Quantitative analysis of multiple fatty acid ethanolamides using ultra-performance liquid chromatography–tandem mass spectrometry

Fatty acid ethanolamides (FAE) represent a group of lipid signaling molecules associated with many physiological and pharmacological actions; however, low FAE tissue levels pose challenges in terms of analytical characterization. The objective was to develop a competent ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for analysis of multiple FAE in animal and human tissue samples. Analytes were extracted using lipid-phase and solid-phase extraction procedures. Chromatographic separation was achieved using a gradient elution in 8 min. FAE were quantified by MS/MS in positive electrospray ionization mode. Linearity was shown in lower and higher FAE concentration ranges, with a limit of quantification (LOQ) ≤0.2 ng/ml for FAE including alpha-linolenoylethanolamide (ALEA), arachidonoylethanolamide (AEA), docosahexaenoylethanolamide (DHEA), linoleoylethanolamide (LEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). Accuracy was shown to be between 92.4% and 108.8%, and precision was <10% for all FAE species. In sum, this sensitive and reproducible method can be used to simultaneously determine multiple FAE at low concentrations in order to facilitate further study of the role of FAE on physiological state.     

The genus Dracunculus--a source of triacylglycerols containing odd-numbered ω-phenyl fatty acids

Reversed phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to identify and quantify triacylglycerols (TAGs) having odd-numbered ω-phenylalkanoic acids from seeds of the flower plant Dracunculus vulgaris, and TAGs from the bacterium Rhodococcus erythropolis prepared by precursor directed biosynthesis from phenylalanine and having the corresponding even-numbered ω-phenylalkanoic acids. Model compounds, which are not commercially available, were prepared by organic synthesis and this allowed us to extend the number of identified natural TAGs to nearly 140 molecular species. Both synthetic and natural compounds containing ω-phenylalkanoic acids were found to have antioxidant and free radical scavenging properties.     

Identification of free fatty acids profiling of type 2 diabetes mellitus and exploring possible biomarkers by GC–MS coupled with chemometrics

Free fatty acids (FFAs), which are considered to be closely related with type 2 diabetes mellitus (T2DM), are not only the main energy source as nutrients, but also signaling molecules in insulin secretion. In this study, gas chromatography–mass spectrometry (GC–MS) coupled with two chemometric resolution methods, heuristic evolving latent projections (HELP) and selective ion analysis (SIA), was successfully applied to investigate plasma FFAs profiling of T2DM. Totally, twenty-three FFAs were identified and quantified. The results showed that HELP and SIA methods could be used to effectively handle overlapping peaks of GC–MS data and hence improve the qualitative and quantitative accuracy. Furthermore, a newly proposed competitive adaptive reweighted sampling (CARS) method coupled with partial least squares linear discriminant analysis (PLS-LDA) was introduced to seek the potential biomarkers. Finally, three fatty acids, oleic acid (OLA C18:1n-9), α-linolenic acid (ALA C18:3n-3), and eicosapentaenoic acid (EPA C20:5n-3), were identified as the potential biomarkers of T2DM for their powerful discriminant ability of T2DM patients from healthy controls. The study indicated that GC–MS combining with chemometric methods was a useful strategy to analyze metabolites and further screen the potential biomarkers of T2DM.     

Measurement of N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography–tandem quadrupole mass spectrometry

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional ¹⁵N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M + 1 isotopomers of each natural AQC–amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M + 1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of ¹⁵N enrichment was confirmed by measuring a series of ¹⁵N-labeled arginine standards. The positional ¹⁵N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional ¹⁵N enrichment in the plasma of rats fed ¹⁵N-labeled glutamine. These results suggest the utility of LC–MS/MS detection of AQC–amino acids for the measurement of isotopic enrichment in ¹⁵N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.     

Higher de novo synthesized fatty acids and lower ω3- and ω6-long-chain polyunsaturated fatty acids in umbilical vessels of women with preeclampsia and high fish intakes

Umbilical veins (UV) and arteries (UA) of preeclamptic women in Curaçao harbor lower long-chain polyunsaturated fatty acids (LCP). The present aim was to test these findings in Mwanza (Tanzania), whose inhabitants have high LCPω3 and LCPω6 intakes from Lake Victoria fish. Women with preeclampsia (n=28) in Mwanza had lower PUFA and higher 20:0 in UV and UA, compared with normotensive/non-proteinuric controls (n=31). Their UV 22:6ω3, 22:4ω6, LCPω6, ω6, and LCPω3+ω6 were lower, while saturated FA, potentially de novo synthesized FA (Σde novo) and (Σde novo)/(LCPω3+ω6) ratio were higher. Their UA had higher 16:1ω7, ω7, 18:0, and 16:1ω7/16:0. Umbilical vessels in Mwanza had higher 22:6ω3, LCPω3, ω3, and 16:0, and lower 22:5ω6, 20:2ω6, 18:1ω9, and ω9, compared to those in Curaçao. Preeclampsia in both Mwanza and Curaçao is characterized by lower LCP and higher Σde novo. An explanation of this might be placental dysfunction, while the similarity of umbilical vessel FA-abnormalities in preeclamptic and diabetic pregnancies suggests insulin resistance as a common denominator.     

Reaction of thiyl radicals with alcohols,ethers and polyunsaturated fatty acids: A possible role of thiyl free radicals in thiol mutagenesis?

Summary Possible reactions of thiyl free radicals in biological environment are reviewed. In particular hydrogen transfer processes from model C–H compounds like alcohols and ethers as well as from polyunsaturated fatty acids to thiyl radicals are described to proceed with reasonably high rate constants (10₃ – 10⁴ and 10⁶ – 10⁷ M^–1 s^–1, respectively). Thiyl radicals have thus to be considered as potentially hazardous species especially with respect to DNA damage and lipid peroxidation.Paper given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.1990     

Determination of phosphoserine/threonine by nano ultra-performance liquid chromatography–tandem mass spectrometry coupled with microscale labeling

Protein phosphorylation is an important regulatory post-translational modification in many biochemical processes. The phosphopeptide analysis strategies developed in this study were all at microscale. After using a standard microwave oven to assist protein digestion, phosphoserine and phosphothreonine were tagged with chemical analogues, such as 2-mercaptoethanol and 3-mercapto-1-propanol, to enable simultaneously relative quantitation and identification. This method enabled the use of thio alcohols for direct labeling of phosphorylated sites (not labeled at the mercapto, amino, hydroxyl, or carboxyl groups) of phosphopeptides. Various digestion parameters (e.g., microwave power, reaction time, NH₄HCO₃ concentration) and derivatization efficiency parameters (e.g., reaction time, labeling tag concentration) were studied and optimized. In both control and experimental samples, microwave-assisted digestion coupled with relative quantitation using analogue tags enabled calculation of phosphopeptide ratios in the same sequence. A non-labeling method was also established for quantifying phosphopeptides in human plasma by using the abundant protein albumin as an internal control for normalizing relative quantities of phosphopeptides. Nano ultra-performance liquid chromatography (nanoUPLC) was combined with LTQ Orbitrap to enable simultaneous protein relative quantitation and identification. These strategies proved to be effective for quantifying phosphopeptides in biological samples.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Δ6-Desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated fatty acids

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and α-linolenic acid. EFAs are converted into ω3- and ω6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of Δ6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2−/− mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood–testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2−/− mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.     

Comprehensive analysis of the mouse renal cortex using two-dimensional HPLC – tandem mass spectrometry

Background  

Proteomic methodologies increasingly have been applied to the kidney to map the renal cortical proteome and to identify global changes in renal proteins induced by diseases such as diabetes. While progress has been made in establishing a renal cortical proteome using 1-D or 2-DE and mass spectrometry, the number of proteins definitively identified by mass spectrometry has remained surprisingly small. Low coverage of the renal cortical proteome as well as our interest in diabetes-induced changes in proteins found in the renal cortex prompted us to perform an in-depth proteomic analysis of mouse renal cortical tissue.     

Determination of six hydroxyalkyl mercapturic acids in human urine using hydrophilic interaction liquid chromatography with tandem mass spectrometry (HILIC–ESI-MS/MS)

Mercapturic acids are increasingly used as biomarkers for exposure to certain carcinogenic substances. Glycidol, ethylene oxide, propylene oxide, acrolein and 1,3-butadiene are important intermediates of toxicological concern used in the industrial production of various chemicals. The main urinary metabolites of these alkylating substances are hydroxyalkyl mercapturic acids. Therefore, we developed and validated an analytical method for the simultaneous determination of six hydroxyalkyl mercapturic acids in human urine after solid phase extraction. The mercapturic acids were separated using hydrophilic interaction liquid chromatography (HILIC) and quantified by tandem mass spectrometry using isotopically labelled internal standards. The developed method enables for the first time the determination of 2,3-dihydroxypropyl mercapturic acid (DHPMA), a metabolite of glycidol, in human urine. Additionally, the mercapturic acids of ethylene oxide (hydroxyethyl mercapturic acid, HEMA), propylene oxide (2-hydroxypropyl mercapturic acid, 2-HPMA), acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA) as well as of 1,3-butadiene(3,4-dihydroxybutyl mercapturic acid, DHBMA and monohydroxy-3-butenyl mercapturic acid, MHBMA) can be determined. The limits of detection range from 3.0 to 7.0 μg/L. Intra- and inter-day precision was determined to range from 1% to 9%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for the determination of occupational exposure to alkylating substances as well as for the determination of background concentrations of the respective mercapturic acids in the general population.     

Identification of Nocardia species using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

Background

The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC).

Results

The newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level.

Conclusions

The results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9078-5) contains supplementary material, which is available to authorized users.     

Quantification of lysophosphatidic acids in rat brain tissue by liquid chromatography–electrospray tandem mass spectrometry

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological functions. A highly selective and sensitive liquid chromatography–tandem mass spectrometry (LC/MS/MS) method was developed for the determination of LPAs (16:0 LPA, 18:0 LPA, 18:1 LPA, 20:4 LPA) in rat brain cryosections. After partitioning the LPAs from other lipophilic material present in the tissue with a liquid–liquid extraction, a reversed-phase column and ion pair technique was used for separating analytes with a gradient elution. An internal standard (17:0 LPA) was included in the analysis. Detection and quantification of the LPAs were carried out with a triple quadrupole mass spectrometer using negative electrospray ionization (ESI) and multiple reaction monitoring (MRM). The artificial formation of LPAs from lysophosphatidylcholines during the sample preparation procedure and instrumentation was carefully studied during the method development. The method was validated; acceptable selectivity, accuracy, precision, recovery, and stability were obtained for concentrations within the calibration curve range of 0.02–1.0 μM for LPAs. The quantification limit of the assay was 54 fmol injected into column for each LPAs. The method was applied to comparative studies of LPA levels in rat brain cryosections after the various chemical pre-treatments of the sections.     

Hydroxyl and 1-hydroxyethyl free radical detection using spin traps followed by derivatization and gas chromatography—mass spectrometry

Summary

Detection of hydroxyl free radicals is frequently performed by electron spin resonance (ESR) following spin trapping of the radical using 5,5-dimethylpyrroline N-oxide (DMPO) to generate a stable free radical having a characteristic ESR spectrum. The necessary ESR equipment is expensive and not readily available to many laboratories. In the present study, a specific and sensitive gas chromatography—mass spectrometry (GC/MS) method for detection of hydroxyl and hydroxyethyl free radicals is described. The DMPO or N-t-butyl—α—phenylnitrone (PBN) radical adducts are extracted and derivatized by trimethylsylilation and analyzed by GC/MS. To standardize the method, ^.OH and 1-hydroxyethyl radicals were generated in two different systems: 1) a Fenton reaction in a pure chemical system in the absence or presence of ethanol and 2) in liver microsomal suspensions where ethanol is metabolized in the presence of NADPH. In the Fenton system both radicals were easily detected and specifically identified using DMPO or PBN. In microsomal suspensions DMPO proved better for detection of ^.OH radicals and PBN more suitable for detection of 1-hydroxyethyl radicals. The procedure is specific, sensitive and potentially as useful as ESR.     

Structural analysis of mycolic acids from phenol-degrading strain of Rhodococcus erythropolis by liquid chromatography–tandem mass spectrometry

We used reversed phase liquid chromatography?Celectrospray ionization tandem mass spectrometry for direct analysis of mycolic acids (MAs) from four different cultivations of Rhodococcus erythropolis. This technique enabled us to identify and quantify the specific molecular species of MAs directly from lipid extracts of the bacterium, including the determination of their basic characteristics such as retention time and mass spectra. We identified a total of 60 molecular species of MAs by means of LC/MS. In collision-induced dissociation tandem mass spectrometry, the [M-H]^? ions eliminated two residues, i.e., meroaldehyde and carboxylate anions containing ??-alkyl chains. The structural information from these fragment ions affords structural assignment of the mycolic acids, including the lengths and number of double bond(s). Two strains, i.e., R. erythropolis CCM 2595 and genetically modified strain CCM 2595 pSRK 21 phe were cultivated on two different substrates (phenol and phenol with addition of humic acids as a sole carbon source). The addition of humic acids showed that there is a marked increase of unsaturated mycolic acids, mostly in the range of 20?C100?%. This effect is more pronounced in the R. erythropolis CCM 2595 strain.     

Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Occurrence of Curtobacterium sp. possessing ω-cyclohexyl fatty acids in soil with zinc added

A bacterial strain, Curtobacterium sp., isolated from a soil with zinc added possessed -cyclohexyl fatty acids. -Cyclohexyl undecanoic acid made up 47% of the total fatty acids; it was the most abundant fatty acid in the strain grown in tryptone medium. 12-Methyl tetradecanoic acid (23%) and 14-methyl hexadecanoic acid (22%) were also major fatty acids. The proportion of -cyclohexyl undecanoic acid increased as the pH of the medium decreased and as the culture temperature increased.The bacteria grew almost normally in zinc-enriched medium, and -cyclohexyl undecanoic acid increased with zinc concentration. Zinc added to the medium was not abundant in the cell fraction, and the ratio of increase of zinc in the cells was not so high as in the culture medium. These results suggested that -cyclohexyl fatty acids are related to the zinc tolerance of the isolated strain, and that this tolerance depends on low permeability of the membrane to zinc.     

Microscale analysis of amino acids using gas chromatography–mass spectrometry after methyl chloroformate derivatization

To conduct studies of stable isotope incorporation and dilution in growing plants, a rapid microscale method for determination of amino acid profiles from minute amounts of plant samples was developed. The method involves solid-phase ion exchange followed by derivatization and analysis by gas chromatography–mass spectrometry (GC–MS). The procedure allowed the eluent to be derivatized directly with methyl chloroformate without sample lyophilization or other evaporation procedures. Sample extraction and derivatization required only ca. 30 min and quantification of the 19 amino acids eluted from the cation exchange solid-phase extraction step from a single cotyledon (0.4 mg fresh weight) or three etiolated 7-day-old Arabidopsis seedlings (0.1 mg fresh weight) was easily accomplished in the selected ion monitoring mode. This method was especially useful for monitoring mass isotopic distribution of amino acids as illustrated by Arabidopsis seedlings that had been labeled with deuterium oxide and ¹⁵N salts. Sample preparation was facile, rapid, economical, and the method is easily modified for integration into robotic systems for analysis with large numbers of samples.     

Acyl coenzyme A: Phospholipid acyltransferases in porcine platelets discriminate between ω-3 and ω-6 unsaturated fatty acids

The properties of porcine platelet acyltransferases which catalyze the incorporation of unsaturated fatty acids into the 2 positions of phospholipids were compared with those of porcine liver microsomes and rat liver microsomes. There were significant differences in the relative rates of incorporation of acyl groups into phospholipids as catalyzed by the membranes from different species and organs. The 1-acylglycerophosphate acyltransferase system showed relatively broad specificity for saturated and unsaturated fatty acids, with 14- to 20-carbon chains, while unsaturated acyl-CoAs with 18- and 20-carbon chains were generally good substrates in the acylations of 1-acylglycerophosphocholine and 1-acylglycerophosphoinositol. ω-3 and ω-6 unsaturated fatty acids were recognized differently by different acyltransferase systems in platelets. When activities for combinations of ω-3 and ω-6 unsaturated acyl-CoAs with the same number of carbons and with similar number of double bonds were compared, ω-6 fatty acids were relatively more preferred substrates than ω-3 fatty acids for the 1-acylglycerophosphoinositol acyltransferase system as compared with 1-acylglycerophosphocholine acyltransferase system.