Chemical Ionization Tandem Mass Spectrometry Research Articles

Single-use porous thin film extraction with gas chromatography atmospheric pressure chemical ionization tandem mass spectrometry for high-throughput analysis of 16 PAHs

The need for high throughput, reliable analytical methods for environmental analysis has driven our development of novel sampling technologies that can be used in microextraction devices and integrated into chromatographic systems for fast analysis. Here we report a new method of water analysis for 16 EPA priority polycyclic aromatic hydrocarbons (PAHs) that relies on a tailor made porous polymeric film for extraction with analysis by gas chromatography with atmospheric pressure chemical ionization-tandem mass spectrometry (APGC-MS/MS). The extraction device is user friendly, single-use and gives exhaustive extraction from a relatively small volume (20 mL). The films show good stability, with excellent analytical performance. Critical factors affecting the extraction efficiency such as desorption solvent, desorption time, extraction agitation, and extraction time were studied and optimized. These thin films are cost effective and efficient for single use analysis, but carry-over studies showed that the films can be reused at least 10 times without extensive washing and with no loss in performance. The 16 PAHs were measured with excellent limits of detection ranging from 1 pg mL−1 for BaA to 100 pg mL−1 for Flu and linearity (R2 > 0.997) over the 1–50,000 pg mL−1 concentration range. This high throughput method (1-h extractions, which can be completed simultaneously) showed good reproducibility (<20%) with recoveries in the range of 19.0–40.6% and concentration factors varying between 37.9 and 81.1. Analysis of real seawater and river water samples using matrix-matched calibration in synthetic seawater and river water showed only small matrix effects maintaining high sensitivity (LODs:1–100 pg mL−1), demonstrating that the films are suitable for analysis of real samples without the need for standard addition.

Quantitative Analysis of Eight Triterpenoids and Two Sesquiterpenoids in Rhizoma Alismatis by Using UPLC-ESI/APCI-MS/MS and Its Application to Optimisation of Best Harvest Time and Crude Processing Temperature.

Rhizoma Alismatis (RA), widely known as “Ze-Xie” in China, is the tuber of Alisma orientale (Sam.) Juzep (Alismaceae), a Chinese herbal medicine that has been used to treat hyperlipidemia, diabetes, hypertension, dysuria, and inflammation. In this study, a sensitive and reliable method based on an ultra-performance liquid chromatography (UPLC) couple with two ionisation modes, including electrospray ionisation (ESI) and atmospheric pressure chemical ionisation (APCI) tandem mass spectrometry (MS), namely, UPLC-ESI/APCI-MS/MS was developed and validated to simultaneously determine 8 triterpenoids (ESI mode) and 2 sesquiterpenoids (APCI mode) in RA. Ten marker compounds were analysed with a Waters' CORTECS UPLC C18 column (200 mm × 2.1 m, 1.6 μm) and gradient elution with water (contained 0.1% formic) and acetonitrile within 7 min. The established method was validated for linearity, intra- and interday precisions, accuracy, recovery, and stability. The calibration curve for 10 marker compounds showed good linear regression (r > 0.9971). The limits of detection and quantification for analytes were 0.14–1.67 ng/mL and 0.44–5.65 ng/mL, respectively. The relative standard deviations (RSD, %) and accuracy (RE, %) of intra- and interday precisions were less than 3.83% and 1.21% and 3.22% and 1.46%, repeatability and stability for real samples were less than 2.78% and 3.19%, respectively. All recoveries of the 10 marker compounds ranged from 97.24% to 102.49% with RSDs less than 4.05%. The developed method efficiently determined the 10 marker compounds in RA and was subsequently applied to optimise harvest time and crude processing temperature. The result indicated the 90% wilted phase and 70°C (or lower) may be the best harvest time and the processing temperature of RA.

Endocrine Hormone Beta-estradiol and Anti-estrogen Letrozole Modulate 20:3 Isomer Production from 20:2n-6 in Human Cancer Cells (P08-119-19)

ObjectivesThe product of linoleic acid elongation, 20:2n-6, is the only known substrate for both FADS1 and FADS2. FADS1 catalyzes 20:2n-6 à 5Z,11Z,14Z-20:3 (sciadonic acid, SA), whereas FADS2 action yields 8Z,11Z,14Z-20:3 (DGLA). In certain cancer cell types, non-functional FADS2 activity unmasks 18:2n-6 elongation to 20:2n-6 and Δ5 desaturation by FADS1 to yield SA. Estrogen is known to inhibit Δ5-desaturation activity. Our objective was to examine the effect of 17-β-estradiol (estrogen) and the antiestrogen drug letrozole on the modulation of fatty acid desaturation and SA levels in vitro. MethodsEstrogen doses (0 to 200 ppm) and letrozole doses (0 to 100 ppm) studies were performed using stably transformed MCF7 cells with either FADS1 or FADS2 or empty vector and five different (MCF7, HepG2, SK-N-SH, Y79 and Caco2) wild type cancer cells. Fatty acid methyl esters (FAME) were prepared from harvested cells and analyzed quantitatively by gas chromatograph-flame ionization detector (GC-FID). The peak structures were positively identified by GC-covalent adduct chemical ionization tandem mass spectrometry (GC-CACI-MS/MS). ResultsIncreasing estrogen caused a decrease in the 5-desaturation product 5,11,14–20:3 in a dose-response manner in all cell types, but had no effect on DGLA. Letrozole exerted its effects most prominently in MCF7 cells. In response to increasing letrozole doses, DGLA increased, and SA decreased. ConclusionsWe provide the first biochemical evidence demonstrating letrozole enhancing FADS2-mediated DGLA levels in MCF7 cells. In all cell types estrogen reduced FADS1 mediated synthesis of SA. Both product fatty acids have anti-inflammatory activity, DGLA as a precursor to PGE1 and SA in displacing the largely proinflammatory arachidonic acid. Funding SourcesNIH grant R01 AT007003.

Development and validation of a liquid chromatography-tandem mass spectrometry method for quantification of Lupeol in plasma and its application to pharmacokinetic study in rats

Lupeol, a phytosterol and triterpene, possesses numerous medicinal properties against cancer, inflammation, arthritis, diabetes, heart diseases, etc. A novel, sensitive, specific and reproducible method for quantification of Lupeol in rat plasma using liquid chromatography combined with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (LC-MS/MS) was developed and validated as per regulatory guidelines. Sample preparation was simple and fast which consisted of one-step protein precipitation using acetonitrile. Testosterone was used as an internal standard. HyPurity Advance column was used to develop the chromatography method using 0.1% formic acid in water and acetonitrile as mobile phases by gradient elution. APCI positive ion mode was used for mass spectrometric detection. Multiple reaction monitoring (MRM) transitions of m/z 409.5 [M + H - H2O]+→137.3 for Lupeol and m/z 289.1 [M + H]+→97.1 for Testosterone were used for quantification. The method was validated over a linear concentration range of 5–5000 ng/mL with a correlation coefficient (r2) of ≥ 0.99. This method showed acceptable accuracy (89.52–97.10%), precision (%CV ≤ 10.75%) and recovery with a negligible matrix effect. Lupeol was found to be stable in the stock solution, autosampler condition and also in plasma for four freeze-thaw cycles, 6 h at ambient temperature and 30 days at −20°C. This method was successfully applied to measurement of Lupeol in plasma samples from pharmacokinetic study in rats and can be easily extended to human pharmacokinetic studies.

Background levels of dioxin-like polychlorinated biphenyls (dlPCBs), polychlorinated, polybrominated and mixed halogenated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs, PBDD/Fs & PXDD/Fs) in sera of pregnant women in Accra, Ghana

Human exposure data on dioxins and dioxin-like compounds (DLCs) in Ghana are limited. Based on health risks associated with dioxins and DLCs, the impact of maternal body burdens on foetal exposure is significant. This is the first study that assesses polychlorinated, polybrominated and mixed halogenated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs, PBDD/Fs and PXDD/Fs), and dioxin-like polychlorinated biphenyls (dlPCBs) in sera of primiparous Ghanaians. Our sample selection includes 34 participants from two municipalities (Accra and Tema), and explores contributions from environmental and dietary exposures using questionnaire data. Sample preparation involved C18 solid phase extraction, purification with acidified silica and lipid removal cartridges, and detection with gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. The calculated average toxic equivalent concentration was 5.3 pg TEQ/g lw, with contributions from dlPCBs (1.25 pg TEQ/g lw), PCDD/Fs (3.10 pg TEQ/g lw), PBDD/Fs (0.49 pg TEQ/g lw) and PXDD/Fs (0.50 pg TEQ/g lw). The calculated total TEQ concentration was lower than background TEQ concentrations reported in sera of pregnant women globally. Positive correlations were obtained for total dioxins and DLC concentrations with age and Body Mass Index (BMI). Dietary intake of seafood and dairy products had a strong influence on PCDD/F and dlPCB concentrations. Statistically significant differences were observed for dioxins and DLCs in participants from Accra (in close proximity to Agbogbloshie e-waste site) and Tema. Given the significant TEQ contribution of PBDD/Fs and PXDD/Fs (~20%), it is essential to explore these classes of dioxins and DLCs in future biomonitoring studies as they may pose health risks, and add extra diagnostic information in source exposure investigations.

Evaluation of gas chromatography-atmospheric pressure chemical ionization tandem mass spectrometry as an alternative to gas chromatography tandem mass spectrometry for the determination of polychlorinated biphenyls and polybrominated diphenyl ethers

A method for the analysis of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in serum was developed using gas chromatography coupled to tandem mass spectrometry with atmospheric pressure chemical ionization (GC-APCI-MS/MS). The ionization and fragmentation performance of APCI were evaluated and compared with those of electron ionization (EI). In contrast to extensive fragmentation caused by EI with high energy, soft ionization achieved by APCI produced mass spectra dominated by molecular ions from the first stage of MS analysis. Better sensitivity and selectivity achieved by the GC-APCI-MS/MS method allowed to analyze serum samples with a low volume (100 μL). The limits of detection (LODs) ranged from 0.067 to 14 pg/mL for the analysis in serum samples. The developed method was evaluated at three spiking levels (0.05, 0.5 and 5 ng/mL for PCBs), showing good recoveries and repeatability. The recoveries ranged from 74.0% to 130.5%, and the relative standard deviations (RSDs) were less than 20%, for all analytes. The determination of PCBs and PBDEs in the human serum samples by GC-APCI-MS/MS was compared with gas chromatography-tandem mass spectrometry with EI (GC-EI-MS/MS). BDE-99 and BDE-100 were successfully quantified by GC-APCI-MS/MS, while these two PBDE congeners were not detected by GC-EI-MS/MS. The GC-APCI-MS/MS method had a clear advantage when analyzing compounds at low levels.

Determination of acrylamide in coffee by ultra performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry

A method using ultra high performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (UHPLC-APCI-MS/MS) was developed for the determination of acrylamide in coffee. The coffee samples spiked with 13C3-acrylamide as the internal standard were extracted with methanol, and cleaned using HLB solid phase extraction (SPE) cartridges. The liquid chromatography separation was performed on a Brownlee validated AQ C18 column with isocratic elution. Methanol and 0.1% (volume percentage) formic acid aqueous solution were used as the mobile phase. Identification of acrylamide was achieved by APCI-MS/MS with multiple reaction monitoring (MRM) in the positive mode. The quantification analysis was performed by the internal standard method. The calibration curve showed good linearity with a correlation coefficient of 0.999 in the range of 0.5-100.0 μg/L. The limit of detection (LOD) was 5.0 μg/kg. The limit of quantification (LOQ) was 10.0 μg/kg. Recovery of acrylamide from coffee sample was evaluated at concentrations of 100.0, 200.0 and 1000.0 μg/kg. The average recoveries of acrylamide were between 94.6%-115.0% with relative standard derivations (RSDs) in the range of 2.8%-3.6% (n=6). This simple, accurate and sensitive method was proven to be suitable for the determination of acrylamide in coffee.

Oxidation evaluation of free astaxanthin and astaxanthin esters in Pacific white shrimp during iced storage and frozen storage.

In this paper, the changes in free astaxanthin (F-AST) and astaxanthin esters (AST-Es) in Litopenaeus vannamei during iced storage and frozen storage were investigated. The liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry method was used to quantify the molecular species of AST-Es in shrimp during storage. Based on the analysis of autoxidation products, apo-12-astaxanthinal and apo-13-astaxanthinone docosahexaenoic acid (DHA) ester were identified as the major oxidation products of F-AST and AST-Es in L. vannamei during storage. The total astaxanthin (T-AST) content decreased by 34.51% after 7 days in iced storage. In contrast, the content of T-AST decreased by 43.76% after 12 weeks in frozen storage. The content of F-AST decreased by 29.99% while 13-cis-astaxanthin increased after 3 days in iced storage, which indicated that degradation of AST was accompanied by isomerization. Total volatile basic nitrogen and T-AST content showed a significant negative correlation while in frozen storage, where the concentration of T-AST might be one indicator to evaluate shrimp freshness. The correlation coefficients between phenol oxidase, lipoxygenase, apo-12-astaxanthinal, and apo-13-astaxanthinone DHA ester were all greater than 0.97 (P < 0.01). This correlation indicates that phenol oxidase and lipoxygenase were the main internal factors to improve oxygenation of astaxanthin in L. vannamei. These results provide a theoretical basis for further study of oxidation and the degradation mechanism in astaxanthin, as well as a new idea for the development and utilization of astaxanthin compounds in Pacific white shrimp. © 2018 Society of Chemical Industry.

Determination of polybrominated diphenyl ethers and novel brominated flame retardants in human serum by gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

The accurate detection of brominated flame retardants (BFRs) in humans is an area of high scientific interest and regulatory need due to their potential toxicity. The instrumental analysis of BFRs was commonly performed on gas chromatography-mass spectrometry (GC-MS) operating in electron ionization (EI) or negative chemical ionization (NCI) modes. However, soft ionization techniques, such as atmospheric pressure chemical ionization (APCI), may be more suitable for the analysis of BFRs because the BFRs show high fragmentation in EI and low selectivity in NCI. Additionally, accurate quantifications of BFRs in complex matrices is challenging due to their low concentrations and therefore, a highly sensitive technique is desperately needed. In this study, a new methodology based on gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry (GC-APCI-MS/MS) analysis was developed for the determination of thirteen BFRs (eight usually monitored polybrominated diphenyl ethers (PBDEs) congeners and five additional novel BFRs) in human serum. The primary task was to evaluate the potential of the GC-APCI-MS/MS technique for the trace analysis of BFRs in human serum. The results of the spiked recovery test using fetal bovine serum showed that mean recoveries of the analytes ranged from 83.4% to 118% with reduced swing differential signaling (RSDs) of ≤21.1%. The methodological limits of detection (mLOD) of the analytes ranged from 0.04 to 30 pg/mL, and these values were at least one order of magnitude lower than those estimated by the authors in a previous study using GC-NCI-MS or GC-EI-MS/MS, indicating that GC-APCI-MS/MS is more sensitive. Specially, compared to GC-NCI-MS and GC-EI-MS/MS, when GC-APCI-MS/MS was used for the detection of highly brominated BFRs, such as BDE-209 and decabromodiphenyl ethane (DBDPE), a notable improvement in sensitivity and reliability was obtained using a deactivated capillary column connected to the analytical column as the transfer line and maintaining a high temperature to improve the chromatographic behaviors. The developed methodology was successfully used for the analysis of BFRs in human serum collected from residents living in a BFR production area and Beijing.

Identification of organic hydroperoxides and peroxy acids using atmospheric pressure chemical ionization–tandem mass spectrometry (APCI-MS/MS): application to secondary organic aerosol

Abstract. Molecules with hydroperoxide functional groups are of extreme importance to both the atmospheric and biological chemistry fields. In this work, an analytical method is presented for the identification of organic hydroperoxides and peroxy acids (ROOH) by direct infusion of liquid samples into a positive-ion atmospheric pressure chemical ionization–tandem mass spectrometer ((+)-APCI-MS/MS). Under collisional dissociation conditions, a characteristic neutral loss of 51 Da (arising from loss of H2O2+NH3) from ammonium adducts of the molecular ions ([M + NH4]+) is observed for ROOH standards (i.e. cumene hydroperoxide, isoprene-4-hydroxy-3-hydroperoxide (ISOPOOH), tert-butyl hydroperoxide, 2-butanone peroxide and peracetic acid), as well as the ROOH formed from the reactions of H2O2 with aldehydes (i.e. acetaldehyde, hexanal, glyoxal and methylglyoxal). This new ROOH detection method was applied to methanol extracts of secondary organic aerosol (SOA) material generated from ozonolysis of α-pinene, indicating a number of ROOH molecules in the SOA material. While the full-scan mass spectrum of SOA demonstrates the presence of monomers (m∕z = 80–250), dimers (m∕z = 250–450) and trimers (m∕z = 450–600), the neutral loss scan shows that the ROOH products all have masses less than 300 Da, indicating that ROOH molecules may not contribute significantly to the SOA oligomeric content. We anticipate this method could also be applied to biological systems with considerable value.

Ionization of tocopherols and tocotrienols in atmospheric pressure chemical ionization.

Tocopherols and tocotrienols are chemical compounds insusceptible to the ionization process under atmospheric pressure conditions. Therefore, the selection of the optimal ion source settings for their quantification requires special attention. The aim of this study was to analyse the influence of the APCI source parameters on the response of tocochromanols and two related compounds. Standard solutions of target compounds were injected on the high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (HPLC/APCI-MS/MS) system separately and analysed with 30 randomly selected ion source settings. The obtained responses were modelled by multivariate linear regression with least absolute shrinkage and selection operator. The developed models were used to choose the best APCI conditions. Multivariate linear models were built for eight tocochromanols, trolox and BHT. The APCI settings derived from the models did not increase the peak areas obtained for T and T3 during the ionization process. Ionization conditions based on models for trolox and BHT improved analytical responses by 12-36% and 4-32%, respectively. The application of the ion source settings optimal for trolox and BHT to tocochromanols did not result in better analytical responses. The ionization pattern of tocochromanols in the APCI source is problematic and should be further investigated. Modelling methodology for response improvement presented in this study can be applied in similar studies.

Enantiomeric separation of triacylglycerols containing very long chain fatty acids

Enantiomers of triacylglycerols (TAGs) containing any combination of very long chain fatty acids (VLCFAs) and/or very long chain polyunsaturated fatty acids (VLCPUFAs) with diolein, dilinolein and didocosahexaenoin were synthesized. Gradient non-aqueous reversed-phase high-performance liquid chromatography/high resolution atmospheric pressure chemical ionization-tandem mass spectrometry (NARP-HPLC/HRMS2-APCI) and chiral liquid chromatography were used for the separation and identification of molecular species of these TAGs. Further, NARP-LC and chiral LC were used to separate natural mixtures of TAGs obtained from four natural sources, i.e. ximenia oil (Ximenia americana), green alga (Botryococcus braunii), breweŕs yeast (Saccharomyces pastorianus) and a dinoflagellate (Amphidinium carterae). The ratio of regioisomers and enantiomers in individual samples was determined and a hypothesis has been confirmed on the biosynthetic pathway of natural TAGs, which is based on the preferential representation of VLCFAs and VLCPUFAs in the sn-1 position of the glycerol backbone.

N-nitrosamines migrating from food contact materials into food simulants: analysis and quantification by means of HPLC-APCI-MS/MS

ABSTRACTIt was the aim of the work described here to develop a validated analytical method for the determination of N-nitrosamines in food simulants. Here, we present the validation of a liquid chromatography–atmospheric pressure chemical ionisation–tandem mass spectrometry method for the determination of 13 N-nitrosamines in the food simulants deionised water, 3% acetic acid and 10% ethanol. Method validation encompassed linearity, LOD, LOQ, recovery, precision and stability of the N-nitrosamines. The method was found to be sufficiently rugged and suitable for routine analysis. In addition, the developed method is on average 10 times more sensitive than the gas chromatography–thermal energy analyser (GC-TEA) method that is currently stipulated in Recommendation XXI for commodities based on natural and synthetic rubber issued by the German Federal Institute for Risk Assessment (BfR). The developed method was applied to determine the N-nitrosamine contents in migration solutions of 12 elastomer samples covering a range of different elastomer types. In 10 out of 12 samples, N-nitrosamines were determined. In most samples, the guidance value of 1 µg/dm2 specified in BfR Recommendation XXI was not exceeded. In conclusion, the analytical method presented here offers a useful alternative to the GC-TEA method currently stipulated in BfR Recommendation XXI. This is of wider relevance, since BfR recommendations are used for compliance assessment of Article 3, paragraph 1 a of the Regulation (EC) No. 1935/2004 with respect to their health safety.

Relevance of 4-F4t-neuroprostane and 10-F4t-neuroprostane to neurological diseases.

F4-neuroprostanes (F4-NeuroPs) are non-enzymatic oxidized products derived from docosahexaenoic acid (DHA) and are suggested to be oxidative damage biomarkers of neurological diseases. However, 128 isomers can be formed from DHA oxidation and among them, 4(RS)-4-F4t-NeuroP (4-F4t-NeuroP) and 10(RS)-10-F4t-NeuroP (10-F4t-NeuroP) are the most studied. Here, we report the identification and the clinical relevance of 4-F4t-NeuroP and 10-F4t-NeuroP in plasma of four different neurological diseases, including multiple sclerosis (MS), autism spectrum disorders (ASD), Rett syndrome (RTT), and Down syndrome (DS). The identification and the optimization of the method were carried out by gas chromatography/negative-ion chemical ionization tandem mass spectrometry (GC/NICI-MS/MS) using chemically synthesized 4-F4t-NeuroP and 10-F4t-NeuroP standards and in oxidized DHA liposome. Both 4-F4t-NeuroP and 10-F4t-NeuroP were detectable in all plasma samples from MS (n = 16), DS (n= 16), ASD (n= 9) and RTT (n = 20) patients. While plasma 10-F4t-NeuroP content was significantly higher in patients of all diseases as compared to age and gender matched healthy control subjects (n= 61), 4-F4t-NeuroP levels were significantly higher in MS and RTT as compared to healthy controls. Significant positive relationships were observed between relative disease severity and 4-F4t-NeuroP levels (r = 0.469, P <0.0001), and 10-F4t-NeuroP levels (r = 0.757, P < 0.0001). The study showed that the plasma amount ratio of 10-F4t-NeuroP to 4-F4t-NeuroP and the plasma amount as individual isomer can be used to discriminate between different brain diseases. Overall, by comparing the different types of disease, our plasma data indicates that 4-F4t-NeuroP and 10-F4t -NeuroP: i) are biologically synthesized in vivo and circulated, ii) are related to clinical severity of neurological diseases, iii) are useful to identify shared pathogenetic pathways in distinct brain diseases, and iv) appears to be distinctive for different neurological conditions, thus representing potentially new biological disease markers. Our data strongly suggest that in vivo DHA oxidation follows preferential chemical rearrangements according to different brain diseases.

The role of fatty acid desaturase (FADS) genes in oleic acid metabolism: FADS1 Δ7 desaturates 11-20:1 to 7,11-20:2

IntroductionIn mammals, FADS2 catalyzes “front-end” Δ4-, Δ6-, and Δ8-desaturation of fatty acyl chains, whereas FADS1 has Δ5-desaturase activity. Eighteen and 20-carbon precursors to highly unsaturated n-3 and n-6 fatty acids are the usual substrates for FADS1 and FADS2. Our main objective was to characterize the metabolic fate of oleic acid (OA) due to action of FADS gene products. MethodsMCF-7 cells were stably transformed with either FADS1 or FADS2 or empty vector. A series of dose-response experiments were conducted with albumin-bound fatty acid substrates (18:1n-9 and 20:1n-9) provided in concentrations up to 100µM. Cells were harvested after 24h, after which FAME were prepared and analyzed by GC-FID and covalent adduct chemical ionization tandem mass spectrometry (CACI-MS/MS). ResultsWhen stably transformed cells were incubated with 18:1n-9, FADS1 and control cells elongated 18:1n-9 → 20:1n-9 (11-20:1), while FADS2 cells Δ6 desaturated, elongated, and then Δ5 desaturated via FADS1 coded activity leading to Mead acid, 9-18:1 → 6,9-18:2 → 8,11-20:2 (20:2n-9) → 6,8,11-20:3 (20:3n-9). Surprisingly, FADS1 cells Δ7 desaturated 11-20:1 → 7,11-20:2, the latter detected at low levels in control and FADS2 cells. Our results imply three pathways operate on 18:1n-9: 1) 18:1n-9 → 18:2n-9 → 20:2n-9 → 20:3n-9; 2) 18:1n-9 → 20:1n-9 → 20:2n-9 → 20:3n-9 and 3) 18:1n-9 → 20:1n-9 → 7,11-20:2. ConclusionAlternative pathways for oleic acid metabolism exist depending on FADS2 or FADS1 activities, we present the first evidence of Δ7 desaturation via the FADS1 gene product

Simultaneous determination of ethyl carbamate and urea in Korean rice wine by ultra-performance liquid chromatography coupled with mass spectrometric detection.

In this study, a rapid method for simultaneous detection of ethyl carbamate (EC) and urea in Korean rice wine was developed. To achieve quantitative analysis of EC and urea, the conditions for Ultra-performance liquid chromatography (UPLC) separation and atmospheric-pressure chemical ionization tandem mass spectrometry (APCI-MS/MS) detection were first optimized. Under the established conditions, the detection limit, relative standard deviation and linear range were 2.83μg/L, 3.75-5.96%, and 0.01-10.0mg/L, respectively, for urea; the corresponding values were 0.17μg/L, 1.06-4.01%, and 1.0-50.0μg/L, respectively, for EC. The correlation between the contents of EC and its precursor urea was determined under specific pH (3.5 and 4.5) and temperature (4, 25, and 50°C) conditions using the developed method. As a result, EC content was increased with greater temperature and lower pH. In Korean rice wine, urea was detected 0.19-1.37mg/L and EC was detected 2.0-7.7μg/L. The method developed in this study, which has the advantages of simplified sample preparation, low detection limits, and good selectivity, was successfully applied for the rapid analysis of EC and urea.

Non-aqueous reversed-phase liquid-chromatography of tocopherols and tocotrienols and their mass spectrometric quantification in pecan nuts

An easy and effective analytical method was developed for the simultaneous quantification of four tocopherols (Ts) and four tocotrienols (T3s) in three pecan nut cultivars (Stuart, Sioux, and Pawnee). The analytes were separated on a C30 column kept at 15°C, under isocratic non-aqueous reversed-phase (NARP) conditions, in only 18min and detected by atmospheric pressure chemical ionization–tandem mass spectrometry (APCI-MS/MS). The HPLC-APCI-MS/MS method was validated, according to the main FDA guidelines, and then applied for the characterization of the real samples. Analytes were extracted by cold saponification with recoveries greater than 87%. The limits of detection (LOD) and limits of quantification (LOQ) were in the range of 0.3–10μg/100mg and 1–30μg/100mg, respectively. Compared to other nuts, vitamin E composition of pecan nuts (Carya illinoinensis) has only partially been elucidated. Results have evidenced the prevalence of γ-forms for both Ts and T3s and clear quantitative differences of the identified vitamers among the studied cultivars. The richest variety in vitamin E was Sioux with a total content of about∼32mg/100g wet weight, followed by Stuart (∼16mg/100g) and Pawnee (∼9mg/100g).

Simultaneous Quantification of Free Cholesterol, Cholesteryl Esters, and Triglycerides without Ester Hydrolysis by UHPLC Separation and In-Source Collision Induced Dissociation Coupled MS/MS

We demonstrate the application of in-source nitrogen collision-induced dissociation (CID) that eliminates the need for ester hydrolysis before simultaneous analysis of esterified cholesterol (EC) and triglycerides (TG) along with free cholesterol (FC) from human serum, using normal phase liquid chromatography (LC) coupled to atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). The analysis requires only 50 μL of 1:100 dilute serum with a high-throughput, precipitation/evaporation/extraction protocol in one pot. Known representative mixtures of EC and TG species were used as calibrators with stable isotope labeled analogs as internal standards. The APCI MS source was operated with nitrogen source gas. Reproducible in-source CID was achieved with the use of optimal cone voltage (declustering potential), generating FC, EC, and TG lipid class-specific precursor fragment ions for multiple reaction monitoring (MRM). Using a representative mixture of purified FC, CE, and TG species as calibrators, the method accuracy was assessed with analysis of five inter-laboratory standardization materials, showing –10% bias for Total-C and –3% for Total-TG. Repeated duplicate analysis of a quality control pool showed intra-day and inter-day variation of 5% and 5.8% for FC, 5.2% and 8.5% for Total-C, and 4.1% and 7.7% for Total-TG. The applicability of the method was demonstrated on 32 serum samples and corresponding lipoprotein sub-fractions collected from normolipidemic, hypercholesterolemic, hypertriglyceridemic, and hyperlipidemic donors. The results show that in-source CID coupled with isotope dilution UHPLC-MS/MS is a viable high precision approach for translational research studies where samples are substantially diluted or the amounts of archived samples are limited.Graphical ᅟ

Identification of triacylglycerols in coix seed extract by preparative thin layer chromatography and liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry

Here we reported a methodology for identification of triacylglycerols (TAGs) and diacylglycerols (DAGs) in coix seed by preparative thin layer chromatography (prep-TLC) and non-aqueous reversed-phase liquid chromatography (NARP LC)-atmospheric pressure chemical ionization (APCI) tandem mass spectrometry (MS/MS). Lipid components were extracted from coix seed by reflux extraction using n-hexane for 3 hr. TAGs and DAGs in coix seed extract were effectively purified and isolated from matrix interferences by prep-TLC and then analyzed by LC-APCI-MS and MS/MS for identification. TAGs were effectively identified taking into consideration of their LC retention behavior, APCI-MS spectra patterns, and MS/MS spectra of [DAG] + ions. In MS/MS spectra of TAGs, diacylglycerol-like fragment [DAG] + ions were useful to identify TAGs with isobaric fragment ions. Based on an established method, 27 TAGs and 8 DAGs were identified in coix seed extract. Among them, 15 TAGs and 8 DAGs were for the first time observed in coix seed. Interestingly, some of TAGs isolated by prep-TLC were partly converted into DAGs through probably photolysis process during storing in room temperature. Thus, degradation phenomenon of TAGs should be considered in the quality evaluation and nutritional property of coix seed. LC-APCI-MS/MS combined with prep-TLC will be practical method for precise TAG and DAG analysis of other herbal plants.

Plasma coenzyme Q10 concentration, antioxidant status, and serum N-terminal pro-brain natriuretic peptide concentration in dogs with various cardiovascular diseases and the effect of cardiac treatment on measured variables.

OBJECTIVE To determine the plasma total antioxidant capacity, erythrocyte superoxide dismutase activity, whole blood glutathione peroxidase activity, and plasma coenzyme Q10 (CoQ10) concentration in dogs with various stages of cardiovascular diseases and in healthy dogs; assess the influence of cardiac treatment on the levels of antioxidant variables, plasma CoQ10 concentration, and serum N-terminal pro-brain natriuretic peptide (NT-proBNP) concentration, and determine any correlation between the disease severity (NT-proBNP concentration) and antioxidant variables or CoQ10 concentration. ANIMALS 43 dogs with various types and stages of cardiovascular diseases (congenital and acquired) and 29 healthy dogs. PROCEDURES Blood samples were collected from all dogs for spectrophotometric assessment of antioxidant variables. Plasma CoQ10 concentration was determined with a high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry method. Serum NT-proBNP concentration was measured with an ELISA. RESULTS Values for antioxidant variables did not differ among groups of dogs with cardiovascular diseases, regardless of disease stage or treatment. Plasma CoQ10 concentration was significantly increased in treated dogs with congestive heart failure (CHF), compared with untreated patients. However, plasma CoQ10 concentration did not differ among heart failure classes. A significant, negative correlation between serum NT-proBNP and plasma CoQ10 concentrations was identified in treated CHF-affected dogs, suggesting that low plasma CoQ10 concentration may be associated with increased severity of CHF. CONCLUSIONS AND CLINICAL RELEVANCE The antioxidant variables evaluated were not altered in dogs with CHF, regardless of cardiac disease stage or treatment. Further investigation into the possible effects of CoQ10 supplementation in dogs with advanced stages of CHF is warranted.