Stable Isotope Dilution Mass Spectrometry Research Articles

Abstract 128: Blood TMAO Predicts Abdominal Aortic Aneurysm Growth Rate And Need For Surgical Intervention

Background: Patients known to have abdominal aortic aneurysms (AAAs) undergo surveillance imaging to predict both the risk for acute aortic syndromes including aortic dissection and aortic rupture, and the need for surgical intervention. Aortic rupture is a feared consequence of rapidly expanding AAAs. Aims: A blood biomarker that predicts risk for AAA and distinguishes slow from fast AAA growth does not exist but could be a valuable tool for risk stratification. We recently reported systemic levels of the gut microbe-generated metabolite trimethylamine N-oxide (TMAO) are associated with the presence of AAA in humans, and directly contribute to AAA progression in animal models. Methods: We leveraged access 805 patients in two cohorts from Europe (n=237) and the United States (n=658) undergoing serial aortic imaging surveillance. Blood TMAO concentration was measured by stable isotope dilution mass spectrometry. Results: TMAO was positively associated with larger baseline and follow-up abdominal aortic diameter beyond traditional factors (Adjusted OR 3.87, 95% CI, 2.70-5.58, P<0.001). In addition, TMAO predicted rapid AAA growth (HR 1.81, 95%CI, 1.23-2.67, P=0.0029). When TMAO was added to a predictive model of traditional risk factors for AAA, we saw a significant improvement in the net reclassification index (NRI, 0.43, P < 0.001; C statistic, 0.77 (0.72–0.81) with TMAO vs. NRI 0.73 (0.69–0.78) without TMAO, P =0.005). Finally, including TMAO in a predictive model for assessing need for surgical intervention among AAA patients significantly improved the prognostic performance of the model (NRI, 0.44, P < 0.001; C statistic, 0.78 (0.73–0.82) with TMAO vs. 0.75 (0.70–0.79) without TMAO, P =0.004) (Fig. 1). Conclusions: Elevated TMAO levels predict increased risk of AAA, and identify patients with fast-growing AAA and those who may benefit from early surgical intervention to prevent acute aortic syndromes.

Quantification of the immunometabolite protein modifications S-2-succinocysteine and 2,3-dicarboxypropylcysteine.

The tricarboxylic acid (TCA) cycle metabolite fumarate nonenzymatically reacts with the amino acid cysteine to form S-(2-succino)cysteine (2SC), referred to as protein succination. The immunometabolite itaconate accumulates during lipopolysaccharide (LPS) stimulation of macrophages and microglia. Itaconate nonenzymatically reacts with cysteine residues to generate 2,3-dicarboxypropylcysteine (2,3-DCP), referred to as protein dicarboxypropylation. Since fumarate and itaconate levels dynamically change in activated immune cells, the levels of both 2SC and 2,3-DCP reflect the abundance of these metabolites and their capacity to modify protein thiols. We generated ethyl esters of 2SC and 2,3-DCP from protein hydrolysates and used stable isotope dilution mass spectrometry to determine the abundance of these in LPS-stimulated Highly Aggressively Proliferating Immortalized (HAPI) microglia. To quantify the stoichiometry of the succination and dicarboxypropylation, reduced cysteines were alkylated with iodoacetic acid to form S-carboxymethylcysteine (CMC), which was then esterified. Itaconate-derived 2,3-DCP, but not fumarate-derived 2SC, increased in LPS-treated HAPI microglia. Stoichiometric measurements demonstrated that 2,3-DCP increased from 1.57% to 9.07% of total cysteines upon LPS stimulation. This methodology to simultaneously distinguish and quantify both 2SC and 2,3-DCP will have broad applications in the physiology of metabolic diseases. In addition, we find that available anti-2SC antibodies also detect the structurally similar 2,3-DCP, therefore "succinate moiety" may better describe the antigen recognized.NEW & NOTEWORTHY Itaconate and fumarate have roles as immunometabolites modulating the macrophage response to inflammation. Both immunometabolites chemically modify protein cysteine residues to modulate the immune response. Itaconate and fumarate levels change dynamically, whereas their stable protein modifications can be quantified by mass spectrometry. This method distinguishes itaconate and fumarate-derived protein modifications and will allow researchers to quantify their contributions in isolated cell types and tissues across a range of metabolic diseases.

Atlas of gut microbe-derived products from aromatic amino acids and risk of cardiovascular morbidity and mortality.

Precision microbiome modulation as a novel treatment strategy is a rapidly evolving and sought goal. The aim of this study is to determine relationships among systemic gut microbial metabolite levels and incident cardiovascular disease risks to identify gut microbial pathways as possible targets for personalized therapeutic interventions. Stable isotope dilution mass spectrometry methods to quantitatively measure aromatic amino acids and their metabolites were used to examine sequential subjects undergoing elective diagnostic cardiac evaluation in two independent cohorts with longitudinal outcome data [US (n = 4000) and EU (n = 833) cohorts]. It was also used in plasma from humans and mice before vs. after a cocktail of poorly absorbed antibiotics to suppress gut microbiota. Multiple aromatic amino acid-derived metabolites that originate, at least in part, from gut bacteria are associated with incident (3-year) major adverse cardiovascular event (MACE) risks (myocardial infarction, stroke, or death) and all-cause mortality independent of traditional risk factors. Key gut microbiota-derived metabolites associated with incident MACE and poorer survival risks include: (i) phenylacetyl glutamine and phenylacetyl glycine (from phenylalanine); (ii) p-cresol (from tyrosine) yielding p-cresol sulfate and p-cresol glucuronide; (iii) 4-OH-phenyllactic acid (from tyrosine) yielding 4-OH-benzoic acid and 4-OH-hippuric acid; (iv) indole (from tryptophan) yielding indole glucuronide and indoxyl sulfate; (v) indole-3-pyruvic acid (from tryptophan) yielding indole-3-lactic acid and indole-3-acetyl-glutamine, and (vi) 5-OH-indole-3-acetic acid (from tryptophan). Key gut microbiota-generated metabolites derived from aromatic amino acids independently associated with incident adverse cardiovascular outcomes are identified, and thus will help focus future studies on gut-microbial metabolic outputs relevant to host cardiovascular health.

Stable isotope dilution mass spectrometry quantification of hydrogen sulfide and thiols in biological matrices

BackgroundHydrogen sulfide (H2S), a gaseous signaling molecule that impacts multiple physiological processes including aging, is produced via select mammalian enzymes and enteric sulfur-reducing bacteria. H2S research is limited by the lack of an accurate internal standard-containing assay for its quantitation in biological matrices. MethodsAfter synthesizing [34S]H2S and developing sample preparation protocols that avoid sulfide contamination with the addition of thiol-containing standards or reducing reagents, we developed a stable isotope-dilution high performance liquid chromatography tandem-mass spectrometry (LC-MS/MS) method for the simultaneous quantification of Total H2S and other abundant thiols (cysteine, homocysteine, glutathione, glutamylcysteine, cysteinylglycine) in biological matrices, conducted a 20-day analytical validation/normal range study, and then both analyzed circulating Total H2S and thiols in plasma from 400 subjects, and within 20 volunteers before and after antibiotic-induced suppression of gut microbiota. ResultsUsing the new assay, all analytes showed minimal interference, no carryover, and excellent intra- and inter-day reproducibility (≤7.6%, and ≤12.7%, respectively), linearity (r2 > 0.997), recovery (90.9%–110%) and stability (90.0%–100.5%). Only circulating Total H2S levels showed significant age-associated reductions in both males and females (p < 0.001), and a marked reduction following gut microbiota suppression (mean 33.8 ± 17.7%, p < 0.001), with large variations in gut microbiota contribution among subjects (range 6.0–66.7% reduction with antibiotics). ConclusionsA stable-isotope-dilution LC-MS/MS method is presented for the simultaneous quantification of Total H2S and multiple thiols in biological matrices. We then use this assay panel to show a striking age-related decline and gut microbiota contribution to circulating Total H2S levels in humans.

Profiling the Plasma Apolipoproteome of Normo- and Hyperlipidemic Mice by Targeted Mass Spectrometry.

Atherosclerotic cardiovascular disease is the leading cause of death worldwide. For decades, mouse modeling of atherosclerosis has been the mainstay for preclinical testing of genetic and pharmacological intervention. Mouse models of atherosclerosis depend on supraphysiological levels of circulating cholesterol carried in lipoprotein particles. Lipoprotein particles vary in atherogenicity, and it is critical to monitor lipoprotein levels during preclinical interventions in mice. Unfortunately, the small plasma volumes typically harvested during preclinical experiments limit analyses to measuring total cholesterol and triglyceride levels. Here we developed a high-throughput, low-cost targeted multiple reaction monitoring (MRM) stable isotope dilution (SID) mass spectrometry assay for simultaneous relative quantification of nine apolipoproteins using a few microliters of mouse plasma. We applied the MRM assay to investigate the plasma apolipoproteome of two atherosclerosis models: the widely used ApoE knockout model and the emerging recombinant adeno-associated virus-mediated hepatic Pcsk9 overexpression model. By applying the assay on size-exclusion chromatography-separated plasma pools, we provide in-depth characterization of apolipoprotein distribution across lipoprotein species in these models, and finally, we use the assay to quantify apolipoprotein deposition in mouse atherosclerotic plaques. Taken together, we report development and application of an MRM assay that can be adopted by fellow researchers to monitor the mouse plasma apolipoproteome during preclinical investigations.

M263 Development of a stable isotope dilution mass spectrometry method for absolute quantification of fecal pancreatic elastase

M263 Development of a stable isotope dilution mass spectrometry method for absolute quantification of fecal pancreatic elastase

Stereochemical Assignment and Absolute Abundance of Nonproteinogenic Amino Acid Homoarginine in Marine Sponges.

Together with arginine, the nonproteinogenic amino acid homoarginine is a substrate for the production of vasodilator nitric oxide in the human body. In marine sponges, homoarginine has been postulated to serve as a precursor for the biosynthesis of pyrrole–imidazole alkaloid and bromotyrosine alkaloid classes of natural products. The absolute abundance of homoarginine, its abundance relative to arginine, and its stereochemical assignment in marine sponges are not known. Here, using stable isotope dilution mass spectrometry, we quantify the absolute abundances of homoarginine and arginine in marine sponges. We find that the abundance of homoarginine is highly variable and can far exceed the concentration of arginine, even in sponges where incorporation of homoarginine in natural products cannot be rationalized. The [homoarginine]/[arginine] ratio in marine sponges is greater than that in human analytes. By derivatization of sponge extracts with Marfey’s reagent and comparison with authentic standards, we determine the l-isomer of homoarginine to be exclusively present in sponges. Our results shed light on the presence of the high abundance of homoarginine in marine sponge metabolomes and provide the foundation to investigate the biosynthetic routes and physiological roles of this nonproteinogenic amino acid in sponge physiology.

Application of Stable Isotope Dilution and Liquid Chromatography Tandem Mass Spectrometry for Multi-Mycotoxin Analysis in Edible Oils

Abstract Background: Mycotoxin contamination in oils remains an important food safety issue. To monitor the occurrence of mycotoxins in edible oils, it is important to develop analytical methods that can determine multiple mycotoxins in oil products. A stable isotope dilution LC-tandem MS (LC-MS/MS) method for the simultaneous determination of 12 mycotoxins in five edible oil matrixes (canola, corn, olive, peanut, and soybean oil) was developed and validated. Methods: Prior to extraction, the oil samples were fortified with ¹³C uniformly labeled internal standards (¹³C-IS) for 12 target mycotoxins, followed by extraction and LC-MS/MS analysis. Quantitation was achieved using solvent-only calibration standards, relative response factors of ¹³C-IS, and target mycotoxins. Results: The majority of recoveries in oil for ochratoxin A and aflatoxins B1, B2, G1, and G2 fortified at 1, 10, and 100 ng/g as well as deoxynivalenol; fumonisins B1, B2, and B3; T-2 toxin; HT-2 toxin; and zearalenone fortified at 10, 100, and 1000 ng/g ranged from 80 to 120% with RSDs of &amp;lt;20%. The method LOQs ranged from 0.1 ng/g (aflatoxin B1) to 6.4 ng/g (zearalenone). Among 16 U.S. market samples, zearalenone was detected in three corn oil samples at 37, 185, and 317 ng/g, respectively. T-2 toxin was found in two corn oil samples at 7 and 10 ng/g, respectively. Conclusions: The method provides sufficient selectivity, sensitivity, accuracy, and repeatability to screen edible oils for regulated mycotoxins such as aflatoxins at low nanogram per gram concentrations without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

Application of Stable Isotope Dilution and Liquid Chromatography Tandem Mass Spectrometry for Multi-Mycotoxin Analysis in Edible Oils.

Background: Mycotoxin contamination in oils remains an important food safety issue. To monitor the occurrence of mycotoxins in edible oils, it is important to develop analytical methods that can determine multiple mycotoxins in oil products. A stable isotope dilution LC-tandem MS (LC-MS/MS) method for the simultaneous determination of 12 mycotoxins in five edible oil matrixes (canola, corn, olive, peanut, and soybean oil) was developed and validated. Methods: Prior to extraction, the oil samples were fortified with ¹³C uniformly labeled internal standards (¹³C-IS) for 12 target mycotoxins, followed by extraction and LC-MS/MS analysis. Quantitation was achieved using solvent-only calibration standards, relative response factors of ¹³C-IS, and target mycotoxins. Results: The majority of recoveries in oil for ochratoxin A and aflatoxins B1, B2, G1, and G2 fortified at 1, 10, and 100 ng/g as well as deoxynivalenol; fumonisins B1, B2, and B3; T-2 toxin; HT-2 toxin; and zearalenone fortified at 10, 100, and 1000 ng/g ranged from 80 to 120% with RSDs of <20%. The method LOQs ranged from 0.1 ng/g (aflatoxin B1) to 6.4 ng/g (zearalenone). Among 16 U.S. market samples, zearalenone was detected in three corn oil samples at 37, 185, and 317 ng/g, respectively. T-2 toxin was found in two corn oil samples at 7 and 10 ng/g, respectively. Conclusions: The method provides sufficient selectivity, sensitivity, accuracy, and repeatability to screen edible oils for regulated mycotoxins such as aflatoxins at low nanogram per gram concentrations without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

Characterizing Sirtuin 3 Deacetylase Affinity for Aldehyde Dehydrogenase 2

Mitochondrial aldehyde dehydrogenase (ALDH2) plays a central role in the detoxification of reactive aldehydes generated through endogenous and exogenous sources. The biochemical regulation of enzyme activity through post-translational modification provides an intricate response system regulating mitochondrial detoxification pathways. ALDH2 is a known target of lysine acetylation, which arises as a consequence of mitochondrial bioenergetic flux and sirtuin deacetylase activity. The mitochondrial deacetylase Sirtuin 3 (SIRT3) has been reported to alter ALDH2 lysine acetylation status, yet the mechanism and consequence of this interaction remain unknown. The in vitro results presented here provide a novel biochemical approach using stable-isotope dilution mass spectrometry to elucidate which lysine residues are targeted by SIRT3 for deacetylation. Furthermore, HPLC-MS/MS and computational modeling elucidate a potential role for acetyl-Lys369 on ALDH2 in perturbing normal β-nicotinamide adenine dinucleotide (NAD+) cofactor binding.

Quantification of dimethylsulfoniopropionate (DMSP) in Acropora spp. of reef-building coral using mass spectrometry with deuterated internal standard.

Dimethylsulfoniopropionate (DMSP) in scleractinian coral is usually analysed indirectly as dimethylsulfide (DMS) using gas chromatography (GC) with a sulfur-specific detector. We developed a headspace GC method for mass spectral analysis of DMSP in branching coral where hexa-deuterated DMSP (d 6 -DMSP) was added to samples and standards to optimise the analytical precision and quantitative accuracy. Using this indirect HS-GC-MS method, we show that common coral sample handling techniques did not alter DMSP concentrations in Acropora aspera and that endogenous DMS was insignificant compared to the store of DMSP in A. aspera. Field application of the indirect HS-GC-MS method in all seasons over a 5-year period at Heron Island in the southern Great Barrier Reef indicated that healthy colonies of A. aspera ordinarily seasonally conserve their branch tip store of DMSP; however, this store increased to a higher concentration under extended thermal stress conditions driven by a strong El Niño Southern Oscillation event. A liquid chromatography mass spectral method (LC-MS) was subsequently developed for direct analysis of DMSP in branching coral, also utilising the d 6 -DMSP internal standard. The quantitative comparison of DMSP in four species of Acropora coral by indirect HS-GC-MS and direct LC-MS analyses gave equivalent concentrations in A. aspera only; in the other three species, HS-GC-MS gave consistently higher concentrations, indicating that indirect analysis of DMSP may lead to artificially high values for some coral species. Graphical Abstract Dimethylsulfoniopropionate (DMSP) was quantified in Acropora spp. of branching coral using deuterated stable isotope dilution mass spectrometry.

Microquantification of phospholipid classes by stable isotope dilution and nanoESI mass spectrometry.

A new method for microquantification of phospholipid classes by nanoelectrospray mass spectrometry and stable isotope dilution is presented. The method covers the sum of phosphatidylcholine and sphingomyelin and in addition selectively quantifies phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. A phospholipid class is quantified together with its corresponding lyso-species due to the presence of a common head group. Phospholipids are extracted from tissue lysates, hydrolysed by hydrofluoric acid, and the liberated polar head groups choline, ethanolamine, serine, and inositol are quantified by nanoelectrospray mass spectrometry using deuterium-labeled analogs of the head groups as internal standards. The method is applied to tissue samples of a gastrointestinal tumor and of corresponding non-affected control tissue. In the tumor sample, the abovementioned phospholipids were found at roughly threefold elevated concentrations with a virtually unaltered relative abundance profile.

Long-Term Ursodeoxycholic Acid Therapy Does Not Alter Lithocholic Acid Levels in Patients with Cystic Fibrosis with Associated Liver Disease

To evaluate the fasting and postprandial serum bile acid composition in patients with cystic fibrosis-associated liver disease (CFLD) after chronic administration of ursodeoxycholic acid (UDCA) (20mg/kg/day). The aim was to specifically focus on the extent of biotransformation of UDCA to its hepatotoxic metabolite, lithocholic acid, because of recent concerns regarding the safety of long-term, high-dose UDCA treatment for CFLD. Twenty patients with CFLD (median age 16years, range: 2.4-35.0) prescribed UDCA therapy for at least 2years were studied. Total and individual serum bile acids were measured by stable-isotope dilution mass spectrometry, in fasting and 2-hour postprandial samples taken during chronic UDCA (20mg/kg/day) administration. During chronic UDCA administration (median duration 8years, IQR: 6-16), UDCA became the predominant serum bile acid in all patients (median, IQR: 3.17, 1.25-5.56μmol/L) and chenodeoxycholic acid concentrations were greater than cholic acid (1.86, 1.00-4.70μmol/L vs 0.40, 0.24-2.71μmol/L). The secondary bile acids, deoxycholate and lithocholate, were present in very low concentrations in fasted serum (<0.05μmol/L). After UDCA administration, 2-hour postprandial concentrations of both UDCA and chenodeoxycholic acid significantly increased (P<.01), but no significant changes in serum lithocholic acid concentrations were observed. These data do not support recent suggestions that enhanced biotransformation of UDCA to the hepatotoxic secondary bile acid lithocholic occurs when patients with CFLD are treated with relatively high doses of UDCA.

Utilizing immunoaffinity chromatography (IAC) cross-reactivity in GC–MS/MS exemplified at the measurement of prostaglandin E1 in human plasma using prostaglandin E2-specific IAC columns

Immunoaffinity chromatography (IAC) is an elegant and highly efficient method to isolate a particular compound from biological samples for measurement by mass spectrometry coupled to GC, CE, or LC. The utility of IAC for the quantitative determination of several prostaglandins including prostaglandin E2 (PGE2) by GC-MS/MS and LC-MS/MS has been demonstrated. The aim of the present work was to test whether the cross-reactivity of the antibody immobilized on an insoluble support can be utilized for the quantitative determination of biomolecules by stable-isotope dilution mass spectrometry. In this communication, we provide evidence that this is indeed possible for prostaglandin E1 (PGE1) in human plasma by GC-MS/MS using commercially available Sepharose 4-based IAC columns with immobilized mouse anti-PGE2 monoclonal antibody with a declared cross-reactivity of about 19% toward PGE1. Endogenous PGE1 and the internal standard [3,3',4,4'-(2)H4]-PGE1 (d4-PGE1) externally added to human plasma samples were extracted by IAC, converted to their pentafluorobenzyl ester-methoxime-trimethylsilyl ether derivatives and analyzed by GC-MS/MS in the electron-capture negative-ion chemical ionization mode. Quantification was performed by selected-reaction monitoring of the mass transition m/z 526→m/z 258 for PGE1 and m/z 530→m/z 262 for d4-PGE1. By this method we measured PGE1 concentrations in EDTA plasma samples (1mL) of six healthy volunteers in the range 10-25pg/mL (29-72pM). PGE1 plasma concentration showed a trend for positive correlation with plasma parameters such as low density lipoprotein (LDL)-cholesterol, total cholesterol and glucose. The method described here provides a novel tool to study the potential link of PGE1 formation to dyslipidemia, insulin resistance and related metabolic disorders.

Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization

BackgroundA high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100μl human serum for each measurement (triplicates consume 300μl) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform. MethodsSteroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women. ResultsThe LOD, LOQ, sLOQ was (0.1pg, 0.3pg, 1pg/ml) for estrone, (0.3pg, 1pg, 3pg/ml) for estradiol, and (0.3pg, 1pg, 30pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels. ConclusionsWe have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry.

A new calibration curve calculation method for absolute quantification of drug metabolizing enzymes in human liver microsomes by stable isotope dilution mass spectrometry

A new calibration curve calculation method in stable isotope dilution mass spectrometry for absolute quantification of drug metabolizing enzymes in human liver microsomes.

Determining mycotoxins in baby foods and animal feeds using stable isotope dilution and liquid chromatography tandem mass spectrometry.

We developed a stable isotope dilution assay with liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine multiple mycotoxins in baby foods and animal feeds. Samples were fortified with [(13)C]-uniformly labeled mycotoxins as internal standards ([(13)C]-IS) and prepared by solvent extraction (50% acetonitrile in water) and filtration, followed by LC-MS/MS analysis. Mycotoxins in each sample were quantitated with the corresponding [(13)C]-IS. In general, recoveries of aflatoxins (2-100 ng/g), deoxynivalenol, fumonisins (50-2000 ng/g), ochratoxin A (20-1000 ng/kg), T-2 toxin, and zearalenone (40-2000 ng/g) in tested matrices (grain/rice/oatmeal-based formula, animal feed, dry cat/dog food) ranged from 70 to 120% with relative standard deviations (RSDs) <20%. The method provides sufficient selectivity, sensitivity, accuracy, and reproducibility to screen for aflatoxins at ng/g concentrations and deoxynivalenol and fumonisins at low μg/g concentrations in baby foods and animal feeds, without using conventional standard addition or matrix-matched calibration standards to correct for matrix effects.

Accurate quantification of synthetic peptides by amino acid-stable isotope dilution mass spectrometry

An approach for quantification of peptides by hydrolysis followed by reversed-phase liquid chromatography (RPLC)-stable isotope dilution mass spectrometry (MS) is presented. The purities of all the determined peptides were greater than 99% by using RPLC and over 90% detected by MS, indicating that the method was workable. The hydrolysis conditions optimized were 4-6 h with 6 mol/L HCl at 150 degrees C. Two or more amino acids were chosen to ensure the accuracy of the determined results. The content range of peptides was determined to be 62.07% - 88.18% with the relative standard deviations less than 8% and relative errors less than 5%. Besides Phe, Val and Ile which were commonly used for the analysis of peptide contents, Arg as another amino acid for peptide quantification can be chosen to enhance the popularity of the method. In a word, application of this method for direct determination of peptide content can avoid the side effects in the derivatization of amino acids and the tedious operation in liquid chromatography. This will improve the precision and accuracy of the method and provide an alternative for peptide quantification.

Stable Isotope Dilution Mass Spectrometry for Membrane Transporter Quantitation

This review provides an introduction to stable isotope dilution mass spectrometry (MS) and its emerging applications in the analysis of membrane transporter proteins. Various approaches and application examples, for the generation and use of quantitation reference standards—either stable isotope-labeled peptides or proteins—are discussed as they apply to the MS quantitation of membrane proteins. Technological considerations for the sample preparation of membrane transporter proteins are also presented.

Development of a Simple, Fast, and Accurate Method for the Direct Quantification of Selective Estrogen Receptor Modulators Using Stable Isotope Dilution Mass Spectrometry

A rapid analytical procedure was developed to quantify major selective estrogen receptor modulators (SERMs) simultaneously using stable isotope dilution mass spectrometry (SID-LCMS). Two novel isotopically labeled (SIL) analogues of natural SERMs, genistein and daidzein, were synthesized using a H/D exchange reaction mechanism. Computational chemistry coupled with MS and NMR data confirmed the site and mechanism of deuteration. The SIL analogues, which were mono- and dideutero substituted at the ortho positions, exhibited minimal deuterium isotope effects and were stable under the employed sample preparation protocol and MS analysis. An isotopic overlap correction was successfully employed to improve the accuracy and precision of the analytical method. The developed method, which was found to be sensitive, selective, precise and accurate, could be a valuable tool for research focused on determining the bioavailability of individual SERMs.