Analysis of Phospholipids in Rat Brain Using Liquid Chromatography–Mass Spectrometry

The brain is a lipid-rich organ containing complex polar lipids including phospholipids (PLs) and sphingolipids. These lipids are involved in the structure and function of cell membranes in the brain. We developed a fast and efficient liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to quantify five different classes of PLs [Choline glycerophospholipid (consists of phosphatidyl choline and plasmenyl choline in these samples), ethanolamine glycerophospholipid (consist of phosphatidyl ethanolamine and plasmenyl ethanolamine in these samples), phosphatidyl serine, phosphatidyl inositol, and sphingomyelin] in the brain tissues of 80-day-old Wistar rats. The PLs were extracted from rat brain using chloroform/methanol/water. After separation using a hydrophilic high performance liquid chromatography column, PL-class-specific fragmentation (head group identification) with a tandem mass spectrometer in positive ion mode was utilized to measure changes in the relative concentration of the five PL classes. The advantage of this approach was its improved specificity over previously reported LC–MS methods. The method had good repeatability (coefficient of variation 3–9%, excluding phosphatidyl inositol) and recovery (92–103%) and compared well with more laborious traditional methods.     

Application of Pharmacological Approaches to Plant–Mammal Interactions

The dominant theory in the field of mammalian herbivore–plant interactions is that intake, and therefore tolerance, of plant secondary metabolites (PSMs) is regulated by mechanisms that reduce absorption and increase detoxification of PSMs. Methods designed by pharmacologists to measure detoxification enzyme activity, metabolite excretion, and most recently, drug absorption, have been successfully applied by ecologists to study PSM intake in a variety of mammalian study systems. Here, we describe several pharmacological and molecular techniques used to investigate the fate of drugs in human that have potential to further advance knowledge of mammalian herbivore–plant interactions.     

Combination of TLC Blotting and Gas Chromatography–Mass Spectrometry for Analysis of Peroxidized Cholesterol

We have established a sensitive and convenient method for analysis of cholesterol hydroperoxides (Chol-OOHs) as trimethylsilyloxyl derivatives using diphenylpyrenylphosphine (DPPP)-thin-layer chromatography (TLC) blotting and gas chromatography–electron ionization–mass spectrometry/selected-ion monitoring (GC–EI–MS/SIM). Chol-OOH standards were prepared by photosensitized oxidation and azo radical-induced peroxidation of cholesterol. Trimethylsilyloxyl derivatives of cholesterol 5α-hydroperoxide (Chol 5α-OOH), cholesterol 7α-hydroperoxide (Chol 7α-OOH), and cholesterol 7β-hydroperoxide (Chol 7β-OOH) could be separated from one another in the SIM chromatogram using a fragment ion with elimination of trimethylsilanol from the molecular ion. This method was used to characterize peroxidized cholesterol from azo radical-exposed human low-density lipoprotein and UVA-irradiated human keratinocytes in the presence of hematoporphyrin. Finally, we succeeded in the quantification of each Chol-OOH isomer present in hairless mouse skin with and without UVA irradiation by use of β-sitosterol hydroperoxide as internal standard. The accumulation of Chol 5α-OOH with Chol 7α/βOOH in the skin indicates that singlet molecular oxygen (¹O₂) participated in the peroxidation of skin cholesterol, because Chol 5α-OOH is known to be a ¹O₂ specific cholesterol peroxidation product. We concluded that the combination of DPPP-TLC blotting and GC–EI–MS/SIM is useful for quantifying peroxidized cholesterol in biological samples and confirming the participation of ¹O₂ in oxidative stress.     

Characterization of the Organic Matter of Humic Acids by Pyrolytic Gas Chromatography–Mass Spectrometry

The results of the pyrolytic analysis of products of the organic matter of Sphagnum fuscum, fuscum peat, and humic acids separated from peat with the use of pyrolysis–chromatography–mass spectrometry in the Rock-Eval version are presented. It was shown that Sphagnum fuscum and peat differed only slightly in the degrees of chemical transformation. Benzene, phenol, and their alkyl-substituted homologues predominated in the thermal desorption products of humic acids upon pyrolysis to 400°C. Acetic acid, monohydric phenols, syringol, and guaiacol, which are the basic compounds in the high-temperature fraction, were formed on the pyrolysis humic acids to 700°C from the carbohydrate, phenylpropionic, and guaiacylpropane structural fragments.     

Evaluation of Lysophospholipid Measurement in Cerebrospinal Fluid Samples using Liquid Chromatography–Tandem Mass Spectrometry

A quantification system for lysophospholipids (lysoPL) was developed, especially for blood samples, using liquid chromatography–tandem mass spectrometry (LC–MS/MS). However, the lysoPL measurement in cerebrospinal fluid (CSF) has not been validated. Therefore, the present study aimed to validate the lysoPL measurement using CSF samples and to elucidate the possible clinical significance of the lysoPL measurement in CSF. For the validation, we observed a good precision and linearity in a sample with high lysoPL levels. The concentrations of lysoPL changed after incubation but the changes were smaller than those observed for serum samples. Moreover, we observed that the CSF levels of 16:0, 18:0 lysophosphatidylcholine, and 18:0, 18:1, and 20:4 lysophosphatidic acid were significantly higher in subjects with central nervous system invasion caused by hematological malignancies or carcinoma than in subjects with no abnormal CSF test results. In conclusion, an LC–MS/MS quantification system for lysoPL in CSF might be useful and could be applied to clinical laboratory testing.     

Quantitative Analysis of Phytosterols in Edible Oils Using APCI Liquid Chromatography–Tandem Mass Spectrometry

Previous methods for the quantitative analysis of phytosterols have usually used GC–MS and require elaborate sample preparation including chemical derivatization. Other common methods such as HPLC with absorbance detection do not provide information regarding the identity of the analytes. To address the need for an assay that utilizes mass selectivity while avoiding derivatization, a quantitative method based on LC–tandem mass spectrometry (LC–MS–MS) was developed and validated for the measurement of six abundant dietary phytosterols and structurally related triterpene alcohols including brassicasterol, campesterol, cycloartenol, β-sitosterol, stigmasterol, and lupeol in edible oils. Samples were saponified, extracted with hexane and then analyzed using reversed phase HPLC with positive ion atmospheric pressure chemical ionization tandem mass spectrometry and selected reaction monitoring. The utility of the LC–MS–MS method was demonstrated by analyzing 14 edible oils. All six compounds were present in at least some of the edible oils. The most abundant phytosterol in all samples was β-sitosterol, which was highest in corn oil at 4.35 ± 0.03 mg/g, followed by campesterol in canola oil at 1.84 ± 0.01 mg/g. The new LC–MS–MS method for the quantitative analysis of phytosterols provides a combination of speed, selectivity and sensitivity that exceed those of previous assays.     

Deuterium Tracer Studies and Gas Chromatography–Mass Spectrometry Analysis of Deuterated Products during Fischer–Tropsch Synthesis

Hydrogen is one of the two reagents of Fischer–Tropsch (FT) synthesis. In order to have the information about the role of hydrogen, which is the key to understand the mechanism of the reaction, deuterium tracer experiments are useful. One of the obstacles in conducting deuterium tracer experiments during FT reactions is to accurately determine the amounts of isotopomers of a hydrocarbon in the FT products. Because of an inverse isotope effect for isotopomers on a GC column, the isotopomer with more deuterium atoms will be eluted first. If the difference in deuterium atoms is >3, the quantitative analysis of a mixture of isotopomers can be conducted using GC–FID. However, if the difference in deuterium atoms is <3, an accurate measurement of the relative amounts of isotopomers of a compound requires a determination of the total area of all of the molecular ions by gas chromatography–mass spectrometry (GC–MS). The results determined using this method are close to the true value (by weight) with an error of ±3 %. The deuterium tracer technique and GC–MS analysis of FT products have been used to study the accumulation effects, the role of H₂O, the isotope effect, the role of C₂ species derived from ethanol and ethene, and deuterium enrichment during FT reactions.     

Nontargeted Lipidomic Characterization of Porcine Organs Using Hydrophilic Interaction Liquid Chromatography and Off-Line Two-Dimensional Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Lipids form a significant part of animal organs and they are responsible for important biological functions, such as semi‐permeability and fluidity of membranes, signaling activity, anti‐inflammatory processes, etc. We have performed a comprehensive nontargeted lipidomic characterization of porcine brain, heart, kidney, liver, lung, spinal cord, spleen, and stomach using hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI/MS) to describe the representation of individual lipid classes in these organs. Detailed information on identified lipid species inside classes are obtained based on relative abundances of deprotonated molecules [M?H]^? in the negative‐ion ESI mass spectra, which provides important knowledge on phosphatidylethanolamines and their different forms of fatty acyl linkage (ethers and plasmalogens), phosphatidylinositols, and hexosylceramides containing nonhydroxy‐ and hydroxy‐fatty acyls. The detailed analysis of identified lipid classes using reversed‐phase liquid chromatography in the second dimension was performed for porcine brain to determine more than 160 individual lipid species containing attached fatty acyls of different acyl chain length, double‐bond number, and positions on the glycerol skeleton. The fatty acid composition of porcine organs is determined by gas chromatography with flame ionization detection after the transesterification with sodium methoxide.     

Identification of Plasmalogen Cardiolipins from Pectinatus by Liquid Chromatography–High Resolution Electrospray Ionization Tandem Mass Spectrometry

High resolution electrospray ionization tandem mass spectrometry (HR-ESI–MS/MS) was used to analyze cardiolipins (Ptd₂Gro) including their plasmalogen forms from three species of the anaerobic beer-spoilage bacterial genus Pectinatus. Cardiolipins including their plasmalogens were analyzed by HR-ESI–MS/MS on Orbitrap and almost 100 cardiolipins (i.e. tetra-acyl—Ptd₂Gro, plasmenyl-tri-acyl—PlsPtd₂Gro, and di-plasmenyl-di-acyl—Pls₂Ptd₂Gro) of three classes were identified. The structures of the molecular species that consist of various regioisomers and structurally similar compounds were revealed in detail. The high resolution mass spectrometry allowed the unambiguous structural assignment of Ptd₂Gro, PlsPtd₂Gro, and Pls₂Ptd₂Gro in the three species of Pectinatus, which contain predominantly odd numbered fatty acids.     

Rapid Identification of Insect Cuticular Hydrocarbons Using Gas Chromatography—Ion-Trap Mass Spectrometry

Insect cuticular hydrocarbons (CHCs) are not only essential for desiccation resistance, they also play an important role as chemical signals and cues in social as well as solitary insects. The identification of CHCs is, therefore, crucial to an understanding of the chemical communication within and between insect species. We describe a method for rapid, simple, and unambiguous identification of CHCs using gas chromatography—ion-trap mass spectrometry. External ionization configuration in combination with a low ion-trap temperature resulted in dramatically increased intensities of molecular ions for alkanes, alkenes and alkadienes, and in high-mass fragmentation patterns with intense ions characteristic for methyl-branched hydrocarbons comparable to those obtained with quadrupole instruments. Additionally, we present an external chemical ionization—tandem mass-spectrometric method that allows for the determination of double-bond positions in alkenes and alkadienes without the need for derivatization prior to analysis.     

Determination of Tertiary-butylhydroquinone and Its Metabolites in Rat Serum by Liquid Chromatography–Ion Trap Mass Spectrometry

A new method applying sensitive and selective liquid chromatography coupled with mass spectrometry (LC/MS/MS) for analyzing tertiary-butylhydroquinone (TBHQ) and its metabolites in rat serum was validated. Using an extracted ion chromatogram (EIC) of m/z 149, free TBHQ was observed in rat serum after dosing TBHQ at 350 mg/kg to male and female Sprague–Dawley (SD) rats. Four major metabolites of TBHQ were identified—a TBHQ-sulfate, two TBHQ-sulfate-derived substances and a TBHQ-glucuronide through MSⁿ spectra. Besides its simplicity, the sample treatment allows one to obtain a very good recovery of analysts, namely around 95%. This result suggests that the method described here is useful for the analysis of TBHQ and its metabolites in rat serum. Moreover, the metabolism of TBHQ was investigated using the method. After oral administration, TBHQ appear to be more completely absorbed and bio-transformed by males than females, which may result in higher acute oral toxicity of TBHQ for males than females.     

Highly Sensitive Determination of Diverse Ceramides in Human Hair Using Reversed-Phase High-Performance Liquid Chromatography–Electrospray Ionization Mass Spectrometry

Since ceramides (CERs) play roles in signal transduction and cell regulation, CERs of human hair might be responsible for apoptosis during keratinization, in addition to their structural barrier and water-holding functions. Although, we previously developed a method for comprehensive profiling of the CERs in hair, that method was too insensitive to quantitatively characterize the CERs in a small amount of hair samples. The aim of this study was to develop a novel method for the highly sensitive determination of the diverse CERs. The method developed is negative ion electrospray ionization mass spectrometry (ESI-MS) coupled to reversed-phase high-performance liquid chromatography (RP-HPLC) using methanol containing 10 mM ammonium acetate as a mobile phase. By this method, 48 peaks derived from 73 kinds of CERs were simultaneously determined in selected ion monitoring measurement using one calibration line of the standard N-palmitoyl dihydrosphigosine, based on extremely small differences in the molar responses among different species of CERs, followed by the calculation of the actual levels using corrections for ¹³C and ²H effects. This method had extremely high sensitivity as indicated in the limit of quantification being in the femtomolar range. Other quantitative validation data, such as reproducibility, linearity and recoveries, were all sufficient. The quantitative levels of CERs determined by RP-HPLC–ESI-MS were comparable with those determined by thin-layer chromatography. This method was successfully applied to the characterization of levels of CERs in only 1-mm pieces derived from a single hair fiber and revealed the presence of interindividual and intraindividual variations of the CER composition. This RP-HPLC–ESI-MS method can be a powerful tool for future research on physicochemical and physiological roles of CERs in hair.     

Analysis of Cyanolipids from Sapindaceae Seed Oils by Gas Chromatography–EI-Mass Spectrometry

As a continuation of our investigation on unusual lipids, in the present work we describe a method based on GC–FID and GC–EI-MS to analyze the molecular composition of intact cyanolipids (CL) from selected Sapindaceae plants. We applied our method to the study of CL of type I (1-cyano-2-hydroxymethyl-prop-2-en-1-ol-diester) from Paullinia cupana var. sorbilis and Allophylus dregeanus and CL type III (1-cyano-2-hydroxymethyl-prop-1-en-3-ol-diester) from A. natalensis and Nephelium lappaceum. Our analytical approach allowed us to obtain useful mass spectra to identify individual isomeric molecular species composing the CL mixtures and resulted in the very sensitive detection and identification of minor CL. Defined CL mass spectra resulted in suitable detection of these phytochemicals in complex plant oil mixtures containing acylglycerols. To the best of our knowledge GC–EI-MS spectra of cyanolipids have never been reported before. Moreover, this study improved previous knowledge of the lipid chemistry of Sapindaceae plants.     

Liquid Chromatography–Light Scattering Detector–Mass Spectrometric Analysis of Digested Oxidized Rapeseed Oil

Rapeseed oil was oxidized chemically and thermally to produce two distinct oxidized oils. These oils, along with unoxidized oils, were subjected to an artificial digestion model to simulate the digestive processes in humans. Lipid digestion involves lipases that break down the intact triacylglycerol (TAG) molecules first to diacylglycerols, and eventually to sn-2-monoacylglycerols (MAG) and free fatty acids. A high performance liquid chromatography-evaporative light scattering detector-electrospray ionization-mass spectrometric (HPLC–ELSD–ESI–MS) method was developed to monitor the lipolysis and the presence of oxidized lipids. The HPLC–ELSD–ESI–MS analysis enabled the separation and detection of nearly all the lipid species present in the sample after TAG hydrolysis. The HPLC–MS analyses of digestion products revealed that oxidized triacylglycerols are hydrolyzed by the digestive enzymes in a manner similar to that of native, unoxidized molecules. Significant amounts of sn-1(3)-MAG were found in all the samples after lipolysis, however, more of these were found in unoxidized rapeseed oil samples than in the oxidized oils. Several oxidized molecules were identified with the aid of synthesized oxylipids. This novel method is scalable to small-scale preparative fractionation of oxidized lipid molecules from a complex digestion sample. Also, the fingerprint-like, diagnostic, MS profiles of oxidized oils, reference compounds, and digestion products may be a great aid in comprehensive analysis of lipid oxidation and lipolysis.     

Analytical Method for Diacylglycerol Kinase ζ Activity in Cells Using Protein Myristoylation and Liquid Chromatography–Tandem Mass Spectrometry

Specific inhibitors of diacylglycerol kinase (DGK) ζ can be promising anticancer medications via the activation of cancer immunity. Although the detection of cellular activities of target enzymes is essential for drug screening in addition to in vitro assays, it is difficult to detect the activity of DGKζ in cells. In the present study, we generated AcGFP-DGKζ cDNA with a consensus N-myristoylation sequence at the 5′ end (Myr-AcGFP-DGKζ) to target DGKζ to membranes. Using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (LC–MS/MS), we showed that Myr-AcGFP-DGKζ, but not AcGFP-DGKζ without the myristoylation sequence, substantially augmented the levels of several phosphatidic acid (PtdOH) species. In contrast to Myr-AcGFP-DGKζ, its inactive mutant did not exhibit an increase in PtdOH production, indicating that the increase in PtdOH production was DGK activity-dependent. This method will be useful in chemical compound selection for the development of drugs targeting DGKζ and can be applicable to various soluble (nonmembrane bound) lipid-metabolizing enzymes, including other DGK isozymes.     

A New Gas Chromatography–Mass Spectrometry Method for Detecting and Quantifying Low Levels of Dimethyl Sulfate in Palm Oil-Based Sulfonated Methyl Esters

Dimethyl sulfate (DMS) is a potential carcinogen that may be formed during the production of α-sulfonated methyl ester (α-SME). To date, there have been no published analytical methods for quantification of DMS in palm oil-based α-SME. The objective of the method development was to establish a simple and reliable method for quantifying and monitoring DMS from palm oil-based α-SME. The sample preparation prior to analysis involved extracting DMS from α-SME using hexane. The gas chromatography–mass spectrometry (GC–MS) separation was performed on an Agilent MS-HP5 column (30 m × 320 μm × 0.25 μm). The MS signal was obtained in the selected ion monitoring (SIM) mode. The method was validated according to the International Conference on Harmonization (ICH) guidelines. The accuracy of the method was measured via percentage recoveries of DMS from spiked α-SME samples at spiking levels of (25, 125, 250, and 500) μg mL⁻¹. The percentage recoveries for all spiking levels were found to be in the range of 88.6–97.4%. The repeatability and interday reproducibility were found to be satisfactory as the relative standard deviations (RSD) were within 11%. The method also has a good linear relationship as indicated by the coefficient of determination of 0.9996 over the range of 1.0–30 μg mL⁻¹. The limits of detection (LOD) and quantification (LOQ) corresponded to 0.8 and 2.3 μg mL⁻¹, respectively. The developed method demonstrated sufficient linearity, intraday and interday precision, sensitivity, and accuracy for determination of DMS in palm oil-based α-SME.     

Liquid Chromatography–Mass Spectrometric Analysis of Lipids Present in Human Meibomian Gland Secretions

The purpose of the study was to qualitatively characterize the major lipid species present in human meibomian gland secretions (MGS) by means of high-performance liquid chromatography with atmospheric pressure ionization mass spectrometric detection of the analytes (NP HPLC-MS). Two different NP HPLC-MS methods have been developed to analyze lipid species that were expected to be present in MGS. The first method was optimized for the analysis of relatively nonpolar lipids [wax esters (WE), di- and triacyl glycerols (DAG and TAG), cholesterol (Chl) and its esters (Chl-E), and ceramides (Cer)], while the second method was designed to separate and detect phospholipids. The major lipid species in MGS were found to be WE, Chl-E, and TAG. A minor amount of free Chl (less then 0.5% of the Chl-E fraction) was detected in MGS. No appreciable amounts of DAG and Cer were found in MGS. The second NP HPLC-MS method, capable of analyzing model mixtures of authentic phospholipids (e.g. phosphatidylglycerol, phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin) in submicrogram/mL concentrations, showed little or no presence of these species in the MGS samples. These observations suggest that MGS are a major source of the nonpolar lipids of the WE and Chl-E families for the tear film lipid layer (TFLL), but not of the previously reported phospholipid components of the TFLL.     

The Application of an Emerging Technique for Protein–Protein Interaction Interface Mapping: The Combination of Photo-Initiated Cross-Linking Protein Nanoprobes with Mass Spectrometry

Protein–protein interaction was investigated using a protein nanoprobe capable of photo-initiated cross-linking in combination with high-resolution and tandem mass spectrometry. This emerging experimental approach introduces photo-analogs of amino acids within a protein sequence during its recombinant expression, preserves native protein structure and is suitable for mapping the contact between two proteins. The contact surface regions involved in the well-characterized interaction between two molecules of human 14-3-3ζ regulatory protein were used as a model. The employed photo-initiated cross-linking techniques extend the number of residues shown to be within interaction distance in the contact surface of the 14-3-3ζ dimer (Gln8–Met78). The results of this study are in agreement with our previously published data from molecular dynamic calculations based on high-resolution chemical cross-linking data and Hydrogen/Deuterium exchange mass spectrometry. The observed contact is also in accord with the 14-3-3ζ X-ray crystal structure (PDB 3dhr). The results of the present work are relevant to the structural biology of transient interaction in the 14-3-3ζ protein, and demonstrate the ability of the chosen methodology (the combination of photo-initiated cross-linking protein nanoprobes and mass spectrometry analysis) to map the protein-protein interface or regions with a flexible structure.     

Determination of Triacylglycerols in Butterfat by Normal-Phase HPLC and Electrospray–Tandem Mass Spectrometry

Here, we report the identification and quantification of the molecular species of long-, medium-, and short-chain triacylglycerols (TAG) in butterfat (BF), including TAG with an odd number of acyl carbons (ACN) and TAG with unidentified molecular species. In the present study, in addition to auto-MS², a large number of methods, each recording MS² for 1–4 ions, were used for identification of TAG species. For the quantification of long-chain, odd ACN TAG, and TAG with unidentified molecular species, molar correction factors (MCF) were calculated from the uncorrected mol% (area mol%) of each ACN:DB (number of double bonds) class in randomized butterfat (RBF), and the respective mol% in the calculated random composition of RBF. The butyrate, caproate, and medium-chain (C₈, C₁₀) TAG were quantified using regio- or acyl-chain-specific MCF calculated from their area mol% in RBF and mol% in the calculated random composition. These methods enabled us to identify ca. 450 TAG species in 184 quantified peaks of 88 ACN:DB classes. The proportions of saturated, monoene, diene, triene, tetraene, pentaene, and hexaene TAG were 40.0, 38.4, 16.2, 4.5, 0.6, 0.1, and 0.03 mol%, respectively. The proportions of TAG with not identified molecular species and odd ACN TAG were 11.8 and 5.7 mol%, respectively. The most abundant short-chain TAG species were butyroyldipalmitoylglycerol + butyroylmyristoylstearoylglycerol (5.25 mol%) and butyroylpalmitoyloleoylglycerol (4.08 mol%).