Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR-based rapid diagnosis of Staphylococcus aureus bacteraemia.

Effective empirical treatment is of paramount importance to improve the outcome of patients with Staphylococcus aureus bacteraemia. We aimed to evaluate a PCR-based rapid diagnosis of methicillin resistance (GeneXpert MRSA) after early detection of S. aureus bacteraemia using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Patients with a first episode of S. aureus bacteraemia identified using MALDI-TOF MS were randomized in a prospective interventional open study between October 2010 and August 2012. In the control group, antibiotic susceptibility testing was performed after MALDI-TOF MS identification on blood culture pellets. In the intervention group, a GeneXpert MRSA was performed after S. aureus identification. The primary outcome was the performance of GeneXpert MRSA directly on blood cultures. We then assessed the impact of early diagnosis of methicillin resistance on the empirical treatment. In all, 197 episodes of S. aureus bacteraemia were included in the study, of which 106 were included in the intervention group. Median time from MALDI-TOF MS identification to GeneXpert MRSA result was 97 min (range 25-250). Detection of methicillin resistance using GeneXpert MRSA had a sensitivity of 99% and a specificity of 100%. There was less unnecessary coverage of MRSA in the intervention group (17.1% versus 29.2%, p 0.09). GeneXpert MRSA was highly reliable in diagnosing methicillin resistance when performed directly on positive blood cultures. This could help to avoid unnecessary prescriptions of anti-MRSA agents and promote the introduction of earlier adequate coverage in unsuspected cases.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, a revolution in clinical microbial identification.

Until recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for the identification of microorganisms remained confined to research laboratories. In the last 2 years, the availability of relatively simple to use MALDI-TOF MS devices, which can be utilized in clinical microbiology laboratories, has changed the laboratory workflows for the identification of pathogens. Recently, the first prospective studies regarding the performance in routine bacterial identification showed that MALDI-TOF MS is a fast, reliable and cost-effective technique that has the potential to replace and/or complement conventional phenotypic identification for most bacterial strains isolated in clinical microbiology laboratories. For routine bacterial isolates, correct identification by MALDI-TOF MS at the species level was obtained in 84.1-93.6% of instances. In one of these studies, a protein extraction step clearly improved the overall valid identification yield, from 70.3% to 93.2%. This review focuses on the current state of use of MALDI-TOF MS for the identification of routine bacterial isolates and on the main difficulties that may lead to erroneous or doubtful identifications.

Determination of plasma levels of citalopram and its demethylated and deaminated metabolites by gas chromatography and gas chromatography-mass spectrometry.

Sensitive and specific methods based on gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) for the determination of levels of citalopram, desmethylcitalopram and didesmethylcitalopram in the plasma of patients treated with citalopram are presented, as well as a GC-MS procedure for the assay of the citalopram propionic acid derivative. After addition of a separate internal standard for each drug, liquid-solvent extraction is used to separate the basic compounds from the acid compounds. The demethylated amines are derivatized with trifluoroacetic anhydride, and the acid metabolite with methyl iodide. GC-MS is performed in the electron impact mode, as mass spectrometry by the (positive-ion) chemical ionization mode (methane and ammonia) appeared to be unsuitable. The limits of quantification were 1 ng/ml for citalopram and desmethylcitalopram and 2 ng/ml for the other metabolites. The correlation coefficients for the calibration curves (range 10-500 ng/ml) were > or = 0.999 for all compounds, whether determined by GC or GC-MS.

Impact of Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry on the Clinical Management of Patients With Gram-negative Bacteremia: A Prospective Observational Study.

Background. Early identification of pathogens from blood cultures using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may optimize the choice of empirical antibiotic therapy in the setting of bloodstream infections. We aimed to assess the impact of this new technology on the use of antibiotic treatment in patients with gram-negative bacteremia. Methods. We conducted a prospective observational study from January to December 2010 to evaluate the sequential and separate impacts of Gram stain reporting and MALDI-TOF bacterial identification performed on blood culture pellets in patients with gram-negative bacteremia. The primary outcome was the impact of MALDI-TOF on empirical antibiotic choice. Results. Among 202 episodes of gram-negative bacteremia, Gram stain reporting had an impact in 42 cases (20.8%). MALDI-TOF identification led to a modification of empirical therapy in 71 of all 202 cases (35.1%), and in 16 of 27 cases (59.3%) of monomicrobial bacteremia caused by AmpC-producing Enterobacteriaceae. The most frequently observed impact was an early appropriate broadening of the antibiotic spectrum in 31 of 71 cases (43.7%). In total, 143 of 165 episodes (86.7%) of monomicrobial bacteremia were correctly identified at genus level by MALDI-TOF. Conclusions. In a low prevalence area for extended spectrum betalactamases (ESBL) and multiresistant gram-negative bacteria, MALDI-TOF performed on blood culture pellets had an impact on the clinical management of 35.1% of all gram-negative bacteremia cases, demonstrating a greater impact than Gram stain reporting. Thus, MALDI-TOF could become a vital second step beside Gram stain in guiding the empirical treatment of patients with bloodstream infection.

Silver-assisted laser desorption ionization for high spatial resolution imaging mass spectrometry of olefins from thin tissue sections.

Silver has been demonstrated to be a powerful cationization agent in mass spectrometry (MS) for various olefinic species such as cholesterol and fatty acids. This work explores the utility of metallic silver sputtering on tissue sections for high resolution imaging mass spectrometry (IMS) of olefins by laser desorption ionization (LDI). For this purpose, sputtered silver coating thickness was optimized on an assorted selection of mouse and rat tissues including brain, kidney, liver, and testis. For mouse brain tissue section, the thickness was adjusted to 23 ± 2 nm of silver to prevent ion suppression effects associated with a higher cholesterol and lipid content. On all other tissues, a thickness of at 16 ± 2 nm provided the best desorption/ionization efficiency. Characterization of the species by MS/MS showed a wide variety of olefinic compounds allowing the IMS of different lipid classes including cholesterol, arachidonic acid, docosahexaenoic acid, and triacylglyceride 52:3. A range of spatial resolutions for IMS were investigated from 150 μm down to the high resolution cellular range at 5 μm. The applicability of direct on-tissue silver sputtering to LDI-IMS of cholesterol and other olefinic compounds presents a novel approach to improve the amount of information that can be obtained from tissue sections. This IMS strategy is thus of interest for providing new biological insights on the role of cholesterol and other olefins in physiological pathways or disease.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

MSight: an image analysis software for liquid chromatography-mass spectrometry.

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Orthogonal organic and aqueous-based washes of tissue sections to enhance protein sensitivity by MALDI imaging mass spectrometry.

Imaging mass spectrometry (IMS) is an emergent and innovative approach for measuring the composition, abundance and regioselectivity of molecules within an investigated area of fixed dimension. Although providing unprecedented molecular information compared with conventional MS techniques, enhancement of protein signature by IMS is still necessary and challenging. This paper demonstrates the combination of conventional organic washes with an optimized aqueous-based buffer for tissue section preparation before matrix-assisted laser desorption/ionization (MALDI) IMS of proteins. Based on a 500 mM ammonium formate in water-acetonitrile (9:1; v/v, 0.1% trifluororacetic acid, 0.1% Triton) solution, this buffer wash has shown to significantly enhance protein signature by profiling and IMS (~fourfold) when used after organic washes (70% EtOH followed by 90% EtOH), improving the quality and number of ion images obtained from mouse kidney and a 14-day mouse fetus whole-body tissue sections, while maintaining a similar reproducibility with conventional tissue rinsing. Even if some protein losses were observed, the data mining has demonstrated that it was primarily low abundant signals and that the number of new peaks found is greater with the described procedure. The proposed buffer has thus demonstrated to be of high efficiency for tissue section preparation providing novel and complementary information for direct on-tissue MALDI analysis compared with solely conventional organic rinsing.

Therapeutic Drug Monitoring of the new targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by LC tandem mass spectrometry.

The treatment of some cancer patients has shifted from traditional, non-specific cytotoxic chemotherapy to chronic treatment with molecular targeted therapies. Imatinib mesylate, a selective inhibitor of tyrosine kinases (TKIs) is the most prominent example of this new era and has opened the way to the development of several additional TKIs, including sunitinib, nilotinib, dasatinib, sorafenib and lapatinib, in the treatment of various hematological malignancies and solid tumors. All these agents are characterized by an important inter-individual pharmacokinetic variability, are at risk for drug interactions, and are not devoid of toxicity. Additionally, they are administered for prolonged periods, anticipating the careful monitoring of their plasma exposure via Therapeutic Drug Monitoring (TDM) to be an important component of patients' follow-up. We have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 100 microL of plasma for the simultaneous determination of the six major TKIs currently in use. Plasma is purified by protein precipitation and the supernatant is diluted in ammonium formate 20 mM (pH 4.0) 1:2. Reverse-phase chromatographic separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2 and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 20 min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<9.6%), overall process efficiency (87.1-104.2%), as well as TKIs short- and long-term stability in plasma. The method is precise (inter-day CV%: 1.3-9.4%), accurate (-9.2 to +9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL). This is the first broad-range LC-MS/MS assay covering the major currently in-use TKIs. It is an improvement over previous methods in terms of convenience (a single extraction procedure for six major TKIs, reducing significantly the analytical time), sensitivity, selectivity and throughput. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of the latest TKIs developed after imatinib and better define their therapeutic ranges in different patient populations in order to evaluate whether a systematic TDM-guided dose adjustment of these anticancer drugs could contribute to minimize the risk of major adverse reactions and to increase the probability of efficient, long lasting, therapeutic response.

Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

New insights in carbohydrate-deficient transferrin analysis with capillary electrophoresis-mass spectrometry

Capillary zone electrophoresis (CZE) with UV detection has been widely used for the determination of carbohydrate-deficient transferrin (CDT), an indirect marker of the chronic alcohol consumption (≥60-80g/day). A commercially available method (CEofix? CDT kit), containing a bilayer anionic coating, allows for the analysis of CDT with a high resolution between transferrin (Tf) glycoforms with reduced protein adsorption onto the capillary wall. Although widely used in routine analysis, this procedure presents some limitations in terms of selectivity and sensitivity which may be overcome with mass spectrometry (MS). However, the available method is not MS-compatible due to the non-volatile coating as well as the phosphate and borate buffers present in the background electrolyte (BGE). This study firstly consisted in developing MS-compatible separation conditions, i.e., coating and BGE compositions. Numerous cationic, neutral, and anionic coatings were evaluated in combination with BGEs covering a broad range of pH values. A bilayer coating composed of a cationic layer of 10% polybrene (m/v) and an anionic layer of 10% dextran sulfate (m/v) combined with a BGE composed of 20mM ammonium acetate at pH 8.5 provided the best results in terms of glycoforms' resolution, efficiency, adsorption reduction, migration times' repeatability, and coating stability. The method was then transferred to CZE-MS after investigations of the electrospray ionization (ESI) source, equipped with a sheath-flow interface, and the time-of-flight (TOF/MS) parameters. A successful MS detection of tetrasialo-Tf was obtained during infusion, while the experiments highlighted the challenges and issues encountered with intact glycoprotein analysis by CZE-ESI-MS.