Quadrupole Tandem Mass Spectrometer Research Articles

Experimental study of the reaction of NO2− ions with CO2 molecules at temperatures and energies relevant to the Martian atmosphere

Comprehensive theoretical models of the Martian ionosphere require reliable kinetic data for ion – molecule reactions over a wide range of temperatures. In situ measurements of the negative ions have not yet been performed and so ion chemical modelling is relied upon, which indicate that nitrite anions, NO2−, are important. Thus, in the present work the gas phase reaction of NO2− with carbon dioxide molecules, CO2, has been studied at the temperatures and energies relevant to the Martian atmosphere. The rate coefficient was measured over the temperature range 220 ± 2 K to 334 ± 2 K using a variable temperature selected ion flow tube (VT-SIFT) instrument. Additionally, a commercial Tandem Quadrupole Mass Spectrometer (TQMS) has been exploited to explore the reaction occurring during NO2−/CO2 collisions over the centre-of-mass energies from near-thermal to 6 eV. Two different channels of the reaction were studied. Under the thermal conditions of the VT-SIFT, the reaction is seen to proceed slowly via ternary association ultimately producing CO3– ions, the measured effective ternary rate coefficient in helium being k3 = (3.8 ± 1.5) × 10−30 cm6 s−1 at 220 ± 2 K and k3 = (0.5 ± 0.2) × 10−30 cm6 s−1 at 334 ± 2 K. The effective ternary rate coefficient can be greater in the Martian atmosphere where CO2 is the stabilizing gas. In the TQMS experiment, CO3– production occurs via a binary reaction above a threshold energy of 1.8 ± 0.2 eV. These experimental data can be used to refine the existing ion-chemical models of the Martian atmosphere.

Abstract 5269: Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular carcinoma

Abstract Introduction: Hepatocellular carcinoma (HCC) is one of the commonest and the leading cause of cancer death globally. The main cause of this high mortality rate is that patients with HCC are usually diagnosed at late stages since they have only subtle clinical presentations at early stages. This late diagnosis is a result of the requirement of high-end imaging modalities, the use of invasive procedures, and the lack of reliable tumour biomarkers. Metabolomics is the study of metabolites in an organism in response to the changes in health status. The purpose of this study was to identify and validate plasma acylcarnitines as potential diagnostic biomarkers of HCC by using techniques in the field of metabolomics. Methods: 228 EDTA-plasma samples of healthy controls, cirrhotic patients, or treatment-naïve patients with HCC, collected from Imperial College Healthcare Tissue Bank, were used as a discovery and training set. Patient factors, including basic patient characteristics, aetiologies of HCC, and Child-Pugh scores, and data relating to tumours, such as the multiplicity, the sizes in diameter, the status of macro-vascular invasion, were collected. A targeted assay was adapted to measure concentrations of carnitine and acylcarnitines with different chain length using the TQ-S tandem quadrupole mass spectrometer. Non-parametric univariate analyses were applied to evaluate the statistical difference between HCC and cirrhosis. Logistic regression was preferred to develop diagnostic models. A Gambian cohort of 165 plasma samples was then used as an external validation. Results: Among sixteen measured carnitine and acylcarnitines, three acylcarnitines, namely acetylcarnitine, succinylcarnitine, and hexanoylcarnitine, statistically increased in HCC patients comparing to cirrhotic patients. A linear model incorporating these three acylcarnitines and alpha-fetoprotein was then developed. The area under the ROC curve of this multivariable regression model was 0.782 (95% C.I., 0.695-0.870) in the training set. When applied to the validation cohort, the sensitivity and specificity of this model at identifying HCC patients from cirrhosis were 82.2% and 67.8%, respectively. Conclusion: This report provides a comprehensive profile of the changes of carnitine and acylcarnitines among HCC, cirrhotic patients and healthy controls, and determines the value of acylcarnitines as diagnostic biomarkers for HCC. Citation Format: Eric Yi-Liang Shen, Abellona U, Simon Taylor-Robinson, Mark Thursz, Elaine Holmes, Jeremy Nicholson. Discovery and validation of plasma acylcarnitines for the early diagnosis of hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5269.

Rapid diagnosis of viral infections using the real-time fluorescence loop-mediated isothermal amplification

Ceftolozane, in combination with the β-lactamase inhibitor tazobactam, is a new option in the pipeline against multidrug-resistant Gram-negative bacilli. As for other β-lactam antibiotics, optimizing the use of ceftolozane-tazobactam is advisable, especially in difficult-to-treat infections. In this regard, therapeutic drug monitoring would be required to guide the treatment of ceftolozane-tazobactam. Thus, we aimed to develop and validate procedures based on UHPLC-MS/MS for measurement of ceftolozane and tazobactam plasma concentrations in clinical practice.Analyses were conducted using an Acquity® UPLC® integrated system coupled to an Acquity® TQD® tandem-quadrupole mass spectrometer. Ceftolozane, tazobactam and their internal standards (ceftazidime-D5 and sulbactam) were detected by electrospray ionization mass spectrometry in positive and negative ion multiple reaction monitoring modes, using transitions of 667.2 → 199.3/139.0 and 551.9 → 467.9 for ceftolozane and ceftazidime-D5, and 299.0 → 138/254.9 and 232.0 → 140.0 for tazobactam and sulbactam. Measurement procedures developed were used for guiding the treatment and adjusting daily dose of ceftolozane-tazobactam in patients with osteoarticular infections.Coefficients of variation and absolute relative biases were <7.9% and 6.5% in all cases. The lower limit of quantification, linearity, normalized-recoveries, normalized-matrix effects and measurement uncertainties for ceftolozane were: 0.97 mg/L, (0.97–125) mg/L, ≤113.6%, ≤108.7%, and ≤ 18.7%, respectively; and for tazobactam: 1.04 mg/L, (1.04–125) mg/L, ≤103.6%, ≤101.9%, and ≤ 20.0%. No interferences and carry-over were observed. Patients plasma concentrations were higher than the recommended 3–4 times the minimal inhibitory concentrations.Our measurement procedures are suitable for therapeutic drug monitoring of ceftolozane-tazobactam in patients with osteoarticular infections.

Evaluation of the pre-analytical ACL TOP 750 module (HIL) for hemolysis assessment

Ceftolozane, in combination with the β-lactamase inhibitor tazobactam, is a new option in the pipeline against multidrug-resistant Gram-negative bacilli. As for other β-lactam antibiotics, optimizing the use of ceftolozane-tazobactam is advisable, especially in difficult-to-treat infections. In this regard, therapeutic drug monitoring would be required to guide the treatment of ceftolozane-tazobactam. Thus, we aimed to develop and validate procedures based on UHPLC-MS/MS for measurement of ceftolozane and tazobactam plasma concentrations in clinical practice.Analyses were conducted using an Acquity® UPLC® integrated system coupled to an Acquity® TQD® tandem-quadrupole mass spectrometer. Ceftolozane, tazobactam and their internal standards (ceftazidime-D5 and sulbactam) were detected by electrospray ionization mass spectrometry in positive and negative ion multiple reaction monitoring modes, using transitions of 667.2 → 199.3/139.0 and 551.9 → 467.9 for ceftolozane and ceftazidime-D5, and 299.0 → 138/254.9 and 232.0 → 140.0 for tazobactam and sulbactam. Measurement procedures developed were used for guiding the treatment and adjusting daily dose of ceftolozane-tazobactam in patients with osteoarticular infections.Coefficients of variation and absolute relative biases were <7.9% and 6.5% in all cases. The lower limit of quantification, linearity, normalized-recoveries, normalized-matrix effects and measurement uncertainties for ceftolozane were: 0.97 mg/L, (0.97–125) mg/L, ≤113.6%, ≤108.7%, and ≤ 18.7%, respectively; and for tazobactam: 1.04 mg/L, (1.04–125) mg/L, ≤103.6%, ≤101.9%, and ≤ 20.0%. No interferences and carry-over were observed. Patients plasma concentrations were higher than the recommended 3–4 times the minimal inhibitory concentrations.Our measurement procedures are suitable for therapeutic drug monitoring of ceftolozane-tazobactam in patients with osteoarticular infections.

The effect of infusion solutions on endothelial cell glycocalyx

Ceftolozane, in combination with the β-lactamase inhibitor tazobactam, is a new option in the pipeline against multidrug-resistant Gram-negative bacilli. As for other β-lactam antibiotics, optimizing the use of ceftolozane-tazobactam is advisable, especially in difficult-to-treat infections. In this regard, therapeutic drug monitoring would be required to guide the treatment of ceftolozane-tazobactam. Thus, we aimed to develop and validate procedures based on UHPLC-MS/MS for measurement of ceftolozane and tazobactam plasma concentrations in clinical practice.Analyses were conducted using an Acquity® UPLC® integrated system coupled to an Acquity® TQD® tandem-quadrupole mass spectrometer. Ceftolozane, tazobactam and their internal standards (ceftazidime-D5 and sulbactam) were detected by electrospray ionization mass spectrometry in positive and negative ion multiple reaction monitoring modes, using transitions of 667.2 → 199.3/139.0 and 551.9 → 467.9 for ceftolozane and ceftazidime-D5, and 299.0 → 138/254.9 and 232.0 → 140.0 for tazobactam and sulbactam. Measurement procedures developed were used for guiding the treatment and adjusting daily dose of ceftolozane-tazobactam in patients with osteoarticular infections.Coefficients of variation and absolute relative biases were <7.9% and 6.5% in all cases. The lower limit of quantification, linearity, normalized-recoveries, normalized-matrix effects and measurement uncertainties for ceftolozane were: 0.97 mg/L, (0.97–125) mg/L, ≤113.6%, ≤108.7%, and ≤ 18.7%, respectively; and for tazobactam: 1.04 mg/L, (1.04–125) mg/L, ≤103.6%, ≤101.9%, and ≤ 20.0%. No interferences and carry-over were observed. Patients plasma concentrations were higher than the recommended 3–4 times the minimal inhibitory concentrations.Our measurement procedures are suitable for therapeutic drug monitoring of ceftolozane-tazobactam in patients with osteoarticular infections.

Advances in LC-MS/MS Methods for Allergen Testing, Meat Speciation, and Gelatin Speciation.

Background: Food authenticity is demanded by the consumer at all times. The consumer places trust in the manufacturer that the food product is genuine in terms of what is recorded on the packaging label. Objective: Recent advancements in LC-tandem MS methodology in the detection of allergens, meat, and gelatin speciation in raw food products and processed foods are detailed in this paper. Method: For each of the three methods, initial proteome analysis and the screening leading to the determination of unique tryptic peptides were conducted using a high-resolution, accurate tandem mass spectrometer. Having identified the unique markers, the method was transferred to a tandem quadrupole mass spectrometer for a higher-sensitivity quantitative study, multiple reaction monitoring transition analysis. Results: For the allergens method a detection limit of at least 10 ppm was attained across the 12 allergen peptides in this workflow. In the gluten workflow the resulting chromatograms show good detection down to 5 ppm, with no interference from the food matrices. The meat speciation method details that signature peptides could be readily identified at 1% w/w with no matrix interference. Conclusions: These single-injection workflows with cycle-time optimization enable wide coverage of analytes to identify multiple species within challenging matrix samples.

Bioanalytical Method Development for the Detection of the Rac/Cdc42 Inhibitor MBQ‐167 in Mouse Tissue

Metastatic breast cancer is the second most common cancer in US women. The mortality rate has decreased approximately 40% in the last 25 years due to advancement in treatment and diagnostics. However, it is estimated that 20–30% of all breast cancer patients will develop metastatic breast cancer. Therefore, there is a need to develop targeted therapeutics to reduce cancer cell migration and subsequent metastasis. The novel small molecule MBQ‐167 inhibits Rac and Cdc‐42, proteins involved in cancer cell growth, migration and invasion. In vivo studies have shown that MBQ‐167 inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90% (Humphries‐Bickley, et al., 2017). However, to continue validating this drug for FDA approval, it is necessary to study the pharmacokinetic profile of the drug, which includes elucidating the tissue distribution of the compound in the body. Hence, it is necessary to develop a rapid and sensitive method to quantify MBQ‐167 in tissues. The purpose of this study was to develop and validate a method for the quantification of MBQ‐167 in mouse tissues using Supercritical fluid chromatography coupled with electrospray ionization tandem mass spectrometry (SFC‐MS/MS). Mouse livers were homogenized, spiked with MBQ‐167, and liquid‐liquid extractions were performed with different organic solvents (heptane, ethyl acetate, 2‐propanol, toluene, and acetonitrile), in order to determine the optimal sample preparation method to selectively separate the drug MBQ‐167 from endogenous material in mouse tissue. Separation was performed on an ACQUITY UPC2BEH (3.0 × 100 mm, 1.7 μm) column at 40 °C with a mobile phase consisting of CO2 and methanol 0.1 % formic acid (95:5, v/v) at a flow rate of 1 mL/min. A tandem triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode. High recovery of MBQ‐167 (&gt; 65%) and lower matrix effects were observed upon extraction with heptane:ethyl acetate (1:1). Consistent recovery was observed in other tissues such as kidney, lungs and heart. The method was sensitive with a lower limit of quantification (LLOQ) of 10 ng/mL. Linearity was observed over the concentration range of 10–1000 ng/mL. The data obtained supports the use of SFC‐MS/MS as a highly specific method to evaluate the tissue distribution of MBQ‐167 in mice.Support or Funding InformationThis study is supported by MARC grant NIH/NIGMS 5T34GM007821‐39 (to GRG) and NIGMS‐RISE 25 GM061838 (to MM).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Nested Case–Control Study on Plasma Vitamin E and Risk of Cancer: Evidence of Effect Modification by Selenium

BackgroundEvidence from epidemiologic studies has been inconsistent regarding the role of vitamin E in cancer incidence risk. ObjectiveThe aim of this study was to evaluate the prospective association between baseline plasma vitamin E levels and subsequent cancer risk in Chinese adults with hypertension, and to identify effect modifiers. DesignA nested, case–control study was conducted from 20,702 hypertensive participants in the China Stroke Primary Prevention Trial, a randomized, double-blind, controlled trial, conducted from May 2008 to August 2013. ParticipantsThe current study included 229 new cancer cases and 229 controls matched for age (±1 year), sex, treatment group, and study site. Main outcome measuresPlasma vitamin E was measured by liquid chromatography with tandem quadrupole mass spectrometers and plasma selenium was measured by inductively coupled plasma mass spectrometry using Thermo Fisher iCAP Q ICP-MS. Statistical analysesOdds ratios (OR) of cancer in relation to plasma concentrations of vitamin E were calculated using conditional logistic regression models. ResultsMedian follow-up duration was 4.5 years. Overall, vitamin E was not associated with subsequent risk of total cancer (per 1-mg/L [2.3 μmol/L] increase: OR 1.01, 95% CI 0.93 to 1.09) and non-gastrointestinal cancer (OR 1.10, 95% CI 0.98 to 1.24). However, there was a significant, inverse association between vitamin E and gastrointestinal cancer (OR 0.86, 95% CI 0.75 to 0.99), particularly esophageal cancer (OR 0.67, 95% CI 0.48 to 0.95). Moreover, high vitamin E decreased the risk of total cancer (OR 0.91, 95% CI 0.84 to 0.99) and gastrointestinal cancer (OR 0.83, 95% CI 0.73 to 0.95) among patients with high selenium levels (median≥83.7 μg/L [1.1 μmol/L]), and increased the risk of total cancer (OR 1.13, 95% CI 1.00 to 1.26) and non-gastrointestinal cancer (OR 1.25, 95% CI 1.03 to 1.50) among those with low selenium levels (<83.7 μg/L [1.1 μmol/L]). ConclusionsThis study suggests that higher levels of plasma vitamin E are associated with reduced risk of gastrointestinal cancer. High vitamin E decreased the risk of total cancer among patients with high selenium levels, but increased the risk of total cancer among those with low selenium levels.

Measurement of ceftolozane and tazobactam concentrations in plasma by UHPLC-MS/MS. Clinical application in the management of difficult-to-treat osteoarticular infections

BackgroundCeftolozane, in combination with the β-lactamase inhibitor tazobactam, is a new option in the pipeline against multidrug-resistant Gram-negative bacilli. As for other β-lactam antibiotics, optimizing the use of ceftolozane-tazobactam is advisable, especially in difficult-to-treat infections. In this regard, therapeutic drug monitoring would be required to guide the treatment of ceftolozane-tazobactam. Thus, we aimed to develop and validate procedures based on UHPLC-MS/MS for measurement of ceftolozane and tazobactam plasma concentrations in clinical practice. Material and methodsAnalyses were conducted using an Acquity® UPLC® integrated system coupled to an Acquity® TQD® tandem-quadrupole mass spectrometer. Ceftolozane, tazobactam and their internal standards (ceftazidime-D5 and sulbactam) were detected by electrospray ionization mass spectrometry in positive and negative ion multiple reaction monitoring modes, using transitions of 667.2 → 199.3/139.0 and 551.9 → 467.9 for ceftolozane and ceftazidime-D5, and 299.0 → 138/254.9 and 232.0 → 140.0 for tazobactam and sulbactam. Measurement procedures developed were used for guiding the treatment and adjusting daily dose of ceftolozane-tazobactam in patients with osteoarticular infections. ResultsCoefficients of variation and absolute relative biases were <7.9% and 6.5% in all cases. The lower limit of quantification, linearity, normalized-recoveries, normalized-matrix effects and measurement uncertainties for ceftolozane were: 0.97 mg/L, (0.97–125) mg/L, ≤113.6%, ≤108.7%, and ≤ 18.7%, respectively; and for tazobactam: 1.04 mg/L, (1.04–125) mg/L, ≤103.6%, ≤101.9%, and ≤ 20.0%. No interferences and carry-over were observed. Patients plasma concentrations were higher than the recommended 3–4 times the minimal inhibitory concentrations. ConclusionsOur measurement procedures are suitable for therapeutic drug monitoring of ceftolozane-tazobactam in patients with osteoarticular infections.

Characterization of Bromethalin and its Degradation Products in Veterinary Toxicology Samples by GC-MS-MS.

Bromethalin is a neurotoxicant with unusual instability and chromatographic behavior that make it difficult to analyze by gas chromatography (GC) in forensic examination of non-target animal deaths. Physicochemical breakdown of bromethalin produced multiple unique products with discernible mass spectra. This paper describes an investigation of the GC electron impact-mass spectrometric properties of bromethalin and its capacity to generate up to twenty heat- or light-induced breakdown products. Two principal breakdown products are isomeric with one another and involve release of both fluorine and methyl groups to develop dehydrofluorodesmethylbromethalin products. These compounds have proven to be excellent surrogate markers in screening forensic samples for bromethalin exposure, particularly in veterinary samples in which the active metabolite desmethylbromethalin has not yet accumulated to any appreciable extent, such as baits and animal stomach contents. The compounds as well as their parent bromethalin were easily monitored by GC interfaced with a tandem-quadrupole mass spectrometer using multiple-reaction monitoring (MRM) modes. Unusual gas chromatographic properties of bromethalin included: (i) specific requirements for a maximum oven temperature; (ii) non-linear increases in detector response on increased injection volumes, hypothesized to result from variable diffusion coefficients. We report here the development of GC strategies that facilitate detection of bromethalin and its breakdown products, as well as their MRM analysis by tandem-quadrupole mass spectrometry. The developed approaches are applicable to feed, baits and stomach contents as well as extracted tissue samples such as liver and kidney.

Determination of 12 commonly found compounds in DUID cases in whole blood using fully automated supported liquid extraction and UHPLC-MS/MS

A high-throughput UHPLC-MS/MS method for the most frequently found compounds; tetrahydrocannabinol (THC), amphetamine, methamphetamine, MDMA, clonazepam, diazepam, nordiazepam, oxazepam, alprazolam, nitrazepam, morphine, and codeine, in driving under the influence of drugs (DUID) cases in whole blood, is presented. Automated sample preparation by 96-well supported liquid extraction (SLE) plates with ethyl acetate + heptane (80 + 20, v/v) as organic solvent was carried out on a Freedom Evo 200 platform from Tecan. An aliquot of 100 μL whole blood was used. Sample preparation time for 96 samples was 1.5 h. Compounds were separated with gradient elution on a C18 column (50 × 2.1 mm, 1.7 μm) with a mobile phase consisting of 5 mM pH 10.2 ammonium formate and methanol. The run time was 4.5 min and 1 μL was injected on an Acquity UPLC I-Class system with a Xevo TQS tandem-quadrupole mass spectrometer in multiple-reaction monitoring mode (MRM) from Waters. Isotope labelled, 13C, internal standards (ISs) were used for all compounds except for alprazolam and morphine, which had deuterated analogs. Quantification was carried out with calibrators without whole blood matrix. Full validation was carried out according to international guidelines, and a new approach for evaluation of process efficiency (PE) has been presented. Linear or quadratic weighted (1/x) calibration curves were used with R2 ≥ 0.999. The method showed satisfactory deviations ±16% when compared to the existing methods, and satisfactory agreement with proficiency testing control samples (z-score −1.6 to 1.8, n = 16 samples). The precision, estimated as the relative standard deviation (RSD) of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤7.2% in antemortem and ≤9.3% in postmortem samples. Recovery was ≥85% for all the compounds, except morphine ≥62% and THC ≥ 50%. PE was satisfactory for all the compounds with low variation in IS response, RSD ≤ 16% (THC 27%) in antemortem samples and ≤34% (THC 66%) in postmortem samples. To the best of our knowledge, this is the first automated 96-well SLE UHPLC-MS/MS method developed for the simultaneous determination of these 12 compounds in whole blood covering the concentration ranges found in forensic samples. The method has been used in routine work during the last ten months, analysing about 9900 antemortem and 1000 postmortem whole blood samples, and has proven to be robust and reliable.

Comparative pharmacokinetics of oxytetracycline in tilapia (Oreochromis spp.) maintained at three different salinities

Environmental factors, such as temperature, pH, and salinity of water may affect the pharmacokinetics (PK) of a drug in aquatic animals and in most instances water salinity is ignored in PK studies. This study compared PK profiles of oxytetracycline (OTC) following a single oral dosage of 50 mg/kg in tilapia (Oreochromis spp.) maintained in three aquatic environments: freshwater (FW: 0 ppt salinity), brackish water (BW: 15 ppt salinity) and salt water (SW: 30 ppt salinity). Water quality parameters between the three systems were similar except salinity. Following OTC administration, blood samples were collected at 23 time points: 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24 h, and 2, 4, 6, 8, 10, 12, 14, 18, 22, 26, 30, 34, 38 and 42 days. At each sampling time, six fish from each group were netted, sedated with buffered MS-222, bled and then euthanized. The OTC was extracted from plasma by Solid Phase Extraction (SPE) and analyzed by Ultra High Pressure Liquid Chromatography coupled by as tandem quadrupole mass spectrometer. The plasma concentration versus time data of OTC for the FW, BW and SW tilapia were subjected to PK analysis using non-compartment methods. Pharmacokinetics of OTC was characterized by rapid absorption and slow excretion in the FW and BW tilapia. Compared to the FW and BW groups, absorption and elimination of OTC was faster in the SW tilapia. The AUC0-∞ of OTC was in order of FW (165 h.μg/mL) > BW (145 h.μg/mL) > SW (55.5 h.μg/mL) group. In SW tilapia, terminal half-life (69 h) of OTC was >2 times shorter than FW (177 h) and BW (155 h) groups. However, AUCs and terminal half-lives of the FW and BW groups were not significantly different. The study indicated that rise in water salinity level increases clearance of OTC in tilapia. It is suggested that OTC residues in tissues will not be the same in tilapia maintained at different water salinity levels. The results confirmed that infectious diseases associated with bacteria having a MIC of 0.5–1.0 μg/mL can be treated with the 50 mg/kg dosage of OTC in the FW and BW group, but the same dosage in the SW tilapia may lead to therapeutic failure and increased risk of resistance emergence.

Determination of Rubidium by ID-ICP-QMS/QMS with Fluoromethane as the Reaction Cell Gas to Separate Spectral Interference from Strontium.

A determination of rubidium (Rb) was carried out by isotope dilution (ID) using an inductively coupled plasma tandem quadrupole mass spectrometer (ICP-QMS/QMS) with an octupole reaction-cell (ORC). Spectral interference of 87Sr with the measurement of 87Rb was effectively removed by using fluoromethane (CH3F) as the reaction cell gas at the optimum flow rate. In comparison to the measurement obtained with a mathematical correction, good precision for the analysis of the Rb isotope could be obtained independent of the concentration of Sr without any chemical separation in advance. The usefulness of the present approach was confirmed by an analysis of Rb in multiple certified reference materials (CRMs) for food and environmental analysis, for which the results agreed with their certified values in the range of expanded uncertainty.

Sensitive quantification of the somatostatin analog AP102 in plasma by ultra-high pressure liquid chromatography-tandem mass spectrometry and application to a pharmacokinetic study in rats.

AP102 is a di-iodinated octapeptide somatostatin agonist (SSA) designed to treat acromegaly and neuroendocrine tumors. A sensitive and selective method was validated for the quantification of AP102 in plasma following the European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. Sample preparation was performed using solid-phase extraction microplates. Chromatographic separation was achieved on an ultra-high pressure liquid chromatography (UHPLC) C18 column in 6.0minutes. The compounds were quantified using multiple reaction monitoring on a tandem quadrupole mass spectrometer with 13 C,15 N-labeled AP102 as internal standard. Calibration ranged from 50 to 10000pg/mL. The lower limit of quantification (LLOQ) was measured at 20pg/mL, and robust analytical performances were obtained with trueness at 99.2%-100.0%, intra-assay imprecision at 2.5%-4.4%, and inter-assay imprecision at 8.9%-9.7%. The accuracy profiles (total error) built on the 3 concentrations levels showed accuracy within the 70%-130% range. AP102 is remarkably stable since no proteolytic fragments were detected on plasma samples analyzed by Orbitrap-MS. Pharmacokinetic studies were conducted in rats, after single dose (1, 3, and 10 μg/kg, sc) and continuous subcutaneous administration (osmotic minipumps for 28days, 3.0 or 10.0 μg/kg/h). AP102 showed a rapid absorption by the subcutaneous route (Tmax : 15-30minutes) and a fast elimination (t1/2 : 33-86minutes). The PK profile of AP102 exhibited a mean clearance of 1.67L/h and a mean distribution volume at steady state of 7.16L/kg, about 10-fold higher than those observed with other SSA or non- and mono-iodinated AP102. LogD7.4 determination confirmed the lipophilic properties of AP102 that might influence its distribution in tissues.

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 µg of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 µg of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Analysis of Fluorine in Drinking Water by ICP-QMS/QMS with an Octupole Reaction Cell.

The analysis of fluorine was carried out by measuring BaF+ ions with an inductively coupled plasma tandem quadrupole mass spectrometer (ICP-QMS/QMS). After optimization, a radio frequency power of 1300 W was found to benefit for the production of BaF+ ions while suppressing the production of BaOH3+ ions. After optimization of the reaction cell gas, it was found that the best performance for measuring BaF+ could be achieved at a flow rate of O2 in the range from 0.65 to 0.75 mL min-1. The signal intensity of BaF+ depended linearly on the concentration of Ba when it was not higher than 100 mg kg-1. The co-existence of metallic cations, such as Na in the sample, might suppress the generation of BaF+ ions in the plasma, while anions might not cause such a kind of interferences. The background equivalent concentration (BEC) and the lower detection limit (LDL) of fluorine were 0.4 and 0.06 mg kg-1, respectively, by adjusting the samples to a 10 mg kg-1 Ba matrix. The concentration of fluorine in a certified reference material (ERM-CA015a) was determined with the present method, for which the observed value was (1.36 ± 0.05)mg kg-1, which agreed with the certified value (1.3 ± 0.1)mg kg-1, where both values were shown as (mean value ± expanded uncertainty) with a coverage factor of (k = 2) for calculating the expanded uncertainty giving a level of confidence of approximately 95%. The present method was applied to the analysis of a tap water sample collected in the laboratory, for which the results of recovery tests gave a recovery around 100% with good reproducibility.

Experimental Confirmation of SrF(CH3F)0-4+ and SrF(H2O)(CH3F)0-3+ Cluster Ions Generated in the Reaction-cell of ICP-QMS/QMS.

Multiple unknown high-order cluster ions were observed as the results of ion-molecule reactions between strontium ions and fluoromethane molecules in the reaction-cell of an inductively coupled plasma tandem quadrupole mass spectrometer (ICP-QMS/QMS). In order to elucidate the structures of these unknown cluster ions, isotope-enriched fluoromethane (CD3F) was used as the reaction-cell gas compared to natural fluoromethane (CH3F). As results, SrF(CH3F)0-4+ and SrF(H2O)(CH3F)0-3+ cluster ions were experimentally confirmed in the present work, while SrF(H2O)(CH3F)0-3+ cluster ions in the reaction-cell of ICP-QMS/QMS were observed and confirmed for the first time in the world.

Steroid Metabolism in Human Prostate Cancer Tissue: Effect of CYP17 Inhibitors

De novo steroidogenesis is a mechanism that develops during castration‐resistant prostate cancer (CRPC). There is increasing evidence of utilization of the backdoor pathway in CRPC where precursors in the classical pathway are converted to alternative steroids and eventually dihydrotestosterone (DHT) through a series of catalytic reactions without the need to form intermediary testosterone. CYP17 is an essential enzyme for de novo androgen metabolism in the classical and backdoor steroidogenesis pathways. Abiraterone is an FDA‐approved CYP17A1 inhibitor for CRPC, whereas VT‐464 is a nonsteroidal CYP17 inhibitor currently undergoing clinical trials. Although prostate cancer primarily develops in the peripheral zone (PZ), ~20–30% tumors also originate in the transition zone (TZ). However, it is unknown if PZ and TZ differ in steroidogenic ability. We compared the 17‐hydroxypregnenolone (17‐OH Preg) or progesterone metabolism in PZ and TZ human prostate tissues obtained following radical prostatectomy from patients with localized prostate cancer. The effect of CYP17 inhibitors on steroid metabolism in prostate tissues was also evaluated.Human prostate tissues were obtained from the Vancouver Prostate Centre tissue bank. Ethical approval (certificate #H09‐01628) was procured from the Clinical Research Ethics Board of University of British Columbia. The prostate cancer patients underwent radical prostatectomy, without any neoadjuvant therapy. Prostate tissues were homogenized with Precellys homogenizer using 100 mM potassium phosphate. In vitro mixture containing human prostate homogenate (HPH; 45 mg/ml) was incubated with 17‐OH Preg or progesterone (2 μg/ml), NADPH‐regenerating system in 100 mM potassium phosphate buffer (pH 7.4) at 37°C for 60 min. Steroids were extracted twice with hexane:ethyl acetate (60:40) and derivatized with 50 mM hydroxylamine. Steroid formation by HPH was measured by Waters Acquity UPLC coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column. CYP17 inhibitors (abiraterone and VT‐464 at 1 μM) were co‐incubated with HPH.Following incubation of 17‐OH Preg with HPH, androstenedione, testosterone and DHT levels decreased due to their metabolism, whereas 17‐OH‐progesterone and androsterone formation was significantly increased after incubation. Interestingly, the formation of pregnan‐3,20‐dione, 5‐pregnan‐3‐ol‐20‐one and 5‐pregnan‐17‐ol‐3,20‐dione, which are backdoor pathway steroids, dramatically increased in both PZ and TZ tissue. Similarly, incubation of HPH with progesterone led to decrease in DHT and increase in pregnan‐3,20‐dione and 5‐pregnan‐3‐ol‐20‐one levels. The formation of androstenedione, 17‐OH‐progesterone, testosterone, androsterone and 17‐OH Preg remained unchanged after incubation with progesterone. Comparison of steroid metabolism following incubation with 17‐OH Preg and progesterone in PZ and TZ revealed no significant differences between two prostate zones. Co‐incubation of substrates with abiraterone and VT‐464 led to decrease in the formation of pregnan‐3,20‐dione and 5‐pregnan‐3‐ol‐20‐one, however, both the CYP17 inhibitors had limited effects on the CYP17‐mediated activities in classical pathway.The de novo tissue steroidogenesis is similar in PZ and TZ tissues and responds to the availability of precursors in tumor microenvironment through backdoor pathway in CRPC. VT‐464 or abiraterone‐mediated modulation of steroidogenesis observed in our study suggests that compensatory pathways are possibly activated as a result of CYP17 inhibition. Steroid metabolism in HPH presents a viable model for screening of steroidogenesis inhibitors.Support or Funding InformationA grant‐in‐aid was provided by Glaxo SmithKline (GSK).

Confirmation of40Ar+related product ions in the octopole reaction cell of an ICP-QMS/QMS with18O2enriched oxygen as the reaction cell gas

40Ar+ related product ions in an octopole reaction cell (ORC) were observed with an inductively coupled plasma tandem quadrupole mass spectrometer (ICP-QMS/QMS). In order to simplify the ion composition of the plasma, the carrier gas was directly connected to the torch injector, i.e. without introduction of the sample solution. The m/z of the first QMS was set to 40 permitting the passage of 40Ar+. Isotope enriched oxygen gas (18O2) was used as the reaction gas in order to separate the spectra of O3+ and Ar2O2+. The intensities of the ions produced in the ORC were measured by the second QMS. As the products of the reactions among 40Ar+ ions and 18O2 molecules in the ORC, the following ions were observed: 18O+, 18O2+, 18O3+, 18O4+, 40Ar18O+, 40Ar18O2+, 40Ar218O2+, and 40Ar18O22+. At a reaction gas flow rate of 0.3 mL min−1, the ionic species with the highest signal intensity was 18O2+, followed by 40Ar+ and 18O+. The maximum signal intensities of different product ions were obtained at different flow rates of the reaction gas. A lower flow rate of the reaction gas benefited the production of 40Ar18O+ and 18O3+, while a higher flow rate of the reaction gas benefited the production of 40Ar18O2+ and 40Ar218O2+. The maximum transfer efficiency from 40Ar+ to the resulting ionic species was 91%, 2.0%, and 0.12% for 18O2+, 18O+, and 40Ar18O+, respectively. The maximum transfer efficiency to the other investigated ionic species was less than 0.023%. By using 18O2 as the reaction gas, the mechanisms of 40Ar+ resulted ionic species interfering with the measurement of 32S16O+ were confirmed. The results showed that the contributions of 16O3+ and 40Ar2O2+ were almost equivalent to each other.