching.ResultsThe HTGC technique showed better detectability of high lipophilic compounds, particularly steroid-fatty acid esters, which generally have poor chromatographic properties on a conventional GC column. The in-house DB search consisted of the retention index and mass spectrum corresponding to each compound selected. The method was applied to tissue samples obtained from cardiac hypertrophy-induced mice. Increased levels of palmitic, linoleic, oleic, and stearic acids and cholesterol were detected and identified.ConclusionsThis data-dependent non-targeted metabolite profiling technique could be more effective in biomarker studies associated with the steroid and lipid metabolism than commercially available DBs.     

A multi-target screening analysis in human plasma using fast liquid chromatography-hybrid tandem mass spectrometry (Part II)

Objectives

Perform a comparison of results obtained with a LC-MS/MS method and a Remedi® instrument on clinical serum samples.

Design and methods

Results obtained on 146 selected plasma samples were compared between the two methods.

Results

On the 336 positive identifications, 89% were obtained using the LC-MS/MS technique and 57% by the LC-DAD. Benzodiazepines were well recognized by LC-MS/MS. For some compounds such as antidepressant agents, sensitivity was improved using LC-MS/MS. Moreover, this method extended the panel of drugs detected in clinical toxicology.

Conclusion

The new software platform developed for screening and identification of small molecules (SmileMS) allows an easy and reproducible detection of drugs and toxic compounds in blood for general unknown screening. It offers automated generation of reports, which makes the LC-MS/MS easier to use without having specialised skills in mass spectrometry.This LC-MS/MS screening method will be a reliable alternative to the Remedi® instrument in the global process of screening in emergency clinical toxicology laboratories.     

Evaluation of the diagnostic performance of an oral fluid screening test device for substance abuse at traffic controls

IntroductionThe aim of this study was to evaluate the analytical performance of the Kite Biotechnology Oral fluid (OF) screening test device, which is used for roadside screening of cannabis, opiates, amphetamines, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), cocaine and benzodiazepines by comparing samples with matched plasma samples, analysed via liquid chromatography–tandem mass spectrometry (LC-MS/MS) for confirmation.MethodsOF and plasma samples were obtained simultaneously from a total of 100 subjects. OF samples were analysed by OF screening test based on immunochromatography. The OF screening test cut-off values were 50 ng/mL for amphetamines (d-amphetamine) and methamphetamine/MDMA (d-methamphetamine), 30 ng/mL for cocaine (benzoylecgonine), 40 ng/mL for opiates (morphine), 20 ng/mL for benzodiazepines (nordazepam), and 25 ng/mL for cannabis (Δ⁹-tetrahydrocannabinol). LC-MS/MS method validation was performed according to the CLSI C62-A recommendations with the following parameters: matrix effect, lower limit of quantification (LLOQ), linearity, intra-day and inter-day precision and accuracy.ResultsThe overall specificity, accuracy and negative predictive values (NPV) were acceptable and met the DRUID standard of >80%. The OF screening test device showed good sensitivity for cocaine, amphetamines and opiates, whereas it indicated poor sensitivity for methamphetamine/MDMA (66.7%) and failed to detect cannabis and benzodiazepines.ConclusionThe present study is the first report to evaluate the Kite Biotechnology OF screening test device. The diagnostic performance of the OF screening test device was acceptable for opiates, cocaine and amphetamines, but it was insufficient for methamphetamine/MDMA, benzodiazepines and cannabis because of sensitivity issues.     

Identification of staphylococcal species based on variations in protein sequences (mass spectrometry) and DNA sequence (sodA microarray)

This report is among the first using sequence variation in newly discovered protein markers for staphylococcal (or indeed any other bacterial) speciation. Variation, at the DNA sequence level, in the sodA gene (commonly used for staphylococcal speciation) provided excellent correlation. Relatedness among strains was also assessed using protein profiling using microcapillary electrophoresis and pulsed field electrophoresis. A total of 64 strains were analyzed including reference strains representing the 11 staphylococcal species most commonly isolated from man (Staphylococcus aureus and 10 coagulase negative species [CoNS]). Matrix assisted time of flight ionization/ionization mass spectrometry (MALDI TOF MS) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC ESI MS/MS) were used for peptide analysis of proteins isolated from gel bands. Comparison of experimental spectra of unknowns versus spectra of peptides derived from reference strains allowed bacterial identification after MALDI TOF MS analysis. After LC-MS/MS analysis of gel bands bacterial speciation was performed by comparing experimental spectra versus virtual spectra using the software X!Tandem. Finally LC-MS/MS was performed on whole proteomes and data analysis also employing X!tandem. Aconitate hydratase and oxoglutarate dehydrogenase served as marker proteins on focused analysis after gel separation. Alternatively on full proteomics analysis elongation factor Tu generally provided the highest confidence in staphylococcal speciation.