Stable Isotope Dilution Liquid Research Articles

Stable isotope dilution liquid chromatography/mass spectrometry analysis of cellular and tissue medium- and long-chain acyl-coenzyme A thioesters

Acyl-Coenzyme A (CoA) thioesters are the principal form of activated carboxylates in cells and tissues. They are employed as acyl carriers that facilitate the transfer of acyl groups to lipids and proteins. Quantification of medium- and long-chain acyl-CoAs represents a significant bioanalytical challenge because of their instability. Stable isotope dilution liquid chromatography/selected reaction monitoring-mass spectrometry (LC/SRM-MS) provides the most specific and sensitive method for the analysis of CoA species. However, relevant heavy isotope standards are not available and they are challenging to prepare by chemical synthesis. Stable isotope labeling by essential nutrients in cell culture (SILEC), developed originally for the preparation of stable isotope labeled short-chain acyl-CoA thioester standards, has now been extended to medium-chain and long-chain acyl-CoAs and used for LC/SRM-MS analyses. Customized SILEC standards with >98% isotopic purity were prepared using mouse Hepa 1c1c7 cells cultured in pantothenic-free media fortified with [(13) C3 (15) N1 ]-pantothenic acid and selected fatty acids. A SILEC standard in combination with LC/SRM-MS was employed to quantify cellular concentrations of arachidonoyl-CoA (a representative long-chain acyl-CoA) in two human colon cancer cell lines. A panel of SILEC standards was also employed in combination LC/SRM-MS to quantify medium- and long-chain acyl-CoAs in mouse liver. This new SILEC-based method in combination with LC/SRM-MS will make it possible to rigorously quantify medium- and long-chain acyl-CoAs in cells and tissues. The method will facilitate studies of medium- and long-chain acyl-CoA dehydrogenase deficiencies as well as studies on the role of medium- and long-chain acyl-CoAs in cellular metabolism.

Determination of regorafenib (BAY 73-4506) and its major human metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in human plasma by stable-isotope dilution liquid chromatography-tandem mass spectrometry.

Regorafenib (BAY 73-4506, commercial name Stivarga(®)) is an oral multikinase inhibitor developed by Bayer Pharma AG (Germany) that targets angiogenic, stromal and oncogenic receptor tyrosine kinases. An isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the simultaneous determination of regorafenib and its two major metabolites BAY 75-7495 (M-2) and BAY 81-8752 (M-5) in lithium-heparinized human plasma. Analysis was performed after protein precipitation on a triple-quadrupole tandem mass spectrometer. The validated concentration range was from 2.00 (lower limit of quantitation) to 2000 µg/l for all the analytes. Selectivity and specificity in the presence of four potential comedications were demonstrated. Interassay accuracy and precision coefficient of variation ranged from 91.2% (at LLOQ of M-5) to 105% and 3.59 to 14.1%, respectively. The method proved to be selective, specific, sufficiently sensitive, highly reproducible and robust for the analysis of large numbers of samples. The method performance met current guidelines on bioanalytical method validation.

Reliable LC-MS/MS assay for the estimation of rilpivirine in human plasma: application to a bioequivalence study and incurred sample reanalysis.

A simple, precise, and rapid stable isotope dilution liquid chromatography-tandem mass spectrometry method has been developed and validated for the quantification of rilpivirine, a non-nucleoside reverse transcriptase inhibitor in human plasma. Rilpivirine and its deuterated analogue, rilpivirine-d6, used as an internal standard (IS) were quantitatively extracted by liquid-liquid extraction with methyl-tert-butyl ether and diethyl ether solvent mixture from 50 μL plasma. The chromatography was achieved on Gemini C18 (150 × 4.6 mm, 5 µm) analytical column in a run time of 2.2 min. The precursor → product ion transitions for rilpivirine (m/z 367.1 → 128.0) and IS (m/z 373.2 → 134.2) were monitored on a triple quadrupole mass spectrometer in the positive ionization mode. The linearity of the method was established in the concentration range of 0.5-200 ng/mL. The mean extraction recovery for rilpivirine (94.9%) and IS (99.9%) from spiked plasma samples was consistent and reproducible. The IS-normalized matrix factors for rilpivirine ranged from 0.98 to 1.02 across three quality controls. Bench top, freeze-thaw, wet extract, and long-term stability of rilpivirine was examined in spiked plasma samples. The application of the method was demonstrated by a bioequivalence study with 25 mg rilpivirine tablet formulation in 40 healthy subjects. The assay reproducibility was shown by reanalysis of 200 study samples and the % change in the concentration of repeat values from the original values was within ±15%.

Malonylglucoside Conjugates of Isoflavones Are Much Less Bioavailable Compared with Unconjugated β-Glucosidic Forms in Rats1–3

Despite considerable interest in the physiologic effects of isoflavones, the in vivo bioavailability of the most common isoflavone forms, malonylglucoside conjugates, has not been determined. Differences in the bioavailability of malonylglucosides compared with the nonconjugated β-glucoside forms may explain the inconsistent findings regarding the physiologic effects of isoflavones. Therefore, our objective was to determine the effect of malonyl- conjugation on isoflavone bioavailability in an animal model. Malonylgenistin and malonyldaidzin, and their corresponding nonconjugated glucosides, were extracted from soy grits and purified using liquid chromatography. Purity of the isolated forms was confirmed by nuclear magnetic resonance analysis. Male rats were gavaged with malonylgenistin, genistin, malonyldaidzin, or daidzin at a dose of 100 μmol/kg body weight. Blood and urine samples were collected at time intervals ranging from 0 to 48 h. Isoflavone metabolites in plasma and urine were determined using stable isotope dilution–liquid chromatography/mass spectrometry. Comparisons of pharmacokinetic variables were made between nonconjugated and conjugated glucosides and over time of plasma collection. The areas under the time-concentration curve of the metabolites in the plasma obtained after the administration of nonconjugated β-glucosides were 1 to 6 times higher than those of their respective malonylglucosides (P ≤ 0.05). Additionally, maximum plasma concentration and urinary excretion of isoflavone metabolites were significantly higher (1–9 times; P ≤ 0.05) after the administration of nonconjugated β-glucosides. To our knowledge, these results demonstrated, for the first time, that nonconjugated β-glucosides are relatively more bioavailable than their respective malonylglucosides. These differences in the bioavailability of conjugated and nonconjugated β-glucosides should be considered in future studies focused on the bioactivity of isoflavones.

Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry

Trimethylamine-N-oxide (TMAO) levels in blood predict future risk for major adverse cardiac events including myocardial infarction, stroke, and death. Thus, the rapid determination of circulating TMAO concentration is of clinical interest. Here we report a method to measure TMAO in biological matrices by stable isotope dilution liquid chromatography tandem mass spectrometry (LC/MS/MS) with lower and upper limits of quantification of 0.05 and >200μM, respectively. Spike and recovery studies demonstrate an accuracy at low (0.5μM), mid (5μM), and high (100μM) levels of 98.2, 97.3, and 101.6%, respectively. Additional assay performance metrics include intraday and interday coefficients of variance of <6.4 and <9.9%, respectively, across the range of TMAO levels. Stability studies reveal that TMAO in plasma is stable both during storage at −80°C for 5years and to multiple freeze thaw cycles. Fasting plasma normal range studies among apparently healthy subjects (n=349) show a range of 0.73–126μM, median (interquartile range) levels of 3.45 (2.25–5.79)μM, and increasing values with age. The LC/MS/MS-based assay reported should be of value for further studies evaluating TMAO as a risk marker and for examining the effect of dietary, pharmacologic, and environmental factors on TMAO levels.

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.

Plasma concentrations of arginine and asymmetric dimethylarginine do not reflect their intracellular concentrations in peripheral blood mononuclear cells

ObjectiveProduction of nitric oxide (NO) from arginine is inhibited by endogenously produced monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA). Elevated levels of ADMA, by limiting NO production, may lead to endothelial dysfunction and cardiovascular disease. Symmetric dimethylarginine (SDMA) and the arginine homolog homoarginine have also been associated with cardiovascular disease. Although NO synthesis, as well as generation of MMA, ADMA, SDMA and homoarginine, occurs intracellularly, these biomarkers are usually measured in plasma. Despite extensive transmembrane transport, it is not clear whether plasma levels of these biomarkers are a valid proxy for their intracellular levels in the cardiovascular system. Since it is difficult to obtain vascular tissue from healthy humans, we explored the relations between concentrations of these biomarkers in plasma and intracellular concentrations in peripheral blood mononuclear cells (PBMC). MethodsIn PBMC and plasma of 27 healthy subjects, concentrations of arginine, MMA, ADMA, SDMA, and homoarginine were determined using stable isotope dilution liquid chromatography tandem mass spectrometry. ResultsIn PBMC, significant positive correlations were observed among arginine and its methylated forms (ρ = 0.43 to 0.81) and these correlations were slightly less pronounced in plasma. Homoarginine was not significantly correlated with (methylated) arginine in either PBMC or plasma. Plasma concentrations of arginine and its methylated forms showed non-significant inverse associations with their respective intracellular concentrations in PBMC and only for homoarginine was a weak positive association observed (ρ = 0.37). ConclusionIn healthy individuals, plasma levels of arginine, MMA, ADMA, and SDMA poorly reflect their intracellular levels in PBMC.

Development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization/selected reaction monitoring/mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of keto-androgens in human serum

Prostate cancer is the most frequently diagnosed form of cancer in males in the United States. The disease is androgen driven and the use of orchiectomy or chemical castration, known as androgen deprivation therapy (ADT) has been employed for the treatment of advanced prostate cancer for over 70 years. Agents such as GnRH agonists and non-steroidal androgen receptor antagonists are routinely used in the clinic, but eventually relapse occurs due to the emergence of castration-resistant prostate cancer. With the appreciation that androgen signaling still persists in these patients and the development of new therapies such as abiraterone and enzalutamide that further suppresses androgen synthesis or signaling, there is a renewed need for sensitive and specific methods to quantify androgen precursor and metabolite levels to assess drug efficacy. We describe the development, validation and application of a stable isotope dilution liquid chromatography electrospray ionization selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for quantification of serum keto-androgens and their sulfate and glucuronide conjugates using Girard-T oxime derivatives. The method is robust down to 0.2–4pg on column, depending on the androgen metabolite quantified, and can also quantify dehydroepiandrosterone sulfate (DHEA-S) in only 1μL of serum. The clinical utility of this method was demonstrated by analyzing serum androgens from patients enrolled in a clinical trial assessing combinations of pharmacological agents to maximally suppress gonadal and adrenal androgens (Targeted Androgen Pathway Suppression, TAPS clinical trial). The method was validated by correlating the results obtained with a hydroxylamine derivatization procedure coupled with tandem mass spectrometry using selected reaction monitoring that was conducted in an independent laboratory.

Determination of Mycotoxins in Milk-Based Products and Infant Formula Using Stable Isotope Dilution Assay and Liquid Chromatography Tandem Mass Spectrometry

A stable isotope dilution assay and liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination of 12 mycotoxins, aflatoxins B₁, B₂, G₁, G₂, and M₁, deoxynivalenol, fumonisins B₁, B₂, and B₃, ochratoxin A, T-2 toxin, and zearalenone, in milk-based infant formula and foods. Samples were fortified with 12 ¹³C uniformly labeled mycotoxins ([¹³C]-mycotoxins) that correspond to the 12 target mycotoxins and prepared by dilution and filtration, followed by LC-MS/MS analysis. Quantitation was achieved using the relative response factors of [¹³C]-mycotoxins and target mycotoxins. The average recoveries in fortified milk, milk-based infant formula, milk powder, and baby yogurt of aflatoxins B₁, B₂, G₁, and G₂ (2, 10, and 50 μg/kg), aflatoxin M₁ (0.5, 2.5, and 12.5 μg/kg), deoxynivalenol, fumonisins B₁, B₂, and B₃ (40, 200, and 1000 μg/kg), ochratoxin A, T-2 toxin, and zearalenone (20, 100, and 500 μg/kg), range from 89 to 126% with RSDs of <20%. The individual recoveries in the four fortified matrices range from 72% (fumonisin B₃, 20 μg/kg, milk-based infant formula) to 136% (T-2 toxin, 20 μg/kg, milk powder), with RSDs ranging from 2 to 25%. The limits of quantitation (LOQs) were from 0.01 μg/kg (aflatoxin M₁) to 2 (fumonisin B₁) μg/kg. Aflatoxin M₁ was detected in two European Reference materials at 0.127 ± 0.013 μg/kg (certified value = 0.111 ± 0.018 μg/kg) and 0.46 ± 0.04 μg/kg (certified value = 0.44 ± 0.06 μg/kg), respectively. In 60 local market samples, aflatoxins B₁ (1.14 ± 0.10 μg/kg) and B₂ (0.20 ± 0.03 μg/kg) were detected in one milk powder sample. Aflatoxin M₁ was detected in three imported samples (condensed milk, milk-based infant formula, and table cream), ranging from 0.10 to 0.40 μg/kg. The validated method provides sufficient selectivity, sensitivity, accuracy, and reproducibility to screen for aflatoxin M₁ at nanograms per kilogram concentrations and other mycotoxins, without using standard addition or matrix-matched calibration to compensate for matrix effects.

Abstract 349: Measurement of Platelet Cyclooxygenase-1 Acetylation Quantifies Interaction of Nonsteroidal Anti-Inflammatory Drugs with Aspirin in vivo

Aspirin prevents heart attack and stroke by irreversibly acetylating serine-529 in human platelet cyclooxygenase-1 (COX-1) and thereby inhibiting the biosynthesis of thromboxane (Tx). We hypothesized that precise quantification of COX-1 acetylation would allow the direct detection of drug-drug interactions between reversible COX inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), and aspirin, which may abrogate its cardio-protective effect. We developed a stable isotope dilution liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) method for measurement of platelet COX-1 acetylation. Serine-529 acetylation reflected faithfully aspirin pharmacology determined with conventional markers (serum TxB2 formation, urinary excretion of 11-dehydro-TxB2, and arachidonic acid induced platelet aggregation), but showed lower technical (CV% 3.7±2.7) and inter-individual variability (CV% 3.7). Oral administration of 325 mg of plain aspirin resulted in complete acetylation of platelet COX-1 within 2 hours and sustained maximal acetylation (71 ± 3% of total COX-1 amount) for up to two days in health volunteers (n=8). Recovery from acetylation, which reflects platelet turn-over, occurred at a rate of 7.2 ± 0.2 % per day. We compared the potential of five NSAIDs to block aspirin from acetylating platelet COX-1 in healthy volunteers (n=7 for each NSAID). Single doses of naproxen (500 mg), ibuprofen (600 mg), and diclofenac (100 mg) but not celecoxib (200 mg) and acetaminophen (1000 mg) prevented complete COX-1 acetylation by 325 mg aspirin administered 2 hours after the NSAID. The competition of NSAIDs with aspirin was conditioned by NSAID pharmacokinetics, potency, and COX isoform selectivity, suggesting variability in the likelihood of the drug-drug interaction between compounds and between patients. Monitoring platelet COX-1 acetylation may have utility in the identification of drug interactions that may limit the cardio-protective effect of low dose aspirin.

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.

Determination of Di-(2-ethylhexyl)phthalate (DEHP) metabolites in human hair using liquid chromatography–tandem mass spectrometry

BackgroundDi-(2-ethylhexyl)phthalate (DEHP) is an endocrine disrupting chemical that is widely used as the major plasticizer for worldwide plastic products. It can cause several toxic effects to human with high dose exposure. In response to the need of human exposure assessment, different biological specimens are taken into account. Compared to blood, urine and other specimens, hair is unique in that it could determine the time period of chemical exposure after several months to years. MethodThe developed method consists of solution incubation, liquid–liquid extraction and stable isotope dilution liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. ResultsA reliable and sensitive analytical method was developed and validated for the determination of 5 metabolites, mono-(2-ethylhexyl)phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (MEHHP), mono-(2-ethyl-5-oxy-hexyl)phthalate (MEOHP), mono-(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono-[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) in human hair. Ten authentic hair specimens were successfully determined and quantitated by the developed method. ConclusionThe developed LC–MS/MS method can successfully determine specific DEHP metabolites in human hair and has a great potential to assess the long term DEHP exposure of human.

Simultaneous determination of asymmetric and symmetric dimethylarginine, l-monomethylarginine, l-arginine, and l-homoarginine in biological samples using stable isotope dilution liquid chromatography tandem mass spectrometry

Production of the endogenous vasodilator nitric oxide (NO) from l-arginine by NO synthase is modulated by l-homoarginine, l-monomethylargine (MMA), asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA). Here we report on a stable isotope dilution liquid chromatography tandem mass spectrometry (LC–MS/MS) method for simultaneous determination of these metabolites in plasma, cells and tissues. After addition of the internal standards (D7-ADMA, D4-l-homoarginine and 13C6-l-arginine), analytes were extracted from the samples using Waters Oasis MCX solid phase extraction cartridges. Butylated analytes were separated isocratically on a Waters XTerra MS C18 column (3.5μm, 3.9mm×100mm) using 600mg/L ammonium formate in water - acetonitrile (95.5:4.5, v/v) containing 0.1vol% formic acid, and subsequently measured on an AB Sciex API 3000 triple quadrupole mass spectrometer. Multiple reaction monitoring in positive mode was used for analyte quantification. Validation was performed in plasma. Calibration lines were linear (r2≥0.9979) and lower limits of quantification in plasma were 0.4nM for ADMA and SDMA and 0.8nM for the other analytes. Accuracy (% bias) was <3% except for MMA (<7%), intra-assay precision (expressed as CV) was <3.5%, inter-assay precision <9.6%, and recovery 92.9–103.2% for all analytes. The method showed good correlation (r2≥0.9125) with our previously validated HPLC-fluorescence method for measurement in plasma, and was implemented with good performance for measurement of tissue samples. Application of the method revealed the remarkably fast (i.e. within 60min) appearance in plasma of stable isotope-labeled ADMA, SDMA, and MMA during infusion of D3-methyl-1-13C-methionine in healthy volunteers.

8-Oxo-2′-deoxyguanosine as a biomarker of tobacco-smoking-induced oxidative stress

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dGuo) is a useful biomarker of oxidative stress. However, its analysis can be challenging because 8-oxo-dGuo must be quantified in the presence of dGuo, without artifactual conversion to 8-oxo-dGuo. Urine is the ideal biological fluid for population studies, because it can be obtained noninvasively and it is less likely that artifactual oxidation of dGuo can occur because of the relatively low amounts that are present compared with hydrolyzed DNA. Stable isotope dilution liquid chromatography–selected reaction monitoring/mass spectrometry (LC-SRM/MS) with 8-oxo-[15N5]dGuo as internal standard provided the highest possible specificity for 8-oxo-dGuo analysis. Furthermore, artifact formation was determined by addition of [13C1015N5]dGuo and monitoring of its conversion to 8-oxo-[13C1015N5]dGuo during the analytical procedure. 8-Oxo-dGuo concentrations were normalized for interindividual differences in urine flow by analysis of creatinine using stable isotope dilution LC–SRM/MS. A significant increase in urinary 8-oxo-dGuo was observed in tobacco smokers compared with nonsmokers either using simple urinary concentrations or after normalization for creatinine excretion. The mean levels of 8-oxo-dGuo were 1.65ng/ml and the levels normalized to creatinine were 1.72μg/g creatinine. Therefore, stable isotope dilution LC–SRM/MS analysis of urinary 8-oxo-dGuo complements urinary isoprostane (isoP) analysis for assessing tobacco-smoking-induced oxidative stress. This method will be particularly useful for studies that employ polyunsaturated fatty acids, in which a reduction in arachidonic acid precursor could confound isoP measurements.

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.

Determination of cyclophosphamide and ifosfamide in sewage effluent by stable isotope-dilution liquid chromatography–tandem mass spectrometry

A reliable and specific method was developed for the determination of the cytotoxic drugs cyclophosphamide and ifosfamide in sewage effluent. The most successful combination was found to be Strata-X solid-phase extraction followed by Florisil ® clean-up with analysis by liquid chromatography–tandem mass spectrometry. Quantification by internal standardisation was achieved using custom synthesised d4-cyclophophosphamide. The mass spectrometer was operated in highly selective reaction monitoring (HSRM) mode, which significantly reduced matrix noise and improved sensitivity. Although it suffered from some ionisation suppression, electrospray ionisation (ESI) was found to give an order of magnitude better sensitivity in terms of limit of detection than atmospheric pressure chemical ionisation (APCI). Using final effluent from two different sewage treatment plants, the method was validated following official European guidelines and shown to be a high performance tool for routine analysis at the sub-nanogram per litre level. Depending on the matrix, the limit of detection for cyclophosphamide was between 0.03 ng/L and 0.12 ng/L and for ifosfamide between 0.05 ng/L and 0.09 ng/L. For cyclophosphamide the accuracy and precision, tested at 1.7 ng/L, were 98–109% and ≤13%, CV respectively. For ifosfamide the accuracy and precision, tested at 1.1 ng/L, were 98–113% and ≤15% CV, respectively. Depending on the sample matrix the absolute recovery of the internal standard was between 57% and 70%. The method was tested by analysis of spot samples taken from the final effluent discharges of two sewage treatment plants; the first using a conventional trickling filter treatment process and second employing activated sludge followed by ultra violet treatment. Cyclophosphamide was detected at 0.19 ng/L at the first plant and at the second detected at 3.7 ng/L and 3.5 ng/L, before and after the UV treatment process; ifosfamide was not detectable at either plant.

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile

RE: Infantile and early childhood masturbation: Sex hormones and clinical profile