Origin and differentiation of human memory CD8 T cells after vaccination.

The differentiation of human memory CD8 T cells is not well understood. Here we address this issue using the live yellow fever virus (YFV) vaccine, which induces long-term immunity in humans. We used in vivo deuterium labelling to mark CD8 T cells that proliferated in response to the virus and then assessed cellular turnover and longevity by quantifying deuterium dilution kinetics in YFV-specific CD8 T cells using mass spectrometry. This longitudinal analysis showed that the memory pool originates from CD8 T cells that divided extensively during the first two weeks after infection and is maintained by quiescent cells that divide less than once every year (doubling time of over 450 days). Although these long-lived YFV-specific memory CD8 T cells did not express effector molecules, their epigenetic landscape resembled that of effector CD8 T cells. This open chromatin profile at effector genes was maintained in memory CD8 T cells isolated even a decade after vaccination, indicating that these cells retain an epigenetic fingerprint of their effector history and remain poised to respond rapidly upon re-exposure to the pathogen.

Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry.

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., ß-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

Development of human serum matrix-based unconjugated estriol-certified reference material by recommended ID-LC-MS/MS reference method.

To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020.

Ecological risk assessment of trace metals in sediments under reducing conditions based on isotopically exchangeable pool.

Determination of potential mobility of toxic trace metals in sediments under changing redox condition is important in ecological risk assessment. Current methods are limited in risk prediction in such dynamic environment. In this study, we have discussed the general disagreement from widely used methods (sediment quality guideline (SQGs), potential ecological risk index (PERI), risk assessment code (RAC) using BCR fraction information). In addition, the stable isotopic dilution method (IDM) was also modified to quantify metal lability in a microcosm experiment mimicking river bank sediment turning into anaerobic. The isotopically exchangeable Cd, Cu, Pb, and Zn quantified by IDM (%E incub) was used in the RAC to reveal the trend of risk during this process. Strong risks from Cd are suggested by the PERI and RAC as a result of high toxicity and mobility of the element, while SQGs suggests medium risk for Cu, Pb, and Zn in certain samples. The disagreement between the results of RAC assessed by metal lability (%E dry) and by BCR metal fractionation reflects the effect of sediment properties and source of metal contamination. The RAC based on the non-residual fractions is likely to overestimate the potential risk for most metals even there is a significant change in sediment Eh. The RAC assessed by %E incub reveals that the variability in risk in response to the reducing Eh is not consistent. Large fluctuation in %E incub for Cd (28.5%, 49.5%), Pb (27.6%, 18.2%), and Cu (14.4%, 24.7%) can shift the risks to a higher level in certain range of Eh in two sediments. In sediment with lower contents of metal binding phases (e.g. mineral oxides, organic matters), the release of metals can be more significant, thus higher ecological risk in changing redox condition.

Inhibitory Covalent Labeling and Clickable-Eu-Tagging-Based ICPMS: Measurement of pH-Dependent Absolute Activities of the Cathepsins in Hepatocyte Lysosomes.

We report an inhibitory covalent labeling and clickable-element-tagging strategy for measuring the absolute activity of a protease in cells using inductively coupled plasma mass spectrometry (ICPMS). Epoxysuccinyl-leucine-tyrosine-6-aminocaproic-lysine-amino-Boc-alkyne (epoxysuccinyl-LYK-alkyne) was designed and synthesized to achieve irreversibly labeling of the cysteine cathepsins, recording their momentary activities. L and Y assisted epoxysuccinyl-LYK-alkyne in accessing the deprotonated -S- of Cys25, located at the bottom of the long cathepsin active domain. Quantitative Eu-tagging was followed using azido-DOTA-Eu through a bioorthogonal 1:1 copper-catalyzed azide-alkyne-cycloaddition click reaction. The Eu tag could be absolutely quantified using 153Eu-species-nonspecific-isotope-dilution ICPMS coupled with HPLC, serving as a Eu ruler and allowing us to simultaneously measure the pH-dependent activities of cathepsins B, L, and S as well as the pH in the lysosomal microenvironment of liver cancerous C7721 and paracancerous C7701 cells. As long as suitable labeling molecules and elemental tags are designed and synthesized, we believe that such a tandem labeling and tagging ICPMS approach can be applied to the measurement of the activities of other proteases in cells, providing more accurate information on the proteases' biofunctions and thus implementing precise clinical diagnoses.

Measurement of Oxidatively Induced DNA Damage in Caenorhabditis elegans with High-Salt DNA Extraction and Isotope-Dilution Mass Spectrometry.

Caenorhabditis elegans is used extensively as a medical and toxicological model organism. However, little is known about background levels of oxidatively induced DNA damage in the nematode or how culturing methods affect DNA damage levels. The tough C. elegans cuticle makes it challenging to extract genomic DNA without harsh procedures that can artifactually increase DNA damage. Therefore, a mild extraction protocol based on enzymatic digestion of the C. elegans cuticle with high-salt phase-separation of DNA has been developed and optimized. This method allows for efficient extraction of >50 µg DNA using a minimum of 250000 nematodes grown in liquid culture. The extracted DNA exhibited acceptable RNA levels (<10% contamination), functionality in polymerase chain reaction assays, and reproducible DNA fragmentation. Gas chromatography/tandem mass spectrometry (GC-MS/MS) with isotope-dilution measured lower lesion levels in high-salt extracts than in phenol extracts. Phenolic extraction produced a statistically significant increase in 8-hydroxyguanine, a known artifact, and additional artifactual increases in 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine, and 8-hydroxyadenine. The high-salt DNA extraction procedure utilizes green solvents and reagents and minimizes artifactual DNA damage, making it more suitable for molecular and toxicological studies in C. elegans. This is, to our knowledge, the first use of GC-MS/MS to measure multiple 8,5'-cyclopurine-2'-deoxynucleosides in a toxicologically important terrestrial organism.

Aflatoxin-Guanine DNA Adducts and Oxidatively Induced DNA Damage in Aflatoxin-Treated Mice in Vivo as Measured by Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution.

Dietary exposure to aflatoxin B1 (AFB1) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB1 exposure leads to the formation of AFB1-N7-guanine (AFB1-N7-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B1 (AFB1-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB1 as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB1-N7-Gua and the two diastereomers of AFB1-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB1-treated mice. Levels varying from 1.5 to 45 lesions/106 DNA bases in AFB1-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB1 treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB1 treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB1-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB1-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB1 exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.

Quantitation of N6-Formyl-lysine Adduct Following Aristolochic Acid Exposure in Cells and Rat Tissues by Liquid Chromatography-Tandem Mass Spectrometry Coupled with Stable Isotope-Dilution Method.

N6-Formyl-lysine (FLys) is an abundant and lasting protein adduct formed when formaldehyde generated by nitrosative/oxidative stress and inflammation reacts with lysine residues. It is believed that the post-translational N6-formylation of lysine is associated with a variety of pathological processes and human diseases. Thus, FLys may serve well as a dosimetric biomarker for exposure to formaldehyde and other oxidative stress-inducing toxicants. However, since current methods for FLys determination are tedious and time-consuming, we developed and validated an aqueous normal phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with isotope-dilution method for the rigorous quantification of FLys with enhanced sensitivity and selectivity. After validating the accuracy and precision of the method with a synthetic peptide containing FLys, the method was applied to quantitate the concentration-dependent formation of FLys in cells exposed to formaldehyde and Fe2+-EDTA, an OH radical-mediated oxidant. The study reveals formaldehyde and Fe2+-EDTA produced FLys at a frequency of 20.2 and 4.1 per 104 lysine per mM, respectively, after correcting for losses during protein digestion steps. The study was further extended to quantitate the concentration-dependent formation of FLys in aristolochic acid I (AA-I) exposed Escherichia coli cells and rat tissues. This study demonstrates for the first time that AA-I exposure induces time- and dose-dependent formation of FLys in cellular proteins. Furthermore, results show AA-I exposure leads to organotropic N6-formylation of lysine, with elevated levels of FLys detectable in the kidney, which is the one of the tumor targeting organs of AAs. Previous studies have also revealed AA exposure induced renal interstitial fibrosis in both laboratory rodents and humans, by a yet to be determined molecular mechanism. These data shed light on the potential caustative role of N6-formylation in the pathophysiology of AA nephrotoxicity and carcinogenicity.

Stable-isotope dilution LC-MS/MS method for quantitative determination of microcystin conjugates with cysteine and glutathione in biotic matrices.

Microcystins are cyclic peptide toxins with hepatotoxic and tumor-promoting properties, which are produced in significant quantities (up to tens of µg/L) in freshwater cyanobacterial water blooms. Several studies reported microcystin accumulation in fish with possible food transfer to humans. These compounds are further metabolized to cysteine and glutathione conjugates which can be present in tissues in significant concentrations. In this study, we focused on the development and evaluation of robust and highly sensitive SPE-LC-MS/MS method for the analysis of microcystin conjugates in fish tissue samples. For the first time, we demonstrate the use of isotopically labeled internal standards which are essential for accurate and precise determination of analytes in complex biotic matrices. LLOQs of respective microcystin conjugates (signal-to-noise ratio; S/N > 10, peak-to-peak method) ranged from 3.3 to 5.0 ng/g of tissue fresh weight (FW). The calibration was linear within a range of concentrations from 1 to 70 ng/mL for all analyzed conjugates. The precision and repeatability of the method were very good with recoveries in the range of 88.5-107.6% and relative standard deviations between 8.8 and 13.2% for all analytes. In the follow-up study, fully validated method was used for the determination of microcystin conjugate levels in common carp exposed to microcystin-containing cyanobacterial biomass under controlled conditions. Significant amounts of microcystin conjugates (up to 55 ng/g) were found in the tissues of fish after 7 weeks of exposure. Our method was shown to be robust, sensitive, selective, and suitable for the determination of trace levels of microcystin conjugates in fish tissues.

Evaluation of the impact of matrix effects in LC/MS measurement on the accurate quantification of neonicotinoid pesticides in food by isotope-dilution mass spectrometry.

The use of isotope-labeled internal standards is the most widely accepted approach to overcome the matrix effects on quantification of pesticides in food by LC/MS. We evaluated the impact of the matrix effects on quantification of six neonicotinoid pesticides, acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam, in food by using deuterated internal standards. The calibration curves for each pesticide were obtained by using matrix-free and matrix-matched calibration solutions with blank brown rice, carrot, and green onion extracts. For brown rice and carrot, the matrix effects were not observed. In contrast, the slopes of calibration curves for each pesticide were influenced by presence of green onion extracts in calibration solutions (variability of the slopes was 4-9%), because the ratios of peak area for native pesticide to those for internal standards were influenced by matrix. The spike-and-recovery test with green onion was also performed. The analytical values obtained by using matrix-free calibration solution were biased from the spiked concentration, whereas those obtained by using matrix-matched calibration solution were comparable to the spiked concentration. These results indicate that matrix-matched calibration solution should be used for accurate quantification of neonicotinoid pesticides in food by LC/MS using deuterated internal standards.

Quantitative Bioimaging of Platinum via Online Isotope Dilution-Laser Ablation-Inductively Coupled Plasma Mass Spectrometry.

A new calibration strategy for elemental bioimaging based on online isotope dilution analysis (IDA) and laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS) was developed and applied for the quantification of platinum in rat kidney tissues. A dry 194Pt spike aerosol was added in a post-cell setup, and the natural 194Pt/195Pt isotope ratio of the sample aerosol from laser ablation was changed accordingly. Spike mass flow determination was carried out based on reversed IDA using a reference standard. Quantitative data obtained by the new approach correlated well with those obtained by external calibration when analyzing parallel tissue slices of rat kidney from cisplatin perfusion studies. The novel quantification approach is traceable to SI units, as IDA is an definitive method. Signal drifts are compensated as the second isotope acts as an internal standard.

A timeline of stable isotopes and mass spectrometry in the life sciences.

This review retraces the role of stable isotopes and mass spectrometry in the life sciences. The timeline is divided into four segments covering the years 1920-1950, 1950-1980, 1980-2000, and 2000 until today. For each period methodic progress and typical applications are discussed. Application of stable isotopes is driven by improvements of mass spectrometry, chromatography, and related fields in sensitivity, mass accuracy, structural specificity, complex sample handling ability, data output, and data evaluation. We currently experience the vision of omics-type analyses, that is, the comprehensive identification and quantification of a complete compound class within one or a few analytical runs. This development is driven by stable isotopes without competition by radioisotopes. In metabolic studies as classic field of isotopic tracer experiments, stable isotopes and radioisotopes were competing solutions, with stable isotopes as the long-term junior partner. Since the 1990s the number of metabolic studies with radioisotopes decreases, whereas stable isotope studies retain their slow but stable upward tendency. Unique fields of stable isotopes are metabolic tests in newborns, metabolic experiments in healthy controls, newborn screening for inborn errors, quantification of drugs and drug metabolites in doping control, natural isotope fractionation in geology, ecology, food authentication, or doping control, and more recently the field of quantitative omics-type analyses. There, cells or whole organisms are systematically labeled with stable isotopes to study proteomic differences or specific responses to stimuli or genetic manipulation. The duo of stable isotopes and mass spectrometry will probably continue to grow in the life sciences, since it delivers reference-quality quantitative data with molecular specificity, often combined with informative isotope effects. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:58-85, 2017.

Phosphorus kinetics in lambs experimentally infected with Trichostrongylus colubriformis with the use of 32P.

This study was conducted to evaluate the metabolism and phosphorus (P) kinetics in lambs experimentally infected with Trichostrongylus colubriformis using the isotope dilution technique and modelling. Fifteen male lambs (21.1 ±â€¯1.50 kg) of the Santa Inês hair breed of approximately six months old, distributed in the treatments infected (I, n = 8) and control (C, n = 7) were used. The infected lambs received serial infections with 5000 T. colubriformis larvae, three times per week, for 3 weeks (45 000 T. colubriformis total larvae). After 66 days of the last inoculation of infective larvae, 6.6 MBq of 32P were injected in each lamb to evaluate the P kinetics. Blood, faeces and urine samples were collected in the following seven days and the slaughter of lambs were carried out on the last day in order to collect bone and soft tissues (Liver, kidney, heart and muscle) samples. To analyse P flows, the biomathematical model with four compartments (C1 - gastrointestinal tract, C2 - plasma, C3 - bone and C4 - soft tissue) was used. Similar P intake (VI) between treatments (C and I) was verified. Lower absorption of endogenous (Vaf) and dietary P (Vaa), as well as, lower amount of endogenous P (from saliva) that reaches the gastrointestinal tract (VIT), consequently, higher excretion of dietary P (VFD) were verified in infected lambs (P < 0.1). Additionally, in infected lambs, the P bioavailability was lower compared to control lambs. With the lower absorption (VaT) of P in infected lambs, there was, consequently, lower distribution to bones and soft tissues (VeD2) and lower P deposition in the bones (VO+D). It was concluded that P metabolism of lambs infected with T. colubriformis was altered, with reduced intestinal absorption and bioavailability, increased faecal loss and reduced P flow to the bone.

Quantitative Analysis of Ingenol in Euphorbia species via Validated Isotope Dilution Ultra-high Performance Liquid Chromatography Tandem Mass Spectrometry.

INTRODUCTION: Various species of the Euphorbia genus contain diterpene ingenol and ingenol mebutate (ingenol-3-angelate), a substance found in the sap of the plant Euphorbia peplus and an inducer of cell death. A gel formulation of the drug has been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the topical treatment of actinic keratosis. OBJECTIVE: To develop a rapid and reliable method for quantification of ingenol in various plant extracts. METHODOLOGY: Methanolic extracts of 38 species of the Euphorbia genus were analysed via ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) after methanolysis and solid-phase extraction (SPE) purification. The 18 O-labelled ingenol analogue was prepared and used as an internal standard for ingenol content determination and method validation. RESULTS: The highest ingenol concentration (547 mg/kg of dry weight) was found in the lower leafless stems of E. myrsinites. The screening confirms a substantial amount of ingenol in species studied previously and furthermore, reveals some new promising candidates. CONCLUSION: The newly established UHPLC-MS/MS method shows to be an appropriate tool for screening of the Euphorbia genus for ingenol content and allows selection of species suitable for raw material production and/or in vitro culture initiation. Copyright © 2017 John Wiley & Sons, Ltd.

A four-compartment compartmental model to assess net whole body protein breakdown using a pulse of phenylalanine and tyrosine stable isotopes in humans.

The stable isotopes of phenylalanine (Phe) and tyrosine (Tyr) are often used to study whole body protein metabolism in humans. Noncompartmental approaches give limited physiological insight in the compartmental characteristics. We therefore developed a compartmental mathematical model of Phe/Tyr metabolism to describe protein fluxes by using stable tracer dynamic data in plasma following intravenous bolus of l-[ring-13C6]Phe and l-[ring-2H4]Tyr in healthy subjects. The model consists of four compartments describing Phe/Tyr kinetics. Because the model is a priori nonidentifiable, it is quantified in terms of two uniquely identifiable submodels representing two limit case scenarios, based on known physiology. The two submodels, identified by using the software SAAM II, fit well the experimental data of all individuals and provide an unbiased overview of the metabolic pathway in terms of intervals of validity of the non-uniquely identifiable variables. The model provides estimates of the flux from Phe to Tyr [4.1 ± 1.0 µmol·kg fat-free mass (FFM)-1·h-1 (mean ± SE)] and intervals of validity of the flux and pool estimates. Our preferred submodel yielded protein breakdown flux (50.5 ± 5.2 µmol·kg FFM-1·h-1), net protein breakdown (4.1 ± 1.0 µmol·kg FFM-1·h-1), Tyr from Phe hydroxylation (~12%), hydroxylated Phe (~8%), and flux ratio of Tyr to Phe arising from protein catabolism (0.68), consistent with available literature. The other submodel suggest that the assumptions made by noncompartmental analysis are consistently underestimated. Our accurate and detailed model for estimating Phe/Tyr metabolic pathways in humans might be essential to applications in a variety of scenarios describing whole body protein synthesis and breakdown in health and disease.

Blood volume measured by ultrasound and radioisotope dilution in critically ill subjects.

BACKGROUND: Accurate bedside assessment of circulating blood volume (BV) continues to challenge clinicians in their attempt to implement goal-directed therapy in the critically ill subject. The aim of this investigation was to comparatively evaluate BV measurements obtained by ultrasound and radioisotope dilution methodologies in adult subjects admitted to a surgical intensive care unit. MATERIALS AND METHODS: Fifty subjects with concurrent central venous catheters and peripheral arterial lines underwent measurement of BV using both ultrasound and radioisotope dilution (BV-RD) methods. The ultrasound dilution method was performed using a 30-mL injectate (BV-UD30) and a 60-mL injectate (BV-UD60) of isotonic saline. RESULTS: There were 24 paired data points for the BV-UD30 and 40 paired data points for the BV-UD60 measurements. Spearman's rank-order correlation demonstrated a positive relationship comparing both the BV-UD30 (r = 0.46, P = 0.0249) and the BV-UD60 (r = 0.80, P < 0.0001) to values obtained by radioisotope measurements. Bland-Altman analysis showed a mean bias of 1329 mL with limits of agreement (LOA) ± 2559 mL comparing BV-RD and BV-UD30, and a mean bias of 62 mL with LOA ±1353 mL for BV-RD and BV-UD60. CONCLUSIONS: This preliminary investigation shows that the BV-UD60 had better agreement with BV-RD, compared with the BV-UD30, but its utility appears limited by a large LOA. As this technology continues to evolve, the ultrasound dilution approach may potentially become a feasible means to calculate BV in critically ill surgical subjects.

Accumulation of intact sulfur mustard in adipose tissue and toxicokinetics by chemical conversion and isotope-dilution liquid chromatography-tandem mass spectrometry.

Sulfur mustard (SM) is a powerful vesicant and one of the most harmful chemical warfare agents. Although having been studied for a long time, it is still difficult to fully elucidate the mechanisms of SM poisoning, and there is no effective antidote or specific treatment for SM injury. The investigations on toxicokinetics and tissue distribution of SM will help to understand its toxicity and provide a theoretical basis for pretreatment and therapy of SM poisoning. But the metabolic trajectory or fate of intact SM in vivo remains unclear, and there are insufficient experimental data to elucidate, due to its high reactivity and difficulty in biomedical sample analysis. In this paper, a sensitive method for the detection and quantification of intact SM in blood or tissues using isotope-dilution LC-MS/MS coupled with chemical conversion was developed. By transforming highly reactive SM into stable derivative product, the real concentration of intact SM in biological samples was obtained accurately. The toxicokinetics and tissue distribution studies of intact SM in rats were successfully profiled by the novel method after intravenous (10 mg/kg) or cutaneous administration (1, 3 and 10 mg/kg). The SM level in blood with peak time at 30-60 min determined in cutaneous exposure experiment was found much higher than previously reported, and the mean residence time in blood extended to 1-1.5 h. A significant accumulation of intact SM was observed in adipose tissues, including the perirenal fat, epididymal fat, subcutaneous fat and brown fat, in which the concentrations of SM were at least 15 times greater than those in non-adipose tissues in cutaneous exposed rats. The recovery of SM in body fat was calculated as 3.3 % of bioavailable SM (the bioavailability after cutaneous exposure was evaluated as 16 %). Thus, the adipose tissue was important for SM distribution and toxicity, which may pioneer a new model for both the prevention and treatment of SM exposure.

Dynamic measurement of newly formed carbonyl compounds in vapors from electronic cigarettes.

Recently, the formation of carbonyl compound within e-cigarettes usage has been reported. The aim of this study was to develop a new analytical method for the direct analysis of carbonyl compounds in vaporized liquids. Two different types of e-cigarettes and different puff's duration have been evaluated, using a modified smoking machine for vapor generation. An isotopic dilution approach, based on deuterated internal standard addition to the e-liquid before filling the e-cigarette tank, has been developed. Carbonyl compounds have been sampled in vapors using a direct, simple, solid-phase microextraction technique with on-fiber derivatization. Related oximes have been analyzed by gas chromatography/mass spectrometry technique. Results confirmed that new carbonyl compounds are formed during the vaping process, and that formation depends both from the heating device and from puffing topography.

A rapid and efficient analytical method for the quantification of a novel anticholinergic compound, R-phencynonate, by stable isotope-dilution LC-MS/MS and its application to bioavailability and dose proportionality studies.

A rapid, specific and high-throughput stable isotope-dilution LC-MS/MS method was developed and validated with high sensitivity for the quantification of R-phencynonate (a eutomer of phencynonate racemate) in rat and dog plasma. Plasma samples were deproteinized using acetonitrile and then separated on a C8 column with an isocratic mobile phase containing acetonitrile-water-formic acid mixture (60:40:0.1, v/v/v) at a flow rate of 0.2 mL/min. Each sample had a total run time of 3 min. Quantification was performed using triple quadrupole mass spectrometry in selected reaction monitoring mode with positive electrospray ionization. The method was shown to be highly linear (r2 > 0.99) and to have a wide dynamic range (0.1-100 ng/mL) with favourable accuracy and precision. No matrix effects were observed. The detailed pharmacokinetic profiles of R-phencynonate at therapeutic doses in rats and dogs were characterized by rapid oral absorption, quick clearance, high volume of distribution and poor absolute bioavailability. R-Phencynonate lacked dose proportionality over the oral dose range, based on the power model. However, the area under concentration-time curve and the maximum plasma concentration increased linearly in a dose-dependent manner in both animal models. The absolute bioavailability of R-phencynonate was 16.6 ± 2.75 and 4.78 ± 1.26% in dogs and rats, respectively.

Stable-Isotope Dilution HPLC-Electrospray Ionization Tandem Mass Spectrometry Method for Quantifying Hydroxyurea in Dried Blood Samples.

BACKGROUND: Sickle cell anemia (SCA) is a life-threatening blood disorder characterized by the presence of sickle-shaped erythrocytes. Hydroxyurea is currently the only US Food and Drug Administration-approved treatment and there is a need for a convenient method to monitor compliance and hydroxyurea concentrations, especially in pediatric SCA patients. METHODS: We describe a novel approach to the determination of hydroxyurea concentrations in dried whole blood collected on DMPK-C cards or volumetric absorptive microsampling (VAMS) devices. Hydroxyurea was quantified by electrospray ionization LC-MS/MS using [13C15N2]hydroxyurea as the internal standard. Calibrators were prepared in whole blood applied to DMPK-C cards or VAMS devices. RESULTS: Calibration curves for blood hydroxyurea measured from DMPK-C cards and VAMS devices were linear over the range 0.5-60 µg/mL. Interassay and intraassay CVs were <15% for blood collected by both methods, and the limit of detection was 5 ng/mL. Whole blood hydroxyurea was stable for up to 60 days on DMPK-C cards and VAMS devices when frozen at -20 °C or -80 °C. Whole blood hydroxyurea concentrations in samples collected on DMPK-C cards or VAMS devices from SCA patients were in close agreement. CONCLUSIONS: This tandem mass spectrometry method permits measurement of hydroxyurea concentrations in small volumes of dried blood applied to either DMPK-C cards or VAMS devices with comparable performance. This method for measuring hydroxyurea from dried blood permits the evaluation of therapeutic drug monitoring, individual pharmacokinetics, and medication adherence using heel/finger-prick samples from pediatric patients with SCA treated with hydroxyurea.