Stable Isotope Dilution Liquid Chromatography-mass Research Articles

Determination of Total and A1-Type β-Casein in Milk and Milk-Derived Ingredients by Liquid Chromatography-Mass Spectrometry Using Characteristic Tryptic Peptides.

Gastrointestinal digestion of A1-type β-casein is conducive to β-casomorphin-7 with potential adverse digestive health effects. Monitoring of A1-type β-casein concentration in milk and milk-derived ingredients used in the formulation of A2-type nutritional products with associated health claims is important from a quality standpoint. New analytical methods were developed and validated for total and A1-type β-casein in milk and milk-derived ingredients. Data on total and A1-type β-casein concentrations in milk, nonfat dry milk, and whey protein concentrate was generated. The methods are based on a bottom-up proteomic approach using tryptic marker peptides and stable isotope dilution liquid chromatography-mass spectrometry. The measurement includes all protein sequences (intact, modified, and partial) which are potential sources of β-casomorphin-7. Total β-casein was quantified using a neat calibration curve. Recovery and between-day precision RSD were 98% and 5.8%, respectively. A1-type β-casein was quantified by the method of standard additions. Between-day precision RSD was 7.2% and limit of quantitation was 0.01% in nonfat dry milk. The mass fraction of A1-type β-casein in the β-casein standard was 0.444. Samples manufactured from A2-type milk contained 0.26-5.0% A1-type β-casein relative to total β-casein. The methods described enable the monitoring of the A1-type β-casein concentration in milk and milk-derived ingredients destined for the manufacture of A2-type products with associated health claims. New methods are presented for the analysis of total and A1-type β-casein in milk and milk-derived ingredients. The mass fraction of A1-type β-casein in a commercial β-casein standard was determined to enable its use as a calibrant.

Determination and quantification of arbutin in plants using stable isotope dilution liquid chromatography–mass spectrometry

Arbutin is a very safe whitening agent for human skin. Since it is more expensive than other agents and has a challenging synthesis, novel methods to obtain this valuable agent are needed. In this study, we developed a precise and accurate method to detect and quantify arbutin using stable isotope dilution liquid chromatography–mass spectrometry (LC–MS). One challenge that needed to be overcome was the matrix effect occurring during the LC–MS analysis due to the analyte ionisation enhancement or suppression in the electrospray ionisation source by co-eluting compounds. Notably, arbutin had different matrix effects in the various sample matrices. A solution to this problem was the use of [d4]-arbutin as a stable isotope-labelled internal standard (SIL-IS), as it compensated the matrix effect of arbutin because it was affected by almost the same matrix effect. The validation of the developed method showed excellent linearity (r2 = 1.000), precision (relative standard deviation ≤ 2.5%), accuracy (recovery, 97.42–98.52%), limit of detection (0.03 μg/mL), and limit of quantification (0.1 μg/mL). Finally, the method of arbutin detection was applied to blueberry leaves to compare the precision and accuracy results obtained by performing stable isotope dilution using LC–MS and gas chromatography–mass spectrometry. The method was applied to strawberry leaves and pear peels, indicating that the SIL-IS method can be expected to find application in the arbutin analysis in other plants.

Synthesis of (13) C2 (15) N2 -labeled anti-inflammatory and cytoprotective tricyclic bis(cyanoenone) ([(13) C2 (15) N2 ]-TBE-31) as an internal standard for quantification by stable isotope dilution LC-MS method.

Tricyclic bis(cyanoenone), TBE-31, one of the most potent activators of the Keap1/Nrf2/antioxidant response element pathway, has been developed as a new anti-inflammatory and cytoprotective agent. (13) C2 (15) N2 -labeled TBE-31 ([(13) C2 (15) N2 ]-TBE-31), which has two (13) C and two (15) N atoms in two cyano groups, was designed to develop a method for quantification of cell, tissue, and plasma levels of TBE-31 that involves chromatography/mass spectrometry coupled with the use of a stable isotope-labeled internal standard. [(13) C2 (15) N2 ]-TBE-31 was successfully synthesized in four steps from a previously reported intermediate, which is prepared in 11 steps from cyclohexanone, by introduction of two (13) C atoms with ethyl [(13) C]formate and two (15) N atoms with hydroxyl[(15) N]amine. The stable isotope dilution liquid chromatography-mass spectrometry method for quantification of TBE-31 was successfully developed using [(13) C2 (15) N2 ]-TBE-31 to compensate for any variables encountered during sample processing and analysis.

Magnetite Nanoparticles Induce Genotoxicity in the Lungs of Mice via Inflammatory Response.

Nanomaterials are useful for their characteristic properties and are commonly used in various fields. Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields, whereas their toxicological properties are not well documented. A safety assessment is thus urgently required for MGT, and genotoxicity is one of the most serious concerns. In the present study, we examined genotoxic effects of MGT using mice and revealed that DNA damage analyzed by a comet assay in the lungs of imprinting control region (ICR) mice intratracheally instilled with a single dose of 0.05 or 0.2 mg/animal of MGT was approximately two- to three-fold higher than that of vehicle-control animals. Furthermore, in gpt delta transgenic mice, gpt mutant frequency (MF) in the lungs of the group exposed to four consecutive doses of 0.2 mg MGT was significantly higher than in the control group. Mutation spectrum analysis showed that base substitutions were predominantly induced by MGT, among which G:C to A:T transition and G:C to T:A transversion were the most significant. To clarify the mechanism of mutation caused by MGT, we analyzed the formation of DNA adducts in the lungs of mice exposed to MGT. DNA was extracted from lungs of mice 3, 24, 72 and 168 h after intratracheal instillation of 0.2 mg/body of MGT, and digested enzymatically. 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and lipid peroxide-related DNA adducts were quantified by stable isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS). Compared with vehicle control, these DNA adduct levels were significantly increased in the MGT-treated mice. In addition to oxidative stress- and inflammation related-DNA adduct formations, inflammatory cell infiltration and focal granulomatous formations were also observed in the lungs of MGT-treated mice. Based on these findings, it is suggested that inflammatory responses are probably involved in the genotoxicity induced by MGT in the lungs of mice.

Biochemical Fractionation and Stable Isotope Dilution Liquid Chromatography-mass Spectrometry for Targeted and Microdomain-specific Protein Quantification in Human Postmortem Brain Tissue

Synaptic architecture and its adaptive changes require numerous molecular events that are both highly ordered and complex. A majority of neuropsychiatric illnesses are complex trait disorders, in which multiple etiologic factors converge at the synapse via many signaling pathways. Investigating the protein composition of synaptic microdomains from human patient brain tissues will yield valuable insights into the interactions of risk genes in many disorders. These types of studies in postmortem tissues have been limited by the lack of proper study paradigms. Thus, it is necessary not only to develop strategies to quantify protein and post-translational modifications at the synapse, but also to rigorously validate them for use in postmortem human brain tissues. In this study we describe the development of a liquid chromatography-selected reaction monitoring method, using a stable isotope-labeled neuronal proteome standard prepared from the brain tissue of a stable isotope-labeled mouse, for the multiplexed quantification of target synaptic proteins in mammalian samples. Additionally, we report the use of this method to validate a biochemical approach for the preparation of synaptic microdomain enrichments from human postmortem prefrontal cortex. Our data demonstrate that a targeted mass spectrometry approach with a true neuronal proteome standard facilitates accurate and precise quantification of over 100 synaptic proteins in mammalian samples, with the potential to quantify over 1000 proteins. Using this method, we found that protein enrichments in subcellular fractions prepared from human postmortem brain tissue were strikingly similar to those prepared from fresh mouse brain tissue. These findings demonstrate that biochemical fractionation methods paired with targeted proteomic strategies can be used in human brain tissues, with important implications for the study of neuropsychiatric disease.

Relative Quantification of Serum Proteins from Pancreatic Ductal Adenocarcinoma Patients by Stable Isotope Dilution Liquid Chromatography–Mass Spectrometry

We report an innovative multiplexed liquidchromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS)-based assay for rapidly measuring a large number of disease specific protein biomarkers in human serum. Furthermore, this approach uses stable isotope dilution methodology to reliably quantify candidate protein biomarkers. Human serum was diluted using a stable isotope labeled proteome (SILAP) standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity removal of the most highly abundant proteins, trypsin digested, and analyzed by LC-MRM/MS. The method was found to be precise, linear, and specific for the relative quantification of 72 proteins when analyte response was normalized to the relevant internal standard (IS) from the SILAP. The method made it possible to determine statistically different concentrations for three proteins (cystatin M, IGF binding protein 7, and villin 2) in control and pancreatic cancer patient samples. This method proves the feasibility of using a SILAP standard in combination with stable isotope dilution LC-MRM/MS analysis of tryptic peptides to compare changes in the concentration of candidate protein biomarkers in human serum.

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers(1,2). These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer(3-7). Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)(1,8). Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products. While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis(9). After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity(10,11). Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids(12). This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites(13). Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products. While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13. Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

Rotenone-mediated changes in intracellular coenzyme A thioester levels: implications for mitochondrial dysfunction.

Rotenone, an organic pesticide and potent mitochondrial complex I inhibitor, causes Parkinson-like neurodegeneration in rodents and is implicated in human Parkinson’s disease. In this rapid report, rotenone induced a dose-dependent decrease in succinyl-coenzyme A (CoA) and increase in β-hydroxybutyryl-CoA in multiple human cell lines (IC50 < 100 nM). Rotenone also inhibited [U-13C6]-glucose-derived [13C]-acetyl-CoA and [13C]-succinyl-CoA biosynthesis in SH-SY5Y neuroblastoma cells. These changes are compatible with a compensatory metabolic rearrangement. Stable isotope dilution liquid chromatography–mass spectrometry and CoA thioester isotopomer analysis provided insight into mechanisms of rotenone toxicity, which will facilitate the development of new biomarkers of mitochondrial dysfunction.

Aldo-keto reductase 1C3 expression in MCF-7 cells reveals roles in steroid hormone and prostaglandin metabolism that may explain its over-expression in breast cancer

Aldo-keto reductase (AKR) 1C3 (type 5 17β-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD 2 to 9α,11β-PGF 2, Δ 4-androstenedione to testosterone, progesterone to 20α-hydroxyprogesterone, and to a lesser extent, estrone to 17β-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1 μM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17β-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor α induced proliferation. MCF-7-AKR1C3 cells also reduced PGD 2, limiting its dehydration to form PGJ 2 products. The AKR1C3 product was confirmed as 9α,11β-PGF 2 and quantified with a stereospecific stable isotope dilution liquid chromatography–mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD 2 on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor γ (PPARγ) signaling by reducing formation of 15-deoxy-Δ 12,14-PGJ 2 (15dPGJ 2).

Quantification of Focal Adhesion Kinase Activation Loop Phosphorylation as a Biomarker of Src Activity

A recently developed stable isotope dilution liquid chromatography-multiple reaction/mass spectrometry method to quantify focal adhesion kinase (FAK) activation loop phosphorylation was used to study endogenous Src kinase activity. This revealed that bis-phosphorylated pTyr(576)/Tyr(577)-FAK was a biomarker of Src activity and inactivation in vitro and in cell culture. Mouse embryonic fibroblasts (MEFs) expressing endogenous Src family kinases contained 65% unmodified Tyr(576)/Tyr(577), 33% mono-phosphorylated-pTyr(576)-FAK, and 6% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. In contrast, MEFs expressing oncogenic Y(529)FSrc contained 38% unmodified Tyr(576)/Tyr(577)-FAK, 29% mono-phosphorylated-pTyr(576)-FAK, and 19% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. This new method has made it possible to accurately determine the absolute amounts of FAK phosphorylation that occur after Src inhibition in cell culture and in vitro with increasing concentrations of the Src inhibitor N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530). Phosphorylation of FAK at Tyr(576)/Tyr(577) was inhibited by AZD0530 in a dose-dependent manner both in cell culture and in vitro. However, there was a substantial difference in the ability of AZD0530 to inhibit Src that was constitutively activated in a cellular context (IC(50) = 2.12 muM) compared with the isolated enzyme (IC(50) = 0.14 muM). When normal MEFs and Y(529)FSrc-expressing MEFs were treated with pervanadate (a global phosphatase inhibitor), pTyr(576)/pTyr(577)-FAK accounted for almost 60% of the total FAK present in the cells. This suggests that activation loop phosphorylation is regulated by tyrosine phosphatases. These results confirm that FAK phosphorylation is a useful biomarker of Src inhibition in vivo. The accuracy and specificity of stable isotope dilution liquid chromatography-mass spectrometry methodology offers significant advantages over current immunochemical approaches for monitoring Src activity.

Phenylalanine and Tyrosine Quantification by Stable Isotope Dilution Liquid Chromatography–Mass Spectrometry from Filter Paper Blood Spots

Phenylketonuria (PKU) is one of the more common inborn errors of metabolism, affecting 1 in 10 000–15 000 individuals (1). Newborn screening for PKU is performed in all 50 states and started with the development of the bacterial inhibition screening assay by Guthrie and Susi (2). Frequent monitoring of blood Phe concentrations is required to maintain the concentration as close as possible to the reference interval [60–120 μmol/L (1–2 mg/dL)] to prevent damage to the brain from increased Phe concentrations and to prevent a negative nitrogen balance from an overly Phe-restricted diet. Moreover, the high risk of birth defects for the fetus of a PKU mother requires extremely frequent monitoring of blood Phe concentrations and would greatly benefit from a rapid and accurate blood spot method. Phe determination from dried blood spots is feasible by various methods: bacterial (2), fluorometric (3), liquid chromatography (LC) (4), and tandem mass spectrometry (MS) (5). Of these methods, only tandem MS allows use of a stable isotope as internal standard. Although tandem MS is the most rapid method available, the extremely high equipment cost and the need for sample derivatization are disadvantages. We developed a rapid and precise blood-spot method for Phe and Tyr determination using stable isotope dilution reversed-phase LC-MS. The advantages of this method are the very short sample preparation without derivatization, a short chromatographic run, the use of selective detection to eliminate interference, and the use of ideal internal standardization (stable isotope). The method described herein is now used routinely in our PKU clinic. l-Phe ( M r 165.19) and l-Tyr ( M r 181.19) were purchased from Sigma Chemical Co. Stable isotopes [ring-d5]-l-phenylalanine ( M r 170.23) …

Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.