Negative Electrospray Ionization Mass Spectrometry Research Articles

Analysis of wines, grape juices and cranberry juices forAlternaria toxins

Sixty six samples of red and white wine from Ontario (VQA), British Columbia (VQA), Québec ("vins artisanaux"), imported wines (from Italy, South America and USA) and Canadian and US grape and cranberry juices were analysed for theAlternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME). After cleanup on aminopropyl SPE columns, AOH and AME were initially determined by reversed phase LC with UV detection. Positive sample extracts were re-analysed by LC-tandem negative ion electrospray mass spectrometry (MS/MS) in multiple reaction mode. Overall mean method recoveries measured by LC-UV were 93% for AOH and 81% for AME. Limits of detection in wine (and juice) by LC-UV for AOH were 0.8 (0.4) ng/ml and for AME were 0.5 (0.4) ng/ml; they were below 0.01 ng/ml by LC-MS/MS. As determined by LC-MS/MS, AOH was found in 13/17 Canadian red wines at levels of 0.03 to 5.02 ng/ml and in 7/7 imported red wines at 0.27-19.4 ng/ml, usually accompanied by lower concentrations of AME. Red grape juices (5 positive/10 samples) contained only sub ng/ml levels of AOH or AME except for one sample (39 ng AME/ml). White wines (3/23 samples), white grape juices (0/4 samples) and cranberry juices (1/5 samples) contained little AOH/AME (≤1.5 ng/ml).

Commercial human albumin preparations for clinical use are immunosuppressive in vitro

We previously reported significant variations in oxidation status and molecular length among sources and lots of human serum albumin (HSA) commercial preparations intended for clinical use. In this report, we investigated what effect the presence of HSA products have on the immune response in vitro. Laboratory study. Trauma research basic science laboratory. Activated human peripheral blood mononuclear cells. Six commercial HSA preparations were tested for their effect on cytokine release from activated human peripheral blood mononuclear cells (PBMCs) and T-lymphocytes. Mass spectrometry analysis of aspartyl-alanyl diketopiperazine (DA-DKP) content of HSA and percentage of HSA having lost its amino terminal dipeptide aspartyl alanyl (HSA-DA) were correlated. Human PBMCs were cultured in the presence of six commercial HSA preparations and activated via the T-cell receptor complex. A cloned T-lymphocyte cell line, activated with specific antigen, was also cultured with both synthetic DA-DKP and small molecular weight extracts from the commercial HSA tested. Supernatants were quantified by enzyme-linked immunosorbent assay for interferon-gamma and tumor necrosis factor-alpha content. DA-DKP was extracted from HSA by centrifugal filters and quantified by anion exchange liquid chromatography coupled to negative electrospray ionization mass spectrometry. HSA species were determined by reverse phase liquid chromatography coupled to positive electrospray ionization, time of flight mass spectrometry. All HSA preparations significantly inhibited the in vitro production of interferon-gamma and tumor necrosis factor-alpha by activated PBMCs. DA-DKP was detected in all HSA sources at concentrations ranging between 42.0 and 79.6 microM. A synthetic form of DA-DKP possessed similar immunosuppressive qualities in a dose-dependent manner on T lymphocytes. DA-DKP was present in significant concentrations in all HSA sources tested and was partially responsible for the immunosuppressive effects of HSA on activated PBMCs and T-lymphocytes in vitro. In view of these findings, administering HSA to immunocompromised critically ill patients might be reevaluated.

ABTS radical-driven oxidation of polyphenols: Isolation and structural elucidation of covalent adducts

The formation of covalent adducts obtained from the reaction of the polyphenols, trans-3,3′,4′,5,7-pentahydroxyflavan (catechin) and 1,3,5-trihydroxybenzene (phloroglucinol), with ABTS radicals is reported. Two adducts derived from (+)-catechin and three adducts from phloroglucinol were isolated and identified using reversed-phase high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The molecular masses of the (+)-catechin-derived adducts (I c and II c) were found to be 802 and 559 Da, respectively, whereas the masses of phloroglucinol-derived adducts (I p, II p, and III p) were 638, 395, and 381 Da, respectively. The initially formed adducts (I c, I p) were unstable and degraded to secondary adducts (II c, II p, and III p) releasing part of the ABTS molecule. The structures of these adducts were elucidated by interpreting the results of MS/MS analysis of prominent ions generated by both positive and negative ion ESI-MS. The adducts were found to scavenge ABTS radicals, an observation that could explain the complex kinetic behaviour manifested by the reactions of ABTS radicals with polyphenols. A mechanism, which accounts for both the formation of the adducts and the degradation products of ABTS radicals, is proposed.

Structural characterization of underivatized arabino-xylo-oligosaccharides by negative-ion electrospray mass spectrometry

Various arabino-xylo-oligosaccharides with known substitution patterns were assessed by negative ESI-Q-TOFMS and ESI-ITMS. The CID spectra of linear xylo-oligosaccharides and of nine isomeric mono- and disubstituted arabino-xylo-oligosaccharides established that structures differing in their substitution pattern can be differentiated by this approach. The negative-ion fragmentation spectra of the deprotonated quasi-molecular ions are mainly characterized by glycosidic cleavage ions from the C-series, which provide sequence informations, and by cross-ring cleavage 0,2A i ions, which provide partial linkage information. When the collision energy increased, the cross-ring cleavage 0,2A i ions underwent consecutive loss of water to produce 0,2A i − 18 fragment ions and glycosidic cleavage ions of the B-series are also produced besides the C i ions. Contrary to linear xylo-oligosaccharides, C i ions, which originate from C-3 monosubstituted xylosyl residues never produce the related cross-ring cleavage 0,2A i ions. Disubstitution at O-2 and O-3 of xylosyl residues appears to enhance the production of the 0,2A i ions compared to monosubstitution. For the differentiation of the mono- and disubstitution patterns of the penultimate xylosyl residue, the relative abundance of the glycosidic cleavage ions at m/ z 263 and 299 found on Q-TOF CID spectra plays a relevant role and appears to be more informative than MS n spectra obtained on a ion trap instrument.

Electrochemically-Induced Reactions of Hexafluorophosphate Anions with Water in Negative Ion Electrospray Mass Spectrometry of Undiluted Ionic Liquids

The influence of water on the observed gas-phase population of negative ions in electrospray mass spectrometry was studied for the undiluted ionic liquid 1,3-butyl-methyl-imidazolium hexafluorophosphate (BMIM+PF6-). During the electrospray process, electrolytic reduction of water enhances the production of tetrafluorophosphate (F4PO-), which undergoes further reactions to produce difluorophosphate (F2PO2-) anions. These anions are observed in addition to the pre-existing hexafluorophosphate anion. The apparent substitution of two fluorine atoms with one oxygen is attributed to a series of reactions initiated by hydrolysis of hexafluorophosphate. This hydrolysis reaction was enhanced by the addition of hydroxide, formed via the hydrolysis of water or through the addition of ammonium hydroxide. The formation of FxPOy- was studied as a function of the electrospray current and solution flow rate. The mass spectral response shows a quantitative logarithmic relationship between SigmaFxPOy- signal intensities (adjusted for mole equivalents of H2O required) and the amount of water present, against which the water content could be rapidly assessed. Results were found to be comparable to Karl Fischer titration data.

Payne rearrangement during analysis of epoxyalcohols of linoleic and α-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry

Hydroperoxides of polyunsaturated fatty acids can be transformed to epoxyalcohols and keto fatty acids by metal enzymes, hematin, and various catalysts. In the current study, we used hematin to transform 9-hydroperoxy-10 E,12 Z-octadecadienoic acid and 13-hydroperoxy-9 Z,11 E-octadecadienoic acid to epoxyalcohols (with trans epoxide configuration) and to keto fatty acids. The products were separated by normal phase high-performance liquid chromatography (NP-HPLC) and analyzed using postcolumn addition of isopropanol/water and online negative ion electrospray ionization mass spectrometry (MS). The tandem MS (MS/MS) spectra were studied using analogs prepared from [9,10,12,13- 2H 4]linoleic acid (18:2 n−6) and from α-linolenic acid (18:3 n−3). We also studied the MS/MS spectra of epoxyalcohols formed from 11-hydroperoxy- and 8-hydroperoxy-9 Z,12 Z-octadecadienoic acids. Results were confirmed by MS/MS analysis of a series of authentic standards. MS/MS ions of 9-keto-10 E,12 Z-octadecadienoic acid and 13-keto-9 Z,11 E-octadecadienoic acid could be explained by keto–enol tautomerism. MS/MS spectra of regioisomeric allylic epoxyalcohols differed in relative intensities of characteristic ions. The MS/MS spectra of the epoxyalcohols with 1-hydroxy-2,3-epoxy-4 Z-pentene or 3-hydroxy-1,2-epoxy-4 Z-pentene elements were virtually identical and showed two characteristic ions that differed by 30 in m/ z values (CH(OH)). The results suggested that epoxide migration (Payne rearrangement) occurred during collision-induced dissociation. We conclude that regioisomeric allylic epoxyalcohols can be identified by their MS/MS spectra, whereas regioisomeric epoxyalcohols can be identified by MS/MS in combination with their retention times on NP-HPLC.

Analytical application of acetate anion in negative electrospray ionization mass spectrometry for the analysis of triterpenoid saponins–ginsenosides

Ginsenosides containing different numbers of glycosyl groups can be easily differentiated based on the formation of characteristic ginsenoside-acetate adduct anions and deprotonated ginsenosides generated by electrospray ionization (ESI) of methanolic solutions of ginsenosides (M) and ammonium acetate (NH4OAc). Ginsenosides containing two glycosyl groups gave a characteristic mass spectral pattern consisting of [M+2OAc]2-, [M-H+OAc]2- and [M-2H]2- ions with m/z values differing by 30 Th, while this mass spectral pattern was not observed for ginsenosides containing one glycosyl group. Formation of [M+2OAc]2- was influenced by the chain length of glycosyl groups and was used to differentiate the ginsenosides containing different combinations of monosaccharide and disaccharide units in the glycosyl groups. Under identical collisional activation conditions, [M+OAc]-, [M-H+OAc]2- and [M+2OAc]2- underwent proton abstractions predominantly to generate [M-H]-, [M-2H]2- and [M-H+OAc]2- ions, respectively. The ion intensity ratios, I[M-H](-/I) [M+OAc]-, I[M-2H](2-/I) [M-H+2OAc]2- and I[M-H+OAc](2-/I) [M+OAc]2-, being sensitive to the structural differences of ginsenosides, could differentiate the isomeric ginsenosides, including (i) Rf, F11 and Rg1, (ii) Rd and Re, and (iii) Rb2 and Rc. Additionally, NH4OAc was found to enhance the sensitivity of detection of ginsenosides in the form of [M-H]- down to the femtomole level.

High-performance liquid chromatography coupled with tandem mass spectrometry applied for metabolic study of ginsenoside Rb 1 on rat

Liquid chromatography coupled with mass spectrometry and tandem mass spectrometry has been applied to investigate the in vivo metabolism of ginsenoside Rb 1 in rat. Both positive electrospray ionization mass spectrometry and negative electrospray ionization mass spectrometry were used to identify the Rb 1 and its metabolites in rat plasma, urine, and feces samples. Oxygenation and deglycosylation were found to be the major metabolic pathways of Rb 1 in rat. A total of nine metabolites were detected in urine and feces samples collected after intravenous and oral administration. Deglycosylated metabolism of Rb 1 generated other ginsenosides as the major metabolites, such as Rd, Rg 3 or F 2, Rh 2, or C-K. This result indicates that the ginsenoside Rb 1 has many pharmacological activities and could be used as a prodrug.

Investigation of zinc binding metallothioneins’ polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs’ polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.

Amino‐catalyzed fragmentation characteristics of polyamides containing N‐methylpyrrole and N‐methylimidazole by electrospray negative ionization mass spectrometry

Different negative fragmentations of synthesized polyamides containing N-methylpyrrole and N-methylimidazole were analyzed by electrospray ionization tandem mass spectrometry with low-energy collision-induced dissociation. An interesting rearrangement derived from amino catalysis of N-methylimidazole was observed. The observation is useful for the study of the correlations between the sequences of polyamides and their mass fragmentation pathways.

Analysis of curcuminoids by positive and negative electrospray ionization and tandem mass spectrometry

The curcuminoids are a group of diarylheptanoid molecules that possess important pharmacological activities, particularly acting as anti-inflammatory agents. The main purpose of this study was to investigate the fragmentation behavior of the three major curcuminoids in ion trap liquid chromatography/tandem mass spectrometry (LC/MS/MS). Both positive and negative mode electrospray ionization in tandem and multidimensional MS(n) experiments in quadrupole ion trap instruments and high-resolution and accurate mass MS and sustained off-resonance irradiation (SORI) MS/MS experiments in a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer were used to elucidate the main fragmentation channels of these compounds. These experiments yielded essentially the same fragmentation results in both ion trap and ICR instruments for all three curcuminoids and for their phenolic monoacetates. Major and diagnostic fragment ions were identified and their origins are proposed.

Fractionation of grape seed extract and identification of gallic acid as one of the major active constituents causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells

The anti-cancer efficacy of grape seed extract (GSE) against prostate cancer (PCA) via its anti-proliferative, pro-apoptotic and anti-angiogenic activities in both cell culture and animal models have recently been described by us. GSE is a complex mixture containing gallic acid (GA), catechin (C), epicatechin (EC) and several oligomers (procyanidins) of C and/or EC, some of which are esterified to GA. To determine which components are most active against PCA, an ethyl acetate extract of GSE was separated by reverse-phase high-performance liquid chromatography (HPLC) into three fractions. Fraction 1 was far more effective than others in causing growth inhibition and apoptotic death of human PCA DU145 cells. Of the components in this fraction, GA showed a very strong dose- and time-dependent growth inhibition and apoptotic death of DU145 cells, but C and procyanidins B1 (EC-C dimer), B2 (EC-EC dimer) and B3 (C-C dimer) were nearly ineffective. Mechanistic studies demonstrated a strong caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavages by GA in DU145 cells. Procyanidin oligomers eluting in HPLC Fractions 2 and 3 were obtained in larger quantities by separating GSE into eight fractions (I-VIII) on a gel filtration column. All fractions were analyzed by HPLC-UV and negative-ion electrospray mass spectrometry. Fractions I-III contained the active compound GA and inactive components C, EC, B1 and B2. Fraction IV contained other dimers and a dimer-GA ester and was also less active than GSE in DU145 cells. Fractions V-VIII, however, caused significant growth inhibition and apoptosis with the highest activity present in the later fractions that contained procyanidin trimers and GA esters of dimers and trimers. Together, these observations identify GA as one of the major active constituents in GSE. Several procyanidins, however, and especially the gallate esters of dimers and trimers also may be efficacious against PCA and merit further investigation.

Host‐defence skin peptides of the Australian Streambank Froglet Crinia riparia: isolation and sequence determination by positive and negative ion electrospray mass spectrometry

A combination of positive and negative ion electrospray mass spectrometry (ES-MS) together with automated Edman sequencing has been used to determine the amino acid sequences of the host-defence peptides from the skin glands of the froglet Crinia riparia. The peptides are called riparins. Of the eight peptides isolated, five are neuropeptides containing intramolecular disulfide linkages; e.g. the major peptide riparin 1.4 (FFLPPCAYKGTC-OH). Positive ion ES-MS identifies the five residues of riparin 1.4 outside the disulfide moiety, but provides no information on the sequence within the disulfide ring. In contrast, the negative ion dissociations of the [M-H]- ion of riparin 1.4 identify the --S-S-- link by loss of H2S2 from the [M-H]- ion, and also provide the sequence within the disulfide unit. Other peptides are riparin 2.1 [(IIEKLVNTALGLLSGL-NH2), a narrow-spectrum antibiotic], signiferin 3.1 [(GIAEFLNYIKSKA-NH2), an nNOS inhibitor] and riparin 5.1 [IVSYPDDAGEHAHKMG-NH2], which shows no neuropeptide, antibiotic or nNOS activity.

Determination of Ginsenoside Rg2 in Rat Plasma by High‐Performance Liquid Chromatography–Mass Spectrometry after Solid‐Phase Extraction

A sensitive liquid chromatography‐mass spectrometric (LC/MS) method for the quantification of ginsenoside Rg2 (Rg2) in rat plasma was developed after solid‐phase extraction (SPE). Chromatographic separation was achieved on a reversed‐phase Kromasil C18 column with the mobile phase of acetonitrile‐ammonium chloride (500 µM/L) and step gradient elution resulted in a total run time of about 9 min. The analytes were detected using electrospray negative ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range (5–2500 ng/mL) (r=0.9999). Limit of quantification (LOQ) was 5 ng/mL and the limit of detection (LOD) was 2 ng/mL using 100 µL plasma sample. Average recoveries ranged from 72.43–84.73% in plasma at the concentrations of 20, 200, and 2000 ng/mL. Intra‐ and interday coefficients of variation for the assay were 4.93–10.87% and 4.06–7.84%, respectively. The method was successfully applied to the analysis of ginsenoside Rg2 in rat plasma. The applicability of this assay was examined in a preliminary pharmacokinetic study of ginsenoside Rg2 in rats.

Characterization of must and wine of six varieties of grapes by direct infusion electrospray ionization mass spectrometry

Samples of must derived from six different varieties of grapes taken during the fermentation process, as well as the respective wine samples directly after the end of the malolactic fermentation, were analyzed by direct infusion negative ion mode electrospray ionization mass spectrometry (ESI-MS). Diagnostic ions for must were different from those of wine samples, although small variations for each of the grape varieties were also detected. The addition of unfermented must or sugar to wine could also be clearly detected. The spectra were acquired in a few minutes per sample, indicating that ESI-MS can be used for high-throughput analysis of samples and should prove useful for quality control during and after the fermentation process.

Preparative separation of cichoric acid from Echinacea Purpurea by pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to the separation of cichoric acid from Echinacea Purpurea (L.) Moench. A 3.0 g quantity of sample was separated using the following two-phase solvent system: MtBE–CH 3CN–water (4:1:5, v/v), 10 mM trifluoroacetic acid in organic stationary phase and 10 mM ammonia in aqueous mobile phase. The obtained fractions were analyzed by high-performance liquid chromatography and negative ion electrospray ionization mass spectrometry. Double separations were performed with the same solvent system yielding 563 mg cichoric acid at 95.6% purity.

Application of ESI/MS, CID/MS and tandem MS/MS to the fragmentation study of eriodictyol 7- O-glucosyl-(1→2)-glucoside and luteolin 7- O-glucosyl-(1→2)-glucoside

A mass spectrometric method based on the combined use of positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry has been applied to the structural characterization of the eriodictyol 7- O-glucosyl-(1→2)-glucoside and luteolin 7- O-glucosyl-(1→2)-glucoside. The low-energy product ion mass spectrum of [M + H] + and [M − H] − ions showed extensive fragmentation of the diglucose moiety, loss of the glycan residue, and fragmentation of the aglycon units that permit characterization of the interglycosidic linkage and the substituents in the A- and B-rings. Both glycosides were shown to yield the 0,2X 0 0,2X 1 ion which can be considered as characteristic of the 1→2 interglycosidic linkage in the glucoglucoside adducts, since it can not be formed in the case of other interglycosidic types. In the case of the eriodictyol diglucoside the 1, 3 fragmentation of the C-ring was observed before those involving the carbohydrates thus allowing the position determination of the diglucoside moiety on the A-ring. In the negative ion mode only the luteolin diglucoside was shown to undergo collision-induced homolytic and heterolytic cleavages of the O-glycosidic bond producing the aglycone radical–anion [Y 0–H] •− and Y 0 − product ions, while this was not observed in the case of eriodictyol glycoside. CID MS/MS analysis of the sodiated molecules gave complementary informations for the structural characterization of the studied compounds. The B 2 + fragment which is useful for establishing that the terminal carbohydrate unit is linked to another carbohydrate and not directly to the aglycone was obtained as base peak. This result is of analytical value for the differentiation of O-diglycosyl and di- O-glycosyl flavonoids.

Quantification of the concentration and 13C tracer enrichment of long-chain fatty acyl-coenzyme A in muscle by liquid chromatography/mass spectrometry

Recent diabetes and obesity research has been focused on the role of intracellular lipids in insulin resistance. Fatty acyl-coenzyme A (CoA) esters play a central role in the trafficking of intracellular lipids, but there has not previously been a method with which to quantify their kinetics using tracer methodology. We have therefore developed a high-performance liquid chromatography (HPLC)–mass spectrometry method to simultaneously measure the 13C stable isotopic enrichment of palmitoyl-acyl-CoA ester and the concentrations of five individual long-chain fatty acyl-CoA esters extracted from muscle tissue samples. The long-chain fatty acyl-CoA can be effectively extracted from frozen muscle tissue samples and baseline separated by a reverse-phase HPLC with the presence of a volatile reagent—triethylamine. Negative ion electrospray mass spectrometry with selected ion monitoring was used to analyze the fatty acyl-CoAs to achieve reliable quantification of their concentrations and 13C isotopic enrichment. Applying this protocol to rabbit muscle samples demonstrates that it is a sensitive, accurate, and precise method for the quantification of long-chain fatty acyl-CoA concentrations and enrichment.

A Biosynthetic Pathway Generating 12-Hydroxy-5,8,14-eicosatrienoic Acid from Arachidonic Acid Is Active in Mouse Skin Microsomes

The epidermis expresses cyclooxygenases, lipoxygenases, and cytochromes P450, which utilize arachidonic acid to generate a diverse array of lipid mediators affecting epidermal cellular differentiation and functions. Recent studies show that mouse epidermis expresses CYP2B19, a keratinocyte-specific epoxygenase that generates 11,12- and 14,15-epoxyeicosatrienoic (EET) acids from arachidonate. We studied CYP2B19-dependent metabolism in mouse epidermal microsomes, reconstituted in the presence of [1-(14)C]arachidonic acid. The majority of the (14)C products formed independently of NADPH, indicative of robust epidermal cyclooxygenase and lipoxygenase activities. We studied two NADPH-dependent products generated in a highly reproducible manner from arachidonate. One of these (product I) coeluted with the CYP2B19 product 14,15-EET on a reversed-phase high-performance liquid chromatography (HPLC) system; there was no evidence for other regioisomeric EET products. Further analyses proved that product I was not an epoxy fatty acid, based on different retention times on a normal-phase HPLC system and failure of product I to undergo hydrolysis in acidic solution. We analyzed purified epidermal (14)C products by liquid chromatography negative electrospray ionization mass spectrometry. Structures of the NADPH-dependent products were confirmed to be 12-oxo-5,8,14-eicosatrienoic acid (I) and 12-hydroxy-5,8,14-eicosatrienoic acid (II). This was the first evidence for a 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway in mouse epidermis. Epidermal microsomes also generated 12-hydroperoxy, 12-hydroxy, and 12-oxo eicosatetraenoic acids from arachidonate, possible intermediates in the 12-hydroxy-5,8,14-eicosatrienoic acid biosynthetic pathway. These results predict that hydroxyeicosatrienoic acids are synthesized from arachidonate in human epidermis. This would have important implications for human skin diseases given the known pro- and anti-inflammatory activities of stereo- and regioisomeric hydroxyeicosatrienoic acids.

Interpretation of High-throughput Liquid Chromatography Mass Spectrometry Data for Quality Control Analysis and Analytical Method Development

An approach to rapidly process and interpret high-throughput liquid chromatography mass spectrometry data is presented. This approach applies an in-house developed computer application to process LC-MS report files containing spectral and chromatographic data from four different detectors (i.e. electrospray positive ionization, electrospray negative ionization mass spectrometry, UV absorption, and evaporative light scattering detection). Properties characteristic of detection and chromatographic retention are extracted and populated into a database. Approaches to applying this analytical information database for quality control analysis of ca. 400,000 samples are presented. Compound quality assessment methods employing average purity and detection data fields are compared to methods employing multiple quality control criteria (e.g. detection, purity, retention, and signal to noise). Structural similarity searches were applied with the analytical information database to identify compounds that may be undetectable by electrospray mass spectrometry. In addition, an approach to applying the database to aid in the selection of analytical detection and chromatography conditions for rapid analytical method development is also discussed.