Positive Ion Detection Mode Research Articles

Sensitive ionization of non‐volatile analytes using protein solutions as spray liquid in desorption electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) is the most popular ambient ionization technique for direct analysis of complex samples without sample pretreatment. However, for many applications, especially for trace analysis, it is of interest to improve the sensitivity of DESI-mass spectrometry (MS). In traditional DESI-MS, a mixture of methanol/water/acetic acid is usually used to generate the primary ions. In this article, dilute protein solutions were electrosprayed in the DESI method to create multiply charged primary ions for the desorption ionization of trace analytes on various surfaces (e.g., filter paper, glass, Al-foil) without any sample pretreatment. The analyte ions were then detected and structurally characterized using a LTQ XL mass spectrometer. Compared with the methanol/water/acetic acid (49:49:2, v/v/v) solution, protein solutions significantly increased the signal levels of non-volatile compounds such as benzoic acid, TNT, o-toluidine, peptide and insulin in either positive or negative ion detection mode. For all the analytes tested, the limits of detection (LODs) were reduced to about half of the original values which were obtained using traditional DESI. The results showed that the signal enhancement is highly correlated with the molecular weight of the proteins and the selected solid surfaces. The proposed DESI method is a universal strategy for rapid and sensitive detection of trace amounts of strongly bound and/or non-volatile analytes, including explosives, peptides, and proteins. The results indicate that the sensitivity of DESI can be further improved by selecting larger proteins and appropriate solid surfaces.

Urinary hydrophilic and hydrophobic metabolic profiling based on liquid chromatography‐mass spectrometry methods: Differential metabolite discovery specific to ovarian cancer

Discovery of novel metabolite biomarker(s) for improved ovarian cancer diagnosis is of great importance. In this paper, the differences of urinary hydrophilic and hydrophobic metabolic profiling between healthy women, benign ovarian tumor, and ovarian cancer patients were studied by metabolomics strategy. Metabolites in urine were analyzed on hydrophilic interaction chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) coupled to MS. Data from HILIC or RPLC, positive or negative ion detection mode were found to be complementary. Data were filtered by orthogonal signal correction (OSC) method, and the three groups were discriminated by partial least squares discrimination analysis (PLS-DA) models. By combining the four datasets, maximum information can be collected, and a PLS-DA model was built after OSC filtering. The model based on combined dataset is superior to the ones based on the separate dataset, and important metabolites were screened based on the combined dataset model. Five metabolites were found to be specific to ovarian cancer and ten metabolites were considered commonly related to ovarian cancer and benign ovarian tumor. Combination of RPLC and HILIC separation, as well as positive and negative ion detection in metabolomic studies show advantages in collecting various metabolites information that helps us better understand the metabolic event.

Fluorescence Probe of 10-Phenyl-acridone-2-sulfonyl Chloride and Its Application for Determination of Free Aliphatic Amines in Environmental Samples by HPLC with Fluorescence Detection and APCI-MS

A simple and sensitive method for the determination of free aliphatic amines using 10-phenyl-acridone-2-sulfonyl chloride (PASC) as a labeling reagent by high-performance liquid chromatography with fluorescence detection and online mass spectrometry identification (HPLC-FLD-MS) has been developed. Derivatization conditions including reagent concentration, buffer pH, reaction time and temperature were optimized. PASC reacted with aliphatic amines at 50 A degrees C for 4 min in aqueous acetonitrile (ACN) in the presence of sodiumtetraborate-NaOH buffer (0.10 mol L-1, pH 9.0) to give high yields of PASC-amine derivatives. Derivatives exhibited intense fluorescence with an excitation maximum at lambda(ex) 265 nm and an emission maximum at lambda(em) 418 nm. The separation of derivatives was performed by a reversed-phase Hypersil BDS C8 column in combination with a gradient elution. The identification of derivatives was carried out by online post-column mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion detection mode. Excellent linear responses were observed with the correlation coefficients of larger than 0.9997, and detection limits (at a signal-to-noise of 3:1) were from 3.0 to 24.3 fmol. Comparing with 10-ethyl-acridine-2-sulfonyl chloride (EASC), PASC exhibited more intense fluorescence and ultraviolet absorbance. The proposed method is sensitive and reproducible for the determination of aliphatic amines from water and soil samples.

DETERMINATION AND IDENTIFICATION OF FATTY ACIDS IN MICROULA SIKKIMENSIS SEED OIL USING 1,2-BENZOCARBAZOLE-9-ETHYL-P-TOLUENESULFONATE AS A NOVEL LABELING REAGENT BY HPLC WITH FLUORESCENCE DETECTION AND APCI-MS

Using 1,2-benzocarbazole-9-ethyl-p-toluenesulfonate (BCETS) as the labeling reagent, a sensitive method for the determination of fatty acids by high performance liquid chromatography (HPLC) with fluorescence detection has been developed. The BCETS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90°C for 30 min in N,N-dimethylformamide solvent. All the tested fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The identification was carried out by post-column atmospheric pressure chemical ionization mass spectrometry (APCI/MS) in positive ion detection mode. The fluorescence excitation and emission wavelengths of the derivatives were at λex 279 nm and λem 380 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are more than 0.9994. Detection limits, at a signal-to-noise ratio of 3:1, were 0.21–0.99 µg/L for the labeled fatty acids. In this study, fatty acids from the extracted Microula sikkimensis Hemsl (M. sikkimensis H.) seed were sensitively determined. It was found that the seed oil mainly contained Oleic acid, Linoleic acid, α-Linolenic acid, Hexadecanoic acid, Stearidonic acid, γ-Linolenic acid, and Eicosenoic acid. M. sikkimensis H. seed oil was rich in unsaturated fatty acids and may be used as high-quality edible oil for its health benefits.

Liquid chromatography–mass spectrometry-electrospray ionisation analysis of Centella asiatica l., Curcuma longa L. and Strobilanthes crispus L. methanol extracts

All the three plants were analysed in liquid chromatography-mass spectrometry-electrospray ionisation to identify plant active principles. Chromatographic separation of the compound of interest was achieved on C-18 column with detection in positive ion mode. The test showed that the most active principles found in Curcuma longa L. were curcuminoids mainly curcumin, demethoxycurcumin, bisdemethoxycurcumin, and dihydrocurcumin. In Centella asiatica L. madecassic acid was found at ion/mz 490. While the active principle in Strobilanthes crispus L. is verbascoside, which was detected at ion/mz 625. As plant active principles were excellently determined, the plant bioactivities such as anti-cancer, antiviral and/or antibacterial could be explored and developed. Key words: Centella asiatica L., Curcuma longa L., Strobilanthes crispus L., liquid chromatography–mass spectrometry-electrospray ionisation (LC-ESI- MS), curcuminoids, madecassic acid, verbascosid.

Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine

Acylglycines play a crucial regulatory and detoxification role in the accumulation of the corresponding acyl CoA esters and are an important class of metabolites in the diagnoses of inborn errors of metabolism. Sensitive quantification of a large number of acylglycines not only improves diagnosis but also enables the discovery of potential new biomarkers of diseases. We report an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC–MS) method for quantifying acylglycines in human urine with high sensitivity. This method is based on the use of a newly developed isotope labeling reagent, p-dimethylaminophenacyl (DmPA) bromide, to label acylglycines to improve detection sensitivity. Eighteen acylglycines, namely acetylglycine, propionylglycine, isobutyrylglycine, butyrylglycine, 4-hydroxyphenylacetylglycine, 2-furoylglycine, tiglylglycine, 2-methybutyrylglycine, 3-methylcrotonylglycine, isovalerylglycine, valerylglycine, hexanoylglycine, phenylacetylglycine, phenylpropionylglycine, glutarylglycine, heptanoylglycine, octanoylglycine and suberylglycine, were measured. This method uses calibration standards prepared in surrogate matrix (un-derivatized urine) and stable-isotope labeled analytes as the internal standards. The analysis was carried out in the positive ion detection mode using multiple reaction monitoring (MRM) survey scans. The calibration curves were validated over the range of 1.0–500nM. The method achieved a lower limit of quantitation (LLOQ) of 1–5nM for all analytes, as measured by the standard derivations associated with calibration curves and confirmed in surrogate matrix; the signal-to-noise ratio at LLOQ ranged from 12.50 to 156.70. Both accuracy (% RE or relative error) and precision (% CV) were <15%. Matrix effects were minimized using the surrogate matrix. All eighteen analytes were stable in urine for at least 5h at room temperature, autosampler (4°C) for 24h, 7 weeks at −20°C and after three freeze/thaw cycles. This surrogate matrix approach was validated using a standard addition experiment. As an example of applications, the endogenous concentrations of all eighteen analytes in urine samples of 20 healthy individuals collected in three consecutive days (i.e., 60 samples) were determined; there was no significant correlation found between the acylglycine profile and gender or body mass indices.

A Novel Labeling Reagent of 2-(12-Benzo[b]acridin-5-(12H)-yl)-acetohydrazide for Determination of Saturated and Unsaturated Fatty Acids in Traditional Chinese Herbs by HPLC-APCI-MS

A new fluorescence labeling reagent 2-(12-benzo[b]acridin-5(12H)-yl)-acetohydrazide (BAAH) has been designed for fatty acids labeling. Eleven fatty acids containing seven saturated and four unsaturated fatty acids were used to evaluate the analytical potential of this reagent. The labeling reaction of BAAH with fatty acids was completed at 85 °C for 60 min using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl) as the condensing agent. Separation of the derivatized fatty acids was carried out on a reversed-phase Thermo Hypersil Gold C18 column (4.6 mm × 250 mm, 5 μm) in combination with a gradient elution with a good baseline resolution. The fluorescence excitation and emission wavelengths were set at λex 280 and λem 510 nm, respectively. The identification was carried out by the online APCI-MS in positive-ion detection mode. Linear correlation coefficients for all fatty acid derivatives were of >0.9994. Detection limits, at a signal-to-noise ratio of 3:1, were 3.89–12.5 nmol L−1 for the labeled fatty acids. The developed method was successfully applied to the accurate determination of fatty acids in five traditional Chinese herbs with satisfactory results.

High-sensitivity liquid chromatography–tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma

A sensitive liquid chromatography–tandem mass spectrometric (LC–MS/MS) method with electrospray ionization was developed for the simultaneous quantitation of five probe drugs and their metabolites in human plasma for assessing the in vivo activities of cytochrome P450 (CYP). CYP isoform specific substrates and their metabolites of CYP1A2 (caffeine), CYP2C9 (losartan), CYP2C19 (omeprazole), CYP2D6 (dextromethorphan) and CYP3A (midazolam) were all simultaneously analyzed using LC–MS/MS after administration of a mixture of five drugs (i.e., a “cocktail approach”) to healthy volunteers. The assay uses propranolol as an internal standard; dual liquid extraction; a Xbridge MS C18 (100mm×2.1mm, 3.5μm) column; a gradient mobile phase of 0.1% formic acid/acetonitrile (7/3→3/7); mass spectrometric detection in positive ion mode. The method was validated from 5 to 500ng/mL for caffeine and paraxanthine, 0.1–40ng/mL for losartan and EXP3174, 0.05–20ng/mL for omeprazole and 5-hydroxyomeprazole, 0.008–0.8ng/mL for dextromethorphan and dextrorphan, 0.01–1.0ng/mL for midazolam, and 0.04–4ng/mL for 1′-hydroxymidazolam. The intra- and inter-day precision over the concentration ranges for all analytes were lower than 12.5% and 13.8% (relative standard deviation, %RSD), and accuracy was between 86.5% and 108.4% and between 87.0% and 107.0%, respectively. This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10–100 times lower than typical therapeutic doses.

Simultaneous Analysis of Strychnine and Brucine and their Major Metabolites by Liquid Chromatography-Electrospray Ion Trap Mass Spectrometry

A liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-ITMS) method was developed for the simultaneous analysis of strychnine, brucine and their major metabolites. Strychnine and brucine were individually incubated with rat liver S9 fraction. The incubation samples were pooled together and analyzed with LC-ESI-ITMS in positive ion and full-scan detection mode. The calibration curves of strychnine and brucine in rat liver showed good linearity in ranges of 0.020 to 8.0 µg/mL for strychnine and 0.020 to 8.5 µg/mL for brucine. The limits of detections were both 0.008 µg/mL and the recoveries were 88.3 and 83.2% for strychnine and brucine, respectively. Two metabolites were identified as strychnine N-oxide and brucine N-oxide by comparing the molecular mass, retention time, full-scan mass spectra, tandem MS and MS(3) spectra with those of strychnine and brucine. The developed method provided high sensitivity and selectivity for the determination of poisonous alkaloids and their major metabolites and can be applied in the determination of samples in forensic and clinically toxicological cases.

Development of a rapid UPLC‐MS/MS method for quantification of saxagliptin in rat plasma and application to pharmacokinetic study

A novel, simple and rapid ultraperformance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) assay was established for quantification of saxagliptin in rat plasma. Plasma samples were processed by liquid-liquid extraction with ethyl acetate and chromatographed on a C₁₈ column (2.1 × 50 mm i.d., 1.7 µm). The mobile phase consisted of methanol and 0.1% formic acid (40:60, v/v). Multiple reaction monitoring transitions were performed for detection in positive-ion mode with an electrospray ionization source. The calibration curve was linear over the concentration range of 0.5-100 ng/mL (R² > 0.99). All accuracy values were between 90.62 and 105.60% relative error and the intra- and inter-day precisions were less than 9.66% relative standard deviation. Extraction recovery was more than 81.01% and the matrix effect ranged from 90.27 to 109.15%. After validation, the method was applied to a pharmacokinetic study where healthy rats were orally given 0.5 mg/kg saxagliptin.

Metabolic differences among melanoma and two prostate cancer cell lines by electrospray ion mobility mass spectrometry

Detecting early stages of cancer has been a challenge for analytical methods. Even with the advances in mammography and prostate specific antigen (PSA) testing the number of false positives and negatives is still significant. Cancer cell lines provide realistic simulations to assess detection technologies for different stages and types of cancer. Metabolomics, the measure of end products of all biological processes, is a recent addition to the field of systems biology and can be targeted towards early disease detection. For this study, the metabolomes of three cancer cell lines, two prostate (DU145 and PC3) and one melanoma (A375), were investigated by an ion mobility mass spectrometer (IMMS) for both positive and negative ion detection mode. IMMS produced multidimensional data that includes m/z, mobility and intensity for each ion detected in the sample. In order to see differences between the cancer cell lines, principal components analysis (PCA), a multi-variant technique used to pull patterns out of the data, was used in conjunction with the IMMS data. The sample patterns from the PCA were observed in the score plot and the individual metabolites that contributed the most to the model were shown in the loadings plot. For positive mode 127 metabolite ions were statistically analyzed and in the negative mode 115 metabolite ions were analyzed. PCA score plots were able to model 98% and 87% of the original data for positive and negative mode, respectively. The metabolite ions from the loadings plot were tentatively identified based on m/z.

Differentiation of human kidney stones induced by melamine and uric acid using surface desorption atmospheric pressure chemical ionization mass spectrometry

Clinically obtained human kidney stones of different pathogenesis were dissolved in acetic acid/methanol solutions and then rapidly analyzed by surface desorption atmospheric pressure chemical ionization mass spectrometry (SDAPCI-MS) without any desalination treatment. The mass spectral fingerprints of six groups of kidney stone samples were rapidly recorded in the mass range of m/z 50-400. A set of ten melamine-induced kidney stone samples and nine uric acid derived kidney stone samples were successfully differentiated from other groups by principal component analysis of SDAPCI-MS fingerprints upon positive-ion detection mode. In contrast, the mass spectra recorded using negative-ion detection mode did not give enough information to differentiate those stone samples. The results showed that in addition to the melamine, the chemical compounds enwrapped in the melamine-induced kidney stone samples differed from other kidney stone samples, providing useful hints for studying on the formation mechanisms of melamine-induced kidney stones. This study also provides useful information on establishing a MS-based platform for rapid analysis of the melamine-induced human kidney stones at molecular levels.

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was developed and validated for the determination of sibutramine and its N-desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t-butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse-phase Luna C18 column with a mobile phase of acetonitrile-10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI-MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05-20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra- and inter-day validation for sibutramine, M1 and M2 were acceptable. This LC-MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human.

Supercritical fluid CO 2 extraction, simultaneous determination of components in ultra-fine powder of Ganoderma sinense by HPLC–ESI-MS method

An off-line mode coupled supercritical fluid extraction (SFE) with high-performance liquid chromatography and electro-spray ion trap mass spectrometry (HPLC–ESI-MS) analytical method was applied to analyze the chemical components of Ganoderma sinense ( G. sinense). The ultra-fine powder of fruit-bodies of G. sinense was extracted by SFE and a HPLC–ESI-MS method was established for simultaneously determining chemical components of G. sinense, and the total extraction yield was 3.75% (w/w). The method of combining diode-array detection (DAD) with MS, was successfully used to detect the chemical components of G. sinense. Most of the peaks exhibited maximum absorptions at 280 and 210 nm, especially at 280 nm. About 20 peaks were acquired in positive and negative ion detection mode, and were analyzed by electro-spray ion trap mass spectrometry (ESI-ITMS). The results indicated that the molecular weight distribution ( m/z: 188–614) of main compounds was identified according to the molecular ions in MS spectra.

Enhanced Detection and Identification of Glycopeptides in Negative Ion Mode Mass Spectrometry

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.

Detection of characteristic distributions of phospholipid head groups and fatty acids on neurite surface by time-of-flight secondary ion mass spectrometry

Neurons have a large surface because of their long and thin neurites. This surface is composed of a lipid bilayer. Lipids have not been actively investigated so far because of some technical difficulties, although evidence from cell biology is emerging that lipids contain valuable information about their roles in the central nervous system. Recent progress in techniques, e.g., mass spectrometry, opens a new epoch of lipid research. We show herein the characteristic localization of phospholipid components in neurites by means of time-of-flight secondary ion mass spectrometry. We used explant cultures of mouse superior cervical ganglia, which are widely used by neurite investigation research. In a positive-ion detection mode, phospholipid head group molecules were predominantly detected. The ions of m/z 206.1 [phosphocholine, a common component of phosphatidylcholine (PC) and sphingomyelin (SM)] were evenly distributed throughout the neurites, whereas the ions of m/z 224.1, 246.1 (glycerophosphocholine, a part of PC, but not SM) showed relatively strong intensity on neurites adjacent to soma. In a negative-ion detection mode, fatty acids such as oleic and palmitic acids were mainly detected, showing high intensity on neurites adjacent to soma. Our results suggest that lipid components on the neuritic surface show characteristic distributions depending on neurite region.

Comparison of positive and negative ion detection of tea catechins using tandem mass spectrometry and ultra high performance liquid chromatography

Tea catechins are an important group of natural compounds associated with health promoting effects and desired commodities for the growing market of dietary supplements and functional foods. Consequently these compounds attract more interest of research groups worldwide. A reliable quantitative analysis of tea catechins is essential for human intervention studies, manufacturers of dietary supplements and quality control by authorities. UHPLC–ESI-MS/MS analytical method was chosen due to rapid runtime, high sensitivity and selectivity. The chromatographic separation of eight tea catechins was achieved within 2.5 min on C 18 BEH analytical column (100 mm × 2.1 mm i.d.; 1.7 μm), whilst the gradient elution mode was employed using water:methanol mobile phase with addition of volatile organic acid. The concentration of organic acids in the mobile phase was optimised within the range of 0.01–0.1% (v/v). High sensitivities were achieved in positive (10.2-16.8 fmol/inj.) and negative ion detection mode (102.1-168.1 fmol/inj.), through accurate and complex tuning of MS parameters. The UHPLC-ESI-MS/MS method was validated in terms of linearity (>0.9997; >0.9990), range (0.02–2.40 mg L −1; 0.15-24.00 mg L −1), LOD (3.0–4.8 μg L −1; 30.1–48.0 μg L −1), LOQ (9.9–15.8 μg L −1; 150.5-240.0 μg L −1), intra-day precision (4.4-7.1% RSD; 3.3-5.1% RSD), accuracy (94.06-113.7%; 89.5-108.4%), retention time repeatability (0.0-0.5% RSD; 0.0-0.6% RSD), and peak area repeatability (1.2-4.0% RSD; 2.4-3.5% RSD) for positive and negative ion detection modes, respectively. The statistical comparison of the quantitative results obtained in positive and negative ion detection mode was performed.

Determination of Free Fatty Acids in Tibet Folk Medicine Potentilla anserina L. Using a New Labeling Reagent by LC with Fluorescence Detection and Identification with Online Atmospheric Chemical Ionization-MS Identification

A sensitive method for the determination of free fatty acids using 1,2-benzo-carbazole-9-ethyl-p-toluenesulfonate (BCETS) as tagging reagent with fluorescence detection has been developed. BCETS could easily and quickly label fatty acids in the presence of the K(2)CO(3) catalyst at 80 A degrees C for 30 min in N,N-dimethylformamide solvent. In this study, fatty acids from the extracted Potentilla anserina L. plant sample were sensitively determined. The corresponding derivatives were separated on a reversed-phase Eclipse XDB-C(8) column by LC in conjunction with gradient elution. The identification was carried out by post-column APCI-MS in positive-ion detection mode. BCETS-fatty acid derivatives gave an intense molecular ion peak at m/z [M+H](+), the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at m/z [M'+CH(2)CH(2)](+), m/z 216.6 and m/z [MH-H(2)O](+) (here, M': corresponding molecular mass of the fatty acids). The fluorescence excitation and emission wavelengths of the derivatives were at lambda (ex) 279 nm and lambda (em) 380 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are more than 0.9994. Detection limits, at a signal-to-noise ratio of 3:1, are 10.79-34.19 fmol for the labeled fatty acids.

HPLC-FLUORIMETRIC METHOD FOR ANALYSIS OF FREE FATTY ACIDS IN STELLERA CHAMAEJASMA L

A sensitive HPLC method for the determination of fatty acids using 2-(2-(pyren-1-yl)-1H-benzo[d]imidazol-1-yl) ethyl-p-toluenesulfonate (PBIOTs) as a novel labeling reagent with fluorescence detection has been developed. PBIOTs could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90°C for 30 min in N,N-dimethylformamide solvent. Fatty acids derivatives were separated on a reversed phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI-MS in positive ion detection mode. PBIOTs-fatty acid derivatives gave an intense molecular ion peak at m/z [M + H]+; with MS/MS analysis, the collision induced dissociation spectra of m/z [M + H]+ produced the specific fragment ions at m/z [M + H–319]+ and m/z 319.0 (here, m/z 319 is the core structural moiety of the PBIOTs molecule). The fluorescence excitation and emission wavelengths of the derivatives were λex = 350 nm and λem = 402 nm, respectively. Linear correlation coefficients for all fatty acid derivatives were ≥0.9985. Detection limits for the labeled fatty acids, at a signal-to-noise ratio of 3:1, were in the range of 10.32– 44.28 fmol. From the extracts of Stellera chamaejasma L, free fatty acids were sensitively determined.

Monitoring dynamic changes in lymph metabolome of fasting and fed rats by electrospray ionization-ion mobility mass spectrometry (ESI-IMMS).

Ambient pressure ion mobility time-of-flight mass spectrometry (IMMS) has recently emerged as a rapid and efficient analytical technique for applications to metabolomics. An important application of metabolomics is to monitor metabolome shifts caused by stress due to toxin exposure, nutritional changes, or disease. The research presented in this paper uses IMMS to monitor metabolic changes in rat lymph fluid caused by dietary stresses over time. Extracts of metabolites found in the lymph fluid collected from dietary stressed rats were subjected to analysis by electrospray (ESI) IMMS operated both in positive and negative ion detection mode. Metabolites detected were tentatively identified based on their mass to charge ratio (m/z). In one sample, 1180 reproducible tentative metabolite ions were detected in negative mode and 1900 reproducible tentative metabolite ions detected in positive mode. Only biologically reproducible ions, defined as metabolite ions that were measured in different rats under the same treatment, were analyzed to reduce the complexity of the data. A metabolite peak list including m/z, mobility, and intensity generated for each metabolome was used to perform principle component analysis (PCA). Dynamic changes in metabolomes were investigated using principle components PC1 and PC2 that described 62% of the variation of the system in positive mode and 81% of the variation of the system in negative mode. Analysis of variance (ANOVA) was performed for PC1 and PC2 and means were statistically evaluated. Profiles of intensities were compared for tentative metabolite ions detected at different times before and after the rats were fed to identify the metabolites that were changing the most. Mobility-mass correlation curves (MMCC) were investigated for the different classes of compounds.