Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

A validated assay by liquid chromatography-tandem mass spectrometry for the simultaneous quantification of elvitegravir and rilpivirine in HIV positive patients

Because of the large variability in the pharmacokinetics of anti-HIV drugs, therapeutic drug monitoring in patients may contribute to optimize the overall efficacy and safety of antiretroviral therapy. An LC–MS/MS method for the simultaneous assay in plasma of the novel antiretroviral agents rilpivirine (RPV) and elvitegravir (EVG) has been developed to that endeavor. Plasma samples (100 μL) extraction is performed by protein precipitation with acetonitrile, and the supernatant is subsequently diluted 1:1 with 20-mM ammonium acetate/MeOH 50:50. After reverse-phase chromatography, quantification of RPV and EVG, using matrix-matched calibration samples, is performed by electrospray ionization–triple quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. The stable isotopic-labeled compounds RPV-13C6 and EVG-D6 were used as internal standards. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effects variability (<6.4%), as well as EVG and RPV short and long-term stability in plasma. Calibration curves were validated over the clinically relevant concentrations ranging from 5 to 2500 ng/ml for RPV and from 50 to 5000 ng/ml for EVG. The method is precise (inter-day CV%: 3–6.3%) and accurate (3.8–7.2%). Plasma samples were found to be stable (<15%) in all considered conditions (RT/48 h, +4°C/48 h, −20°C/3 months and 60°C/1 h). Selected metabolite profiles analysis in patients' samples revealed the presence of EVG glucuronide, that was well separated from parent EVG, allowing to exclude potential interferences through the in-source dissociation of glucuronide to parent drug. This new, rapid and robust LCMS/MS assay for the simultaneous quantification of plasma concentrations of these two major new anti-HIV drugs EVG and RPV offers an efficient analytical tool for clinical pharmacokinetics studies and routine therapeutic drug monitoring service.

Multiplex Liquid Chromatography-Tandem Mass Spectrometry Assay for Simultaneous Therapeutic Drug Monitoring of Ribavirin, Boceprevir, and Telaprevir

New directly acting antivirals (DAAs) that inhibit hepatitis C virus (HCV) replication are increasingly used for the treatment of chronic hepatitis C. A marked pharmacokinetic variability and a high potential for drug-drug interactions between DAAs and numerous drug classes have been identified. In addition, ribavirin (RBV), commonly associated with hemolytic anemia, often requires dose adjustment, advocating for therapeutic drug monitoring (TDM) in patients under combined antiviral therapy. However, an assay for the simultaneous analysis of RBV and DAAs constitutes an analytical challenge because of the large differences in polarity among these drugs, ranging from hydrophilic (RBV) to highly lipophilic (telaprevir [TVR]). Moreover, TVR is characterized by erratic behavior on standard octadecyl-based reversed-phase column chromatography and must be separated from VRT-127394, its inactive C-21 epimer metabolite. We have developed a convenient assay employing simple plasma protein precipitation, followed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the simultaneous determination of levels of RBV, boceprevir, and TVR, as well as its metabolite VRT-127394, in plasma. This new, simple, rapid, and robust HPLC-MS/MS assay offers an efficient method of real-time TDM aimed at maximizing efficacy while minimizing the toxicity of antiviral therapy.

Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

Development of micro-electrospray mass spectrometry for ultra high sensitivity and application in the study of drugs of abuse on endogenous \lbrack Met\rbrack $\sp5$-enkephalin release

A micro-electrospray interface was developed specifically for the neurobiological applications described in this dissertation. Incorporation of a unique nano-flow liquid chromatography micro-electrospray "needle" into the micro-electrospray interface (micro-ES/MS) increased the sensitivity of the mass spectrometric assay by $\sim$1000 fold and thus permitted the first analysis of specific neuroactive compounds in brain extracellular fluid collected by in vivo microdialysis (Md).^ Initial in vivo data presented deals with the pharmacodynamics of a novel GABA$\sb{\rm B}$ antagonist and the availability of the compound in its parent (unmetabolized) form to the brain of the anesthetized rat. Next, the first structurally specific endogenous release of (Met) $\sp5$-enkephalin was demonstrated in unanesthetized freely-moving animals (release of $\sim$6.5 fmole of (Met) $\sp5$-enkephalin into the dialysate by direct neuronal depolarization). The Md/micro-ES/MS system was used to test the acute effects of drugs of abuse on the endogenous release of (Met) $\sp5$-enkephalin from the globus pallidus/ventral pallidum brain region in rats. Four drugs known to be abused by man (morphine, cocaine, methamphetamine and diazepam) were tested. Morphine and cocaine both elicited a two-fold or more increase in the release of (Met) $\sp5$-enkephalin over vehicle controls. Diazepam elicited a small decrease in (Met) $\sp5$-enkephalin levels and methamphetamine showed no significant effect on (Met) $\sp5$-enkephalin. These results imply that (Met) $\sp5$-enkephalin may be involved in the reward pathway of certain drugs of abuse. ^

Multiple inert gas elimination technique by micropore membrane inlet mass spectrometry--a comparison with reference gas chromatography.

The mismatching of alveolar ventilation and perfusion (VA/Q) is the major determinant of impaired gas exchange. The gold standard for measuring VA/Q distributions is based on measurements of the elimination and retention of infused inert gases. Conventional multiple inert gas elimination technique (MIGET) uses gas chromatography (GC) to measure the inert gas partial pressures, which requires tonometry of blood samples with a gas that can then be injected into the chromatograph. The method is laborious and requires meticulous care. A new technique based on micropore membrane inlet mass spectrometry (MMIMS) facilitates the handling of blood and gas samples and provides nearly real-time analysis. In this study we compared MIGET by GC and MMIMS in 10 piglets: 1) 3 with healthy lungs; 2) 4 with oleic acid injury; and 3) 3 with isolated left lower lobe ventilation. The different protocols ensured a large range of normal and abnormal VA/Q distributions. Eight inert gases (SF6, krypton, ethane, cyclopropane, desflurane, enflurane, diethyl ether, and acetone) were infused; six of these gases were measured with MMIMS, and six were measured with GC. We found close agreement of retention and excretion of the gases and the constructed VA/Q distributions between GC and MMIMS, and predicted PaO2 from both methods compared well with measured PaO2. VA/Q by GC produced more widely dispersed modes than MMIMS, explained in part by differences in the algorithms used to calculate VA/Q distributions. In conclusion, MMIMS enables faster measurement of VA/Q, is less demanding than GC, and produces comparable results.

On the interference of Kr during carbon isotope analysis of methane using continuous-flow combustion–isotope ratio mass spectrometry

Stable carbon isotope analysis of methane (delta C-13 of CH4) on atmospheric samples is one key method to constrain the current and past atmospheric CH4 budget. A frequently applied measurement technique is gas chromatography (GC) isotope ratio mass spectrometry (IRMS) coupled to a combustion-preconcentration unit. This report shows that the atmospheric trace gas krypton (Kr) can severely interfere during the mass spectrometric measurement, leading to significant biases in delta C-13 of CH4, if krypton is not sufficiently separated during the analysis. According to our experiments, the krypton interference is likely composed of two individual effects, with the lateral tailing of the doubly charged Kr-86 peak affecting the neighbouring m/z 44 and partially the m/z 45 Faraday cups. Additionally, a broad signal affecting m/z 45 and especially m/z 46 is assumed to result from scattered ions of singly charged krypton. The introduced bias in the measured isotope ratios is dependent on the chromatographic separation, the krypton-to-CH4 mixing ratio in the sample, the focusing of the mass spectrometer as well as the detector configuration and can amount to up to several per mil in delta C-13. Apart from technical solutions to avoid this interference, we present correction routines to a posteriori remove the bias.

Elucidation of Nucleic Acid–Drug Interactions by Tandem Mass Spectrometry

In continuation of the long tradition of mass spectrometric research at the University of Bern, our group focuses on the characterization of nucleic acids as therapeutic agents and as drug targets. This article provides a short overview of our recent work on platinated single-stranded and higher-order nucleic acids. Nearly three decades ago the development of soft ionization techniques opened a whole new chapter in the mass spectrometric analysis of not only nucleic acids themselves, but also their interactions with potential drug candidates. In contrast to modern next generation sequencing approaches, though, the goal of the tandem mass spectrometric investigation of nucleic acids is by no means the complete sequencing of genetic DNA, but rather the characterization of short therapeutic and regulatory oligonucleotides and the elucidation of nucleic acid–drug interactions. The influence of cisplatin binding on the gas-phase dissociation of nucleic acids was studied by the means of electrospray ionization tandem mass spectrometry. Experiments on native and modified DNA and RNA oligomers confirmed guanine base pairs as the preferred platination site and laid the basis for the formulation of a gas-phase fragmentation mechanism of platinated oligonucleotides. The study was extended to double stranded DNA and DNA quadruplexes. While duplexes are believed to be the main target of cisplatin in vivo, the recently discovered DNA quadruplexes constitute another promising target for anti-tumor drugs owing to their regulatory functions in the cell cycle.

Combined Secondary Ion Mass Spectrometry Depth Profiling and Focused Ion Beam Analysis of Cu Films Electrodeposited under Oscillatory Conditions

The recrystallization behavior of Cu films electrodeposited under oscillatory conditions in the presence of plating additives was studied by means of secondary ion mass spectrometry (SIMS) and focused ion beam analysis. When combined with bis-(sodium-sulfopropyl)-disulfide (SPS), Imep levelers (polymerizates of imidazole and epichlorohydrin) show characteristic oscillations in the galvanostatic potential/time transient measurements. These are related to the periodic degradation and restoration of the active leveler ensemble at the interface. The leveler action relies on adduct formation between the Imep and MPS (mercaptopropane sulfonic acid)-stabilized CuI complexes that appear as intermediates of the copper deposition when SPS is present in the electrolyte. SIMS depth profiling proves that additives are incorporated into the growing film preferentially under transient conditions during the structural breakdown of the leveler ensemble and its subsequent restoration. In contrast, Cu films electrodeposited in the presence of a structurally intact Imep–CuI–MPS ensemble remain largely contamination free.

Analytic validation of a gas chromatography-mass spectrometry method for quantification of six amino acids in canine serum samples.

OBJECTIVE To analytically validate a gas concentration of chromatography-mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at -80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at -80°C for 1 week had no effect on GC-MS results.

Wet and dry deposition of 129I in Seville (Spain) measured by accelerator mass spectrometry

Iodine-129 (Full-size image (<1 K)) concentrations have been determined by accelerator mass spectrometry in rainwater samples taken at Seville (southwestern Spain) in 1996 and 1997. This technique allows a reduction in the detection limits for this radionuclide in comparison to radiometric counting and other mass spectrometric methods such as ICP-MS. Typical 129I concentrations range from 4.7×107129I atoms/l (19.2%) to 4.97×109129I atoms/l (5.9%), while 129I depositions are normally in the order of 108–1010 atoms/m2 d. These values agree well with other results obtained for recent rainwater samples collected in Europe. Apart from these, the relationship between 129I deposition and some atmospheric factors has been analyzed, showing the importance of the precipitation rate and the concentration of suspended matter in it.

Characterization and source apportionment of organic aerosol using offline aerosol mass spectrometry

Field deployments of the Aerodyne Aerosol Mass Spectrometer (AMS) have significantly advanced real-time measurements and source apportionment of non-refractory particulate matter. However, the cost and complex maintenance requirements of the AMS make its deployment at sufficient sites to determine regional characteristics impractical. Furthermore, the negligible transmission efficiency of the AMS inlet for supermicron particles significantly limits the characterization of their chemical nature and contributing sources. In this study, we utilize the AMS to characterize the water-soluble organic fingerprint of ambient particles collected onto conventional quartz filters, which are routinely sampled at many air quality sites. The method was applied to 256 particulate matter (PM) filter samples (PM1, PM2:5, and PM10, i.e., PM with aerodynamic diameters smaller than 1, 2.5, and 10 μm, respectively), collected at 16 urban and rural sites during summer and winter. We show that the results obtained by the present technique compare well with those from co-located online measurements, e.g., AMS or Aerosol Chemical Speciation Monitor (ACSM). The bulk recoveries of organic aerosol (60–91 %) achieved using this technique, together with low detection limits (0.8 μg of organic aerosol on the analyzed filter fraction) allow its application to environmental samples. We will discuss the recovery variability of individual hydrocarbon ions, ions containing oxygen, and other ions. The performance of such data in source apportionment is assessed in comparison to ACSM data. Recoveries of organic components related to different sources as traffic, wood burning, and secondary organic aerosol are presented. This technique, while subjected to the limitations inherent to filter-based measurements (e.g., filter artifacts and limited time resolution) may be used to enhance the AMS capabilities in measuring size-fractionated, spatially resolved longterm data sets.

Sensitivity and Fragmentation Calibration of the ROSINA Reflectron-type Time-Of-Flight Mass Spectrometer

The goal of this work was to increase the performance and to calibrate one of the ROSINA sensors, the Reflectron-type Time-Of-Flight mass spectrometer, currently flying aboard the ESA Rosetta spacecraft. Different optimization techniques were applied to both the lab and space models, and a static calibration was performed using different gas species expected to be detected in the vicinity of comet 67P/Churyumov-Gerasimenko. The database thus created was successfully applied to space data, giving consistent results with the other ROSINA sensors.