Analytical and Bioanalytical Chemistry (v.407, #17)

KOSMOS Fellow 2014 and SALSA Visiting Professor. montesmaria@uniovi.es. KOSMOS Summer University 2014 Organizer. Janina.kneipp@chemie.hu-berlin.de. KOSMOS Summer University 2014 Organizer. Ulrich.Panne@BAM.de

Maillard and grilled steak challenge by Hervé This (4873-4875).

Solution to solubility product challenge by Anna Maria Michałowska-Kaczmarczyk; Tadeusz Michałowski (4877-4878).

Effects of pre-analytical processes on blood samples used in metabolomics studies by Peiyuan Yin; Rainer Lehmann; Guowang Xu (4879-4892).
Every day, analytical and bio-analytical chemists make sustained efforts to improve the sensitivity, specificity, robustness, and reproducibility of their methods. Especially in targeted and non-targeted profiling approaches, including metabolomics analysis, these objectives are not easy to achieve; however, robust and reproducible measurements and low coefficients of variation (CV) are crucial for successful metabolomics approaches. Nevertheless, all efforts from the analysts are in vain if the sample quality is poor, i.e. if preanalytical errors are made by the partner during sample collection. Preanalytical risks and errors are more common than expected, even when standard operating procedures (SOP) are used. This risk is particularly high in clinical studies, and poor sample quality may heavily bias the CV of the final analytical results, leading to disappointing outcomes of the study and consequently, although unjustified, to critical questions about the analytical performance of the approach from the partner who provided the samples. This review focuses on the preanalytical phase of liquid chromatography–mass spectrometry-driven metabolomics analysis of body fluids. Several important preanalytical factors that may seriously affect the profile of the investigated metabolome in body fluids, including factors before sample collection, blood drawing, subsequent handling of the whole blood (transportation), processing of plasma and serum, and inadequate conditions for sample storage, will be discussed. In addition, a detailed description of latent effects on the stability of the blood metabolome and a suggestion for a practical procedure to circumvent risks in the preanalytical phase will be given. Graphical Abstract The procedures and potential problems in preanalytical aspects of metabolomics studies using blood samples. Bias in the preanalytical phase may lead to unwanted results in the subsequential studies
Keywords: Metabolomics; Preanalytical; Bias; Blood; Urine; Serum; Plasma; Liquid chromatography–mass spectrometry; Standard operation procedure; Biobank

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry by Javier Alfonso-Garrido; Estefania Garcia-Calvo; Jose L. Luque-Garcia (4893-4905).
Despite enormous advances in the mass spectrometry and proteomics fields during the last two decades, the analysis of membrane proteins still remains a challenge for the proteomic community. Membrane proteins play a wide number of key roles in several cellular events, making them relevant target molecules to study in a significant variety of investigations (e.g., cellular signaling, immune surveillance, drug targets, vaccine candidates, etc.). Here, we critically review the several attempts that have been carried out on the different steps of the sample preparation procedure to improve and modify existing conventional proteomic strategies in order to make them suitable for the study of membrane proteins. We also revise novel techniques that have been designed to tackle the difficult but relevant task of identifying and characterizing membrane proteins.
Keywords: Membrane proteins; Mass spectrometry; Sample preparation; Hydrophobic peptides

Determination of direct alcohol markers: a review by Pamela Cabarcos; Iván Álvarez; María Jesús Tabernero; Ana María Bermejo (4907-4925).
Alcohol is the most popular legal drug used in our society today, and its consumption by pregnant women remains an important public health problem. Gestational alcohol consumption can result in a continuum of adverse fetal outcomes known as fetal alcohol spectrum disorder (FASD). Effective strategies are needed to prevent the increasing adoption of risky drinking behaviors. Because ethanol itself is only measurable for a few hours after ethanol intake in conventional matrices including blood, urine, and sweat, these matrices are only useful to detect recent ethanol exposure. Since approximately early 2000, the non-oxidative ethanol metabolites have received increasing attention because of their specificity and, in some cases, wide time window of detection in non-conventional matrices including hair and meconium. In the attempt to update analytical methods for the determination of non-oxidative markers of alcohol, the objective of this study is to review published studies that measure fatty-acid ethyl esters (FAEE), ethyl glucuronide (EtG), and phosphatidylethanol (PEth) in alternative biological matrices, focusing on the extraction and detection methods and full analytical conditions used.
Keywords: Fatty-acid ethyl ester; Ethyl glucuronide; Phosphatidylethanol; GC–MS; LC–ESI MS–MS; Dispersive liquid–liquid microextraction

Stimuli-responsive materials in analytical separation by Rosa A. Lorenzo; Antonia M. Carro; Angel Concheiro; Carmen Alvarez-Lorenzo (4927-4948).
This review focuses on the fundamentals of stimuli-responsive materials and their applications to three common separation techniques, namely extraction, chromatography, and electrophoresis. Although still little investigated, materials that switch their affinity for the analyte on and off as a function of tiny changes in physical and biochemical variables offer relevant advantages for analyte extraction, concentration, and separation. Temperature and/or pH-responsive polymers in the form of chains or networks, which are dispersed in the sample as free entities or after being grafted onto beads (which may incorporate magnetic cores), enable quantitative capture and/or elution of the analyte under mild conditions and without needing organic solvents. Regarding liquid-chromatography separation, responsive stationary phases enable the implementation of “all-in-water” procedures in which retention times are modulated by means of temperature or pH gradients. Other stimuli that can be externally applied, for example light or magnetic fields, can also be used for efficient extraction or separation of the target substance without altering the composition of the sample matrix. Moreover, stimuli-responsiveness enables straightforward recycling of solid and/or stationary phases for a prolonged lifetime. Improved understanding of the phase transitions of stimuli-responsive materials and design of suitable formats for analytical applications should enable wider and more successful application of stimuli-responsive materials in analytical separations. Graphical Abstract Responsive materials that reversibly switch their affinity for the analyte on and off offer relevant advantages for analyte extraction, concentration, and separation
Keywords: Capillary electrophoresis; HPLC; Extraction (SPE); Stimuli-responsive stationary phases; Green chromatography; MIPs

The availability of national test centers to offer a routine service for analysis and quantitation of some selected steroid hormones [natural estrogens (17-β-estradiol, E2; estrone, E1; estriol, E3), synthetic estrogen (17-α-ethinylestradiol, EE2), androgen (testosterone), and progestogen (progesterone)] in wastewater matrix was investigated; corresponding internationally used chemical- and immuno-analytical test methods were reviewed. The enzyme-linked immunosorbent assay (ELISA) (immuno-analytical technique) was also assessed for its suitability as a routine test method to quantitate the levels of these hormones at a sewage/wastewater treatment plant (WTP) (Darvill, Pietermaritzburg, South Africa), over a 2-year period. The method performance and other relevant characteristics of the immuno-analytical ELISA method were compared to the conventional chemical-analytical methodology, like gas/liquid chromatography–mass spectrometry (GC/LC-MS), and GC-LC/tandem mass spectrometry (MSMS), for quantitation of the steroid hormones in wastewater and environmental waters. The national immuno-analytical ELISA technique was found to be sensitive (LOQ 5 ng/L, LOD 0.2–5 ng/L), accurate (mean recovery 96 %), precise (RSD 7–10 %), and cost-effective for screening and quantitation of these steroid hormones in wastewater and environmental water matrix. A survey of the most current international literature indicates a fairly equal use of the LC-MS/MS, GC-MS/MS (chemical-analytical), and ELISA (immuno-analytical) test methods for screening and quantitation of the target steroid hormones in both water and wastewater matrix. Internationally, the observed sensitivity, based on LOQ (ng/L), for the steroid estrogens E1, E2, EE2, is, in decreasing order: LC-MSMS (0.08–9.54) > GC-MS (1) > ELISA (5) (chemical-analytical > immuno-analytical). At the national level, the routine, unoptimized chemical-analytical LC-MSMS method was found to lack the required sensitivity for meeting environmental requirements for steroid hormone quantitation. Further optimization of the sensitivity of the chemical-analytical LC–tandem mass spectrometry methods, especially for wastewater screening, in South Africa is required. Risk assessment studies showed that it was not practical to propose standards or allowable limits for the steroid estrogens E1, E2, EE2, and E3; the use of predicted-no-effect concentration values of the steroid estrogens appears to be appropriate for use in their risk assessment in relation to aquatic organisms. For raw water sources, drinking water, raw and treated wastewater, the use of bioassays, with trigger values, is a useful screening tool option to decide whether further examination of specific endocrine activity may be warranted, or whether concentrations of such activity are of low priority, with respect to health concerns in the human population. The achievement of improved quantitation limits for immuno-analytical methods, like ELISA, used for compound quantitation, and standardization of the method for measuring E2 equivalents (EEQs) used for biological activity (endocrine: e.g., estrogenic) are some areas for future EDC research. Graphical Abstract ᅟ
Keywords: Endocrine disruptor compound; Steroid hormones; Immuno-analysis; Enzyme-linked immunosorbent assay; Gas/liquid chromatography–tandem mass spectrometry; Wastewater; Bioassay

Lipidomics by Michal Holčapek (4971-4972).
is Professor and Head of Mass Spectrometry Group at the Department of Analytical Chemistry, University of Pardubice. His research interests are mass spectrometry and its coupling with liquid-phase separation techniques with the specialization on lipidomic analysis. In the last few years, he has focused on lipidomic clinical applications with the goal to find lipid biomarkers for some serious human diseases. He is author or co-author of over 110 papers in international peer-reviewed journals with an H-index of 31; member of editorial boards of Analytical and Bioanalytical Chemistry, Analytical Chemistry, Lipids; guest editor of special volumes on Mass spectrometry – innovation and application (Journal of Chromatography A, 2010 and 2012); co-editor of a Virtual issue on Lipidomics (Analytical Chemistry, 2014) and co-editor of the book Extreme Chromatography: Faster, Hotter, Smaller (American Oil Chemical Society, 2011).

Optimizing the lipidomics workflow for clinical studies—practical considerations by Tuulia Hyötyläinen; Matej Orešič (4973-4993).
Lipidomics is increasingly being used in clinical research, offering new opportunities for disease prediction and detection. One of the key challenges of clinical applications of lipidomics is the high sensitivity of measured lipid levels to many analytical, physiological, and environmental factors, which therefore must be taken into account when designing the studies. Here we critically discuss the complete clinical lipidomics workflow, including selection of the subjects, the sample type, the sample preprocessing conditions, and the analytical method and methods for data processing. We also review the lipidomics applications which investigate the confounding factors such as age, gender, fasting time, and handling procedures for measuring blood lipid metabolites.
Keywords: Biomarkers; Lipidomics; Mass spectrometry; Metabolomics; Systems biology; Systems medicine

Applications of ion-mobility mass spectrometry for lipid analysis by Giuseppe Paglia; Michal Kliman; Emmanuelle Claude; Scott Geromanos; Giuseppe Astarita (4995-5007).
The high chemical complexity of the lipidome is one of the major challenges in lipidomics research. Ion-mobility spectrometry (IMS), a gas-phase electrophoretic technique, makes possible the separation of ions in the gas phase according to their charge, shape, and size. IMS can be combined with mass spectrometry (MS), adding three major benefits to traditional lipidomic approaches. First, IMS–MS allows the determination of the collision cross section (CCS), a physicochemical measure related to the conformational structure of lipid ions. The CCS is used to improve the confidence of lipid identification. Second, IMS–MS provides a new set of hybrid fragmentation experiments. These experiments, which combine collision-induced dissociation with ion-mobility separation, improve the specificity of MS/MS-based approaches. Third, IMS–MS improves the peak capacity and signal-to-noise ratio of traditional analytical approaches. In doing so, it allows the separation of complex lipid extracts from interfering isobaric species. Developing in parallel with advances in instrumentation, informatics solutions enable analysts to process and exploit IMS–MS data for qualitative and quantitative applications. Here we review the current approaches for lipidomics research based on IMS–MS, including liquid chromatography–MS and direct-MS analyses of “shotgun” lipidomics and MS imaging.
Keywords: Lipid; Lipidomics; Ion mobility; Mass spectrometry

Lipidome and metabolome analysis of fresh tobacco leaves in different geographical regions using liquid chromatography–mass spectrometry by Lili Li; Xin Lu; Jieyu Zhao; Junjie Zhang; Yanni Zhao; Chunxia Zhao; Guowang Xu (5009-5020).
is a PhD student in the Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. She has majored in high-resolution separation and analysis techniques based on liquid chromatography coupled with mass spectrometry and its application in plant metabolomics. is currently a professor at Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Her main research interest is the characterization and separation of complex mixtures using chromatography in combination with mass spectrometry. is a PhD student in the Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Her research is focused on the study of plant metabolism using capillary electrophoresis–time-of-flight mass spectrometry. is a PhD student in the Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences. She has been studying the development of new metabolomics methods based on liquid chromatography–mass spectrometry technology and their applications in plant organisms. is a PhD student at Dalian Institute of Chemical Physics, Chinese Academy of Science. She began her PhD degree in analytical chemistry under the supervision of Peizhang Lu and Guowang Xu in 2012. Her research is mainly focused on the study of plant metabolism using gas chromatography coupled with mass spectrometry. is an associate professor at Dalian Institute of Chemical Physics, Chinese Academy of Sciences. Her main research interest is the characterization and separation of complex mixtures using liquid chromatography coupled with mass spectrometry. is Director of the Metabonomics Research Center at Dalian Institute of Chemical Physics, Chinese Academy of Sciences. In 2004 he obtained an award for distinguished young scientists from the National Natural Science Foundation of China. His main research fields are chromatography, mass spectrometry and metabolomic applications in disease biomarker discovery, traditional Chinese medicine, and food safety. The combination of the lipidome and the metabolome can provide much more information in plant metabolomics studies. A method for the simultaneous extraction of the lipidome and the metabolome of fresh tobacco leaves was developed. Method validation was performed on the basis of the optimal ratio of methanol to methyl tert-butyl ether to water (37:45:68) from the design of experiments. Good repeatability was obtained. We found that 92.2 % and 91.6 % of the peaks for the lipidome and the metabolome were within a relative standard deviation of 20 %, accounting for 94.6 % and 94.6 % of the total abundance, respectively. The intraday and interday precisions were also satisfactory. A total of 230 metabolites, including 129 lipids, were identified. Significant differences were found in lipidomic and metabolomic profiles of fresh tobacco leaves in different geographical regions. Highly unsaturated galactolipids, phosphatidylethanolamines, predominant phosphatidylcholines, most of the polyphenols, amino acids, and polyamines had a higher content in Yunnan province, and low-unsaturation-degree galactolipids, triacylglycerols, glucosylceramides with trihydroxy long-chain bases, acylated sterol glucosides, and some organic acids were more abundant in Henan province. Correlation analysis between differential metabolites and climatic factors indicated the vital importance of temperature. The fatty acid unsaturation degree of galactolipids could be influenced by temperature. Accumulation of polyphenols and decreases in the ratios of stigmasterols to sitosterols and glucosylstigmasterols to glucosylsitosterols were also correlated with lower temperature in Yunnan province. Furthermore, lipids were more sensitive to climatic variations than other metabolites. Graphical Abstract Lipidomic and metabolomic profiles of tobacco in different regions
Keywords: Lipidome; Metabolome; Fresh tobacco leaves; Climatic factors; Metabolomics

works as a staff scientist at the Sanford-Burnham Medical Research Institute in Orlando, Florida. His research mainly focuses on extending the techniques of multi-dimensional mass-spectrometry-based shotgun lipidomics to trace amounts of lipid classes and their application in biomedical and biological research. is a postdoctoral Associate in Dr Judith Altarejos’ Lab. There she works to delineate the action of Crtc1 on thermogenesis regulated by leptin. is Associate Professor in the department of product processing and nutriology in Oil Crops Research Institute, Chinese Academy of Agricultural Sciences. She has been working in lipidomics in food and nutrition research, including the development of high-throughput analysis methods for analysis of lipids by HPLC–MS–MS and GC–MS. is Professor and director of the department of product processing and nutriology in Oil Crops Research Institute, Chinese Academy of Agricultural Sciences. He works on developing lipidomics approaches and applications for enzymatic modification of lipids and biolipids in oil crops. is Assistant Professor in the Metabolic Disease Program at Sanford-Burnham Medical Research Institute. Her research focuses on understanding how brain circuits modulate peripheral tissue metabolism and developing tools that enable these mechanistic discoveries. is Professor of Cardiovascular Pathobiology at the Sanford-Burnham Medical Research Institute. His research focuses on identification of molecular mechanisms underpinning altered lipid metabolism, trafficking, and homeostasis in disease states. Dr Han is one of the pioneers of lipidomics and one of the inventors of shotgun lipidomics. N-monomethyl phosphatidylethanolamine (MMPE) and N,N-dimethyl phosphatidylethanolamine (DMPE) species are intermediates of phosphatidylcholine (PC) de-novo biosynthesis through methylation of phosphatidylethanolamine (PE). This synthesis pathway for PC is especially important in the liver when choline is deficient in the diet. Despite some efforts focused on the analysis of MMPE and DMPE species, a cost-effective and high-throughput method for determination of individual MMPE and DMPE species, including their regioisomeric structures, is still missing. Therefore we adopted and improved the “mass-tag” strategy for determining these PE-like species by methylating PE, MMPE, and DMPE molecules with deuterated methyl iodide to generate PC molecules with nine, six, and three deuterium atoms, respectively. On the basis of the principles of multidimensional mass-spectrometry-based shotgun lipidomics we could directly identify and quantify these methylated PE species, including their fatty-acyl chains and regiospecific positions. The method provided remarkable sensitivity, with a limit of detection at 0.5 fmol μL−1, high specificity, and a broad linear-dynamics range of >2500 folds. By applying this method to liver samples from streptozotocin (STZ)-induced diabetic mice and controls, we found that the levels of PC species tended to decrease and the amounts of PE species tended to increase in the liver of STZ-induced diabetic mice compared with controls, but no significant changes in MMPE and DMPE species were determined. However, remodeling of fatty-acyl chains in the determined lipids was observed in the liver of STZ-induced diabetic mice, with reduction in 16:1 and increases in 18:2, 18:1, and 18:0 acyl chains. These results indicated the improved method to be a powerful tool to reveal the function of the PC de-novo biosynthesis pathway through methylation of PE species in biological systems. Graphical Abstract Representative mass spectra of PC, MMPE, DMPE, and PE species present in mouse liver samples
Keywords: Diabetes; Liver damage; Mass spectrometry; N-monomethyl phosphatidylethanolamine; N,N-dimethyl phosphatidylethanolamine; Phosphatidylcholine biosynthesis; Shotgun lipidomics

Continuous comprehensive two-dimensional liquid chromatography–electrospray ionization mass spectrometry of complex lipidomic samples by Michal Holčapek; Magdaléna Ovčačíková; Miroslav Lísa; Eva Cífková; Tomáš Hájek (5033-5043).
is currently a Full Professor and Head of the Mass Spectrometry Group in the Department of Analytical Chemistry, University of Pardubice. His research interests are mass spectrometry and its coupling with liquid-phase separation techniques, with specialization in lipidomic analysis and clinical applications. He is the author or coauthor of over 100 articles in international peer-reviewed journals, and has an h index of 30. is a PhD student in the Mass Spectrometry Group in the Department of Analytical Chemistry, University of Pardubice. She is working on the characterization of lipids in biological samples using liquid chromatography–mass spectrometry coupling. is a Research Worker in the Department of Analytical Chemistry, University of Pardubice. He is working mainly on the development of new methods for the analysis of lipids using mass spectrometry and its coupling with high-performance liquid chromatography and supercritical fluid chromatography. is a Research Worker in the Department of Analytical Chemistry, University of Pardubice. She focuses on the development of new mass-spectrometry-based methods for comprehensive lipidomic analysis and applications in biomarker discovery. is an Assistant Professor in the Department of Analytical Chemistry, University of Pardubice. His research interests are the development of online two-dimensional liquid chromatography, and the prediction and optimization of high-performance liquid chromatography separations. A new continuous comprehensive two-dimensional liquid chromatography–electrospray ionization mass spectrometry method has been developed for the lipidomic characterization of complex biological samples. The reversed-phase ultra-high-performance liquid chromatography with a C18 column (150 mm × 1 mm, 1.7 μm) used in the first dimension makes the separation of numerous lipid species differing in their hydrophobic part of the molecule, mainly fatty acyl chain lengths and the number and positions of double bonds, possible. Coeluted lipid species in the first dimension are resolved by the fast hydrophilic interaction liquid chromatography separation (50 mm × 3 mm, 2.7 μm, core–shell particles) of lipid classes according to their different polarities in the second dimension. Retention times in both dimensions, accurate m/z values, and tandem mass spectra provide high confidence in the identification of lipid species. The retention behavior of individual lipids in reversed-phase mode follows the equivalent carbon number pattern, which provides an additional tool for unambiguous identification. This analytical method is applied for the lipidomic characterization of total lipid extracts of human plasma and porcine brain samples, which resulted in the identification of 143 lipid species from four lipid categories and ten lipid classes. Graphical Abstract 2D-LC/ESI-MS of porcine brain lipid extract
Keywords: Lipids; Lipidomics; Comprehensive 2D liquid chromatography; Mass spectrometry; Plasma; Brain

is a postdoctoral fellow in Robert Murphy’s laboratory in the Department of Pharmacology, University of Colorado Denver, where she is working on mass-spectrometric analysis of oxidized phospholipids. She received her PhD degree in 2011 from the Department of Occupational and Environmental Medicine at the University of Gothenburg in Sweden, where she currently holds a position as a research scientist/chemist. The research in Gothenburg is focused on sampling and analysis of exhaled air. is a senior professional research assistant in the laboratory of Robert Murphy, Department of Pharmacology at the University of Colorado Denver. He has many years of experience in the operation of state-of-the-art mass spectrometers and is highly experienced in the analysis of lipids. Most recently, he has worked on advancements in ion mobility mass spectrometry of lipids. is a University of Colorado Distinguished Professor in the Department of Pharmacology at the University of Colorado Denver. He has maintained an active research career for over 40 years using mass spectrometry to study lipid biochemistry. He started his research career in mass spectrometry as a student with Klaus Biemann, has served as President of the American Society for Mass Spectrometry, and is on the editorial boards of numerous scientific journals in biochemistry and mass spectrometry. Lipidomic analysis of the complex mixture of lipids isolated from biological systems can be a challenging process that often involves tandem mass spectrometry and interpretation of both precursor ions and product ions relative to the molecular structure of the lipids. Therefore, detailed understanding of the gas-phase ion chemistry occurring for each class of phospholipids is critically important for an accurate assignment of lipid structure. Some oxidized phosphatidylcholines are known to be biologically active and responsible for pathological events, and are therefore important targets for detection in lipidomic studies. Modification of fatty acyl chains by oxidation may, however, change the behavior of ion formation and decomposition in the mass spectrometer. In this study, we report on the mass-spectrometric behavior of 1-palmitoyl-2-(9′-oxononanoyl)-sn-glycero-3-phosphocholine, a bioactive product of phosphatidylcholine oxidation. In addition to [M−15] and the acetate adduct [M+59], three additional adduct ions, including [M−H], were present in significant abundance in the negative ion electrospray mass spectrum. It was found that this unexpected [M−H] ion was formed by the transfer of a methyl group from the choline residue on the polar head group to the aldehyde functionality of the sn-2 substituent, resulting in a 14-Da increase in the mass of the resulting sn-2 carboxylate anion formed by collisional activation of this ion. These results suggest additional rules for understanding the gas-phase ion chemistry of aldehydic phosphatidylcholine molecular species.
Keywords: Lipidomics; Electrospray ionization; Mass spectrometry; Phosphatidylcholine; Omega-aldehyde phosphatidylcholine; Tandem mass spectrometry

Combining liquid chromatography with ozone-induced dissociation for the separation and identification of phosphatidylcholine double bond isomers by Rachel L. Kozlowski; J. Larry Campbell; Todd W. Mitchell; Stephen J. Blanksby (5053-5064).
Revealing the inherent molecular diversity of lipid biology requires advanced analytical technologies. Distinguishing phospholipids that differ in the position(s) of carbon-carbon double bonds within their acyl chains presents a particular challenge because of their similar chromatographic and mass spectral behaviours. Here—for the first time—we combine reversed-phase liquid chromatography for separation of isomeric phospholipids with on-line mass spectral analysis by ozone-induced dissociation (OzID) for unambiguous double bond position assignment. The customised tandem linear ion-trap mass spectrometer used in our study is capable of acquiring OzID scans on a chromatographic timescale. Resolving the contributions of isomeric lipids that are indistinguishable based on conventional mass spectral analysis is achieved using the combination of liquid chromatography and OzID. Application of this method to the analysis of simple (egg yolk) and more complex (sheep brain) extracts reveals significant populations of the phosphatidylcholine PC 16:0_18:1(n−7) alongside the expected PC 16:0_18:1(n−9) isomer. Graphical Abstract Separation and identification double bond positional isomers of phosphatidylcholines using LC-OzID
Keywords: Mass spectrometry; Liquid chromatography; Phospholipids; Double bond isomers; Ozone-induced dissociation; Collision-induced dissociation

Comprehensive lipid profiling of plasma in patients with benign breast tumor and breast cancer reveals novel biomarkers by Li Yang; Xinge Cui; Ningning Zhang; Min Li; Yu Bai; Xiaohong Han; Yuankai Shi; Huwei Liu (5065-5077).
Abnormal lipid metabolism is a common feature in most solid tumors, and occurs in early stages of the tumor progression. As benign breast tumor is different from malignant tumor of breast cancer, it is particularly important to take benign breast tumor into consideration when investigating cancer biomarkers. In this study, by using a normal-phase/reversed-phase two-dimensional liquid chromatography-mass spectrometry (NP/RP 2D LC-MS) method, we conducted comprehensive lipid profiling in human plasma obtained from six benign breast tumor patients and five breast cancer patients, as well as nine healthy controls. As a result, 512 lipid species were successfully identified. Principal component analysis allowed clear separation of the three groups. Quantitative analysis revealed that many lipid contents were similar in benign and malignant breast tumors compared with controls, and these were proposed as potential breast tumor biomarkers other than breast cancer biomarkers. Two phosphatidylinositol (PI) species, including PI (16:0/16:1) and PI (18:0/20:4), could differentiate between benign and malignant breast tumors, as well as breast cancer patients and healthy controls, indicating that they could be utilized as potential breast cancer biomarkers. In addition, PI (16:0/18:1), phosphatidylglycerol (36:3), and glucosylceramide (d18:1/15:1) were demonstrated to be potential biomarkers to evaluate the level of malignancy of breast tumor. Taken together, our results indicate the usefulness of lipid profiling in the discrimination between patients with breast cancer and non-carcinoma lesions, which might provide assistance in clinical diagnosis. Graphical Abstract ᅟ
Keywords: Breast cancer; Benign breast tumor; Lipidomics; Biomarker; NP/RP 2D LC-MS

Collision-induced dissociation of aminophospholipids (PE, MMPE, DMPE, PS): an apparently known fragmentation process revisited by Ernst Pittenauer; Pavel Rehulka; Wolfgang Winkler; Günter Allmaier (5079-5089).
A new type of low-mass substituted 4-oxazolin product ions of [M + H]+ precursor ions of aminophospholipids (glycerophosphatidylethanolamine, glycerophosphatidyl-N-methylethanolamine, glycerophosphatidyl-N,N-dimethylethanolamine, glycerophosphatidylserine) resulting from high-energy collision-induced dissociation (matrix-assisted laser desorption/ionization time-of-flight/reflectron time-of-flight mass spectrometry) and low-energy collision-induced dissociation (e.g., electrospray ionization quadrupole reflectron time-of-flight mass spectrometry) with accurate mass determination is described; these were previously misidentified as CHO-containing radical cationic product ions. The mechanism for the formation of these ions is proposed to be via rapid loss of water followed by cyclization to an 11-membered-ring transition state for the sn-1 fatty acid substituent and to a ten-membered-ring transition state for the sn-2 fatty acid substituent, and via final loss of monoacylglycerol phosphate, leading to substituted 4-oxazolin product ions. The minimum structural requirement for this interesting skeletal rearrangement fragmentation is an amino group linked to at least one hydrogen atom (i.e., ethanolamine, N-methylethanolamine, serine). Therefore, N,N-dimethylethanolamine derivates do not exhibit this type of fragmentation. The analytical value of these product ions is given by the fact that by post source decay and particularly high-energy collision-induced dissociation achieved via matrix-assisted laser desorption/ionization time-of-flight/reflectron time-of-flight mass spectrometry, the sn-2-related substituted 4-oxazolin product ion is always significantly more abundant than the sn-1-related one, which is quite helpful for detailed structural analysis of complex lipids. All other important product ions found are described in detail (following our previously published glycerophospholipid product ion nomenclature; Pittenauer and Allmaier, Int. J. Mass. Spectrom. 301:90–1012, 2011).
Keywords: High-energy collision-induced dissociation; Matrix-assisted laser desorption/ionization time-of-flight/reflectron time-of-flight mass spectrometry; Low-energy collision-induced dissociation; Electrospray ionization quadrupole reflectron time-of-flight mass spectrometry; Aminophospholipids; Substituted 4-oxazolin product ions

Quantitative lysophospholipidomics in human plasma and skin by LC–MS/MS by Kaisa M. Koistinen; Matti Suoniemi; Helena Simolin; Kim Ekroos (5091-5099).
Lysophospholipids (LPLs) are an essential family of lipids, which serve as bioactive molecules and as precursors and intermediates of the glycerophospholipid and sphingolipid metabolisms. In this work we primarily focused on the subgroup lysoglycerophospholipids that comprise a polar headgroup at the sn-3 position and a fatty acyl group at either the sn-1 or sn-2 position of the glycerol backbone giving rise to the two potential regioisomers 1-acyl-2-LPL and 2-acyl-1-LPL, respectively. We established a quantitative lysophospholipidomics method combining hydrophilic interaction chromatography (HILIC) with the scheduled multiple reaction monitoring (sMRM) algorithm for profiling a vast number of LPLs simultaneously, including the 1-acyl-2-LPL and 2-acyl-1-LPL regioisomers. This approach facilitates baseline separation of monitored lipid classes and regioisomers, including sufficient separation of species having a different degree of unsaturation overcoming the overlapping effect of M + 2 isotopes. The lipid class-based separation improves the quantification of each molecular species as the internal standard elutes together with the endogenous species. The potential of this method is illustrated by analyzing LPLs from human plasma and skin samples. Altogether, 68 molecular lipid species, consisting of 110 regioisomers, were detected in plasma and 43 molecular lipids, consisting of 67 regioisomers, in skin samples. The novel skin LPL profile reveals that most of the lipid species exist as 2-acyl-1-LPL, in comparison to plasma where 1-acyl-2-LPLs are the dominant species. Graphical Abstract Quantitative lysophospholipidomics
Keywords: Lysophospholipids; Lysoglycerophospholipids; Hydrophilic interaction; Liquid chromatography mass spectrometry; Regioisomer; Lipidomics

Dietary uptake of omega-3 fatty acids in mouse tissue studied by time-of-flight secondary ion mass spectrometry (TOF-SIMS) by Peter Sjövall; Martin Rossmeisl; Jörg Hanrieder; Ondrej Kuda; Jan Kopecky; Morten Bryhn (5101-5111).
Dietary intake of omega-3 fatty acids is associated with considerable health benefits, including the prevention of metabolic disorders such as cardiovascular disease and type 2 diabetes. Furthermore, incorporation of the main omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), at the systemic level has been found to be more efficient when these fatty acids are supplied in the form of marine phospholipids compared to triglycerides. In this work, the uptake of omega-3 fatty acids and their incorporation in specific lipids were studied in adipose, skeletal muscle, and liver tissues of mice given high-fat diets with or without omega-3 supplements in the form of phospholipids or triglycerides using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The results demonstrate significant uptake of EPA and DHA, and the incorporation of these fatty acids in specific lipid molecules, in all three tissue types in response to the dietary omega-3 supplements. Moreover, the results indicate reduced concentrations of arachidonic acid (AA) and depletion of lipids containing AA in tissue samples from mice given supplementary omega-3, as compared to the control mice. The effect on the lipid composition, in particular the DHA uptake and AA depletion, was found to be significantly stronger when the omega-3 supplement was supplied in the form of phospholipids, as compared to triglycerides. TOF-SIMS was found to be a useful technique for screening the lipid composition and simultaneously obtaining the spatial distributions of various lipid classes on tissue surfaces. Graphical abstract Lipid-specific analysis of TOF-SIMS spectra was used to study dietary uptake of omega-3 fatty acids in mouse adipose, muscle and liver tissue. Ion images shows the lateral distribution of molecular species, herephosphocholine (red), protein fragment (green) and diacylglycerol (blue), on the tissue surfaces
Keywords: Omega-3; TOF-SIMS; Mouse tissue; Lipids; Imaging; PCA

Nutrition-dependent changes of mouse adipose tissue compositions monitored by NMR, MS, and chromatographic methods by Yulia Popkova; Andrej Meusel; Jana Breitfeld; Dorit Schleinitz; Johannes Hirrlinger; Dirk Dannenberger; Peter Kovacs; Jürgen Schiller (5113-5123).
Many diseases nowadays are assumed to be genetically determined. Therefore, many knockout mouse models have been established and are widely used. Unfortunately, nutrition (in particular the fat content of food) is often neglected in studies on these disease models. In this study the effects of nutrition on the lipid (triacylglycerol, TAG) compositions of different mouse adipose tissues were investigated. Mice were subjected to different diets [high fat (HF) vs. standard diet (SD)] and different adipose tissue samples (brown, visceral, and subcutaneous fat) were isolated after 12 weeks. Subsequent to lipid extraction, the organic extracts were analyzed by mass spectrometry (MALDI and ESI), high-resolution 1H and 31P NMR spectroscopy, high-performance thin-layer chromatography (HPTLC), and gas chromatography (GC). In adipose tissue of mice fed with HF diet, (a) decreased double bond contents and (b) decreased fatty acyl chain lengths of tissue TAGs were observed; this trend could be concomitantly monitored by all methods used. However, the adipose tissue still contained significant amounts of slightly unsaturated fatty acyl residues (18:1). Thus, a certain double bond content seems necessary to maintain the properties of adipose tissues. Graphical Abstract The compositions of different mouse adipose tissues is massively influenced by the composition of the supplied diet. This will be shown by using independent spectroscopic and chromatographic methods.
Keywords: Adipose tissue; Triacylglycerols; Phospholipids; Double bond content; MALDI MS; NMR spectroscopy; Gas chromatography

Quantitative profiling of endocannabinoids in lipoproteins by LC–MS/MS by Mesut Bilgin; Laura Bindila; Juergen Graessler; Andrej Shevchenko (5125-5131).
Endocannabinoids belong to a diverse family of endogenous lipid bioregulators acting as physiological ligands of cannabinoid receptor type 1 and cannabinoid receptor type 2 in the central and peripheral nervous system. They are also present in nmol L−1 concentrations in human blood plasma; however, their association with possible molecular carriers remains poorly characterized. Here we report on the quantification of 46 endogenous molecular species from five major classes of endocannabinoids and endocannabinoid-related compounds in three lipoprotein fractions of human blood plasma: VLDL, LDL, HDL, and in the plasma lipoprotein-free fraction. Although sizable quantities of endocannabinoid-related molecules are associated with lipoproteins, we identified the lipoprotein-free fraction as a major carrier of endocannabinoids in blood circulation with the exception of 2-acylglycerols, which are markedly abundant in VLDL.
Keywords: Endocannabinoids; Lipoproteins; Blood plasma; LC–MS/MS; Quantification

For the most effective analytical strategies, development and validation include optimization of such analytical variables as resolution, detectability, sensitivity, simplicity, cost effectiveness, flexibility, and speed. However, other aspects concerning operator safety and environmental impact are not considered at the same level. The result has been many unintended negative effects of analytical methods developed to investigate different kinds of sample, especially hydrophobic compounds that generate a large amount of chemical waste and have a strong negative environmental impact. In this context, quantification of tocopherols and tocotrienols, i.e. the vitamin E family, is usually achieved by normal-phase liquid chromatography using large volumes of toxic organic solvents, or reversed-phase liquid chromatography using a high percentage of methanol for elution. We propose here a “greener” analytical strategy, including the hyphenation of supercritical-fluid chromatography, using CO2 and ethanol as mobile phase, NH2 as stationary phase, and mass spectrometry for the detection and quantification of vitamin E congeners in soybean oil. An atmospheric-pressure photoionization (APPI) source seemed significantly more sensitive and robust than electrospray or atmospheric-pressure chemical ionization (APCI). This method led to shortened analysis time (less than 5 min) and was revealed to be as sensitive as more traditional approaches, with limits of detection and quantification in the tens of μg L−1. Graphical Abstract SFC-MS analysis of vitamin E compounds
Keywords: Tocopherol; Tocotrienol; Vitamin E; Supercritical-fluid chromatography; Mass spectrometry; Soybean oil

Among the goals of lipidomics applied to triacylglycerols (TAGs) is identification of molecular species, degree and location of unsaturation, and positions of fatty acyl chains (i.e., identification of regioisomers). Toward those ends, we define one, two, and three “Critical Ratios” for Types I, II, and III TAGs that provided different aspects of the desired information. Critical Ratio 1, [MH]+/Σ[DAG]+, is correlated to the degree of unsaturation ([MH]+ is the protonated molecule and Σ[DAG]+ is the sum of diacylglycerol-like ions, [DAG]+); Critical Ratio 2, [AA]+/[AB]+ for Type II TAGs (“ABA/AAB/BAA”) and [AC]+/([AB]++[BC]+) for Type III TAGs (“ABC/CBA/BAC/CAB/ACB/BCA”), is correlated to identification of regioisomers; and Critical Ratio 3, [BC]+/[AB]+, provides information about those [DAG]+ from Type III TAGs. Furthermore, Critical Ratios are used in the Updated Bottom Up Solution (UBUS) to reproduce the mass spectra of TAGs by atmospheric pressure chemical ionization mass spectrometry applied to analysis of soybean oil in a dietary supplement gelcap. We present a new model for the [MH]+/Σ[DAG]+ ratio, quantify regioisomers using the [AA]+/[AB]+ ratio, and describe trends for [BC]+/[AB]+ that have never been reported before. The UBUS is also applied to other classes of molecules, i.e., vitamin D and DAGs. The amount of vitamin D3 in the gelcap fell from 2011 ± 22 when received to 1689 ± 33 just prior to expiration. The Critical Ratios constitute a compact data set that can provide structural information and also act as a library of mass spectra. Graphical Abstract The shape of the Updated Bottom Up Solution for Type II triacylglycerols
Keywords: APCI-MS; Triacylglycerols; Regioisomers; Soybean oil; Lipidomics

Oxidized lipids play a significant role in the pathogenesis of numerous oxidative stress-related human disorders, such as atherosclerosis, obesity, inflammation, and autoimmune diseases. Lipid peroxidation, induced by reactive oxygen and nitrogen species, yields a high variety of modified lipids. Among them, carbonylated lipid peroxidation products (oxoLPP), formed by oxidation of the fatty acid moiety yielding aldehydes or ketones (carbonyl groups), are electrophilic compounds that are able to modify nucleophilic substrates like proteins, nucleic acid, and aminophospholipids. Some carbonylated phosphatidylcholines possess even pro-inflammatory activities. However, little is known about oxoLPP derived from other phospholipid (PL) classes. Here, we present a new analytical strategy based on the mass spectrometry (MS) of PL-oxoLPP derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). Shotgun MS revealed many oxoLPP derived from in vitro oxidized glycerophosphatidylglycerols (PG, 31), glycerophosphatidylcholine (PC, 23), glycerophosphatidylethanolamine (PE, 34), glycerophosphatidylserines (PS, 7), glycerophosphatidic acids (PA, 17), and phosphatidylinositiolphosphates (PIP, 6) vesicles. This data were used to optimize LipidXplorer-assisted identification, and a python-based post-processing script was developed to increase both throughput and accuracy. When applied to full lipid extracts from rat primary cardiomyocytes treated with peroxynitrite donor SIN-1, ten PL-bound oxoLPP were unambiguously identified by LC-MS, including two PC-, two PE-, one PG-, two PS-, and three PA-derived species. Some of the well-known carbonylated PC were detected, while most PL-oxoLPP were shown for the first time. Graphical Abstract Overview of analytical and bioinformatics approach for detection and identification of carbonylated phospholipids.
Keywords: 7-(Diethylamino)coumarin-3-carbohydrazide; Carbonylated lipids; LC-MS; Lipid peroxidation products; LipidXplorer; Shotgun lipidomics

Localization of double bonds in triacylglycerols using high-performance liquid chromatography/atmospheric pressure chemical ionization ion-trap mass spectrometry by Eva Háková; Vladimír Vrkoslav; Radka Míková; Karolina Schwarzová-Pecková; Zuzana Bosáková; Josef Cvačka (5175-5188).
A method for localizing double bonds in triacylglycerols using high-performance liquid chromatography–tandem mass spectrometry with atmospheric pressure chemical ionization (APCI) was developed. The technique was based on collision-induced dissociation or pulsed Q collision-induced dissociation of the C3H5N+• adducts ([M + 55]+•) formed in the presence of acetonitrile in the APCI source. The spectra were investigated using a large series of standards obtained from commercial sources and prepared by randomization. The fragmentation spectra made it possible to determine (i) the total number of carbons and double bonds in the molecule, (ii) the number of carbons and double bonds in acyls, (iii) the acyl in the sn-2 position on the glycerol backbone, and (iv) the double-bond positions in acyls. The double-bond positions were determined based on two types of fragments (alpha and omega ions) formed by cleavages of C–C bonds vinylic to the original double bond. The composition of the acyls and their positions on glycerol were established from the masses and intensities of the ions formed by the elimination of fatty acids from the [M + 55]+• precursor. The method was applied for the analysis of triacylglycerols in olive oil and vernix caseosa. Graphical Abstract The diagnostic fragments in the APCI PQD MS2 spectrum of the [M + 55]+• adduct of triolein
Keywords: Double bond; Gas-phase chemistry; Lipidomics; Olive oil; Vernix caseosa

Rapid evaluation of 25 key sphingolipids and phosphosphingolipids in human plasma by LC-MS/MS by Abdul Basit; Daniele Piomelli; Andrea Armirotti (5189-5198).
We report on a new, sensitive, and fast LC-MS/MS method for the simultaneous determination of 25 key sphingolipid components in human plasma, including phosphorylated sphinganine and sphingosine, in a single 9-min run. This method enables an effective and high-throughput coverage of the metabolic changes involving the sphingolipidome during physiological or pathological states. The method is based on liquid–liquid extraction followed by reversed-phase LC-MS/MS. Exogenous odd-chain lipids are used as cost-effective but reliable internal standards. The method was fully validated in surrogate matrix and naive human plasma following FDA guidelines. Sample stability and dilution integrity were also tested and verified.
Keywords: Ceramides; Sphingomyelins; Sphingosine-1-phosphate; Sphinganine-1-phosphate; LC-MS/MS; Human plasma

Multidimensional mass spectrometry-based shotgun lipidomics analysis of vinyl ether diglycerides by Kui Yang; Christopher M. Jenkins; Beverly Dilthey; Richard W. Gross (5199-5210).
Diglycerides play a central role in lipid metabolism and signaling in mammalian cells. Although diacylglycerol molecular species comprise the majority of cellular diglycerides that are commonly measured using a variety of approaches, identification of extremely low abundance vinyl ether diglycerides has remained challenging. In this work, representative molecular species from the three diglyceride subclasses (diacyl, vinyl ether, and alkyl ether diglycerides; hereafter referred to as diradylglycerols) were interrogated by mass spectrometric analysis. Product ion mass spectra of the synthesized diradylglycerols with varied chain lengths and degrees of unsaturation demonstrated diagnostic fragmentation patterns indicative of each subclass. Multidimensional mass spectrometry-based shotgun lipidomics (MDMS-SL) analysis of mouse brain and heart lipid extracts were performed using the identified informative signature product ions. Through an array of tandem mass spectrometric analyses utilizing the orthogonal characteristics of neutral loss scanning and precursor ion scanning, the differential fragmentation of each subclass was exploited for high-yield structural analyses. Although molecular ion mass spectra readily identified diacylglycerol molecular species directly from the hexane fractions of tissue extracts enriched in nonpolar lipids, molecular ion peaks corresponding to ether-linked diglycerides were not observable. The power of MDMS-SL utilizing the tandem mass spectrometric array analysis was demonstrated by identification and profiling of individual molecular species of vinyl ether diglycerides in mouse brain and heart from their undetectable molecular ion peaks during MS1 analysis. Collectively, this technology enabled the identification and profiling of previously inaccessible vinyl ether diglyceride molecular species in mammalian tissues directly from extracts of biologic tissues. Graphical Abstract ᅟ
Keywords: Plasmalogen; Vinyl ether; Vinyl ether diglycerides; Shotgun lipidomics

Determination of the triacylglycerol fraction in fish oil by comprehensive liquid chromatography techniques with the support of gas chromatography and mass spectrometry data by Marco Beccaria; Rosaria Costa; Giuseppe Sullini; Elisa Grasso; Francesco Cacciola; Paola Dugo; Luigi Mondello (5211-5225).
Fish oil made from menhaden (Brevoortia tyrannus) can be used as a dietary supplement for the presence of high levels of the long-chained omega-3 fatty acids, viz. epentaenoic and docosahexanoic. In this work, for the first time, two different multidimensional approaches were developed and compared, in terms of peak capacity, for triacylglycerol characterization. In particular, silver ion chromatography with a silver-ion column and non-aqueous reverse-phase liquid chromatography with a C18 column were tested in both comprehensive (stop-flow) and off-line modes. The use of mass spectra attained by atmospheric pressure chemical ionization for both LC approaches, and the fatty acids methyl esters profile of menhaden oil obtained by gas chromatography analysis, greatly supported the elucidation of the triacylglycerol content in menhaden oil. The off-line approach afforded a better separation and, thus, higher peak capacity to allow identifying and semiquantifying more than 250 triacylglycerols. Such a huge number has never been reported for a menhaden oil sample.The main disadvantage of such an approach over the stop-flow one was the longer analysis time, mainly attributable to solvent exchange between the two dimensions. Graphical Abstract Top: stop-flow Ag+-LC × RP-LC-APCI-MS plot of the TAGs in menhaden oil. Bottom: modulated detector response. Left: 2D analysis of a selected fraction
Keywords: Triacylglycerols; Polyunsaturated fatty acids; Comprehensive two-dimensional liquid chromatography; Fish oil; Response factors; Quantification

LC–MS/MS-based quantification of cholesterol and related metabolites in dried blood for the screening of inborn errors of sterol metabolism by S. Becker; S. Röhnike; S. Empting; D. Haas; K. Mohnike; S. Beblo; U. Mütze; R. A. Husain; J. Thiery; U. Ceglarek (5227-5233).
Smith–Lemli–Opitz syndrome (SLOS) is an inherited metabolic disease in the cholesterol biosynthesis pathway which is characterised by accumulation of 7- and 8-dehydrocholesterol and by reduced cholesterol concentrations in all tissues and body fluids. With this study, we developed a new, rapid, robust and high-throughput tandem mass spectrometric method as routine application for the selective SLOS screening and therapy monitoring in serum and dried blood. After protein precipitation of 10 μL serum or 4.7 mm dried blood spot, the sum of 7- and 8-dehydrocholesterol (DHC) was analysed by rapid chromatography combined with tandem mass spectrometry. Method comparison with GC–MS was performed for 46 serum samples. A comparison between serum and corresponding dried blood spots for DHC and cholesterol was performed with 40 samples from SLOS patients. Concentrations of DHC and cholesterol were analysed in 2 dried blood samples from newborns with SLOS and 100 unaffected newborns. Intra- and inter-assay variabilities ranged between 3.7 and 17.7 % for serum and dried blood spots. Significant correlations between the new LC–MS/MS method and GC–MS were determined for DHC (r = 0.937, p < 0.001) and for cholesterol (r = 0.946, p < 0.001). Significant coefficients of correlation between serum and dried blood spot samples above 0.8 were calculated for both analytes. A cut-off value of 5.95 for the ratio of DHC/cholesterol (multiplied by 1000) was found to distinguish newborns diagnosed with SLOS from normal newborns in a retrospective analysis after 5 years. The developed method enables a rapid quantification of the sum parameter 7- and 8-DHC in newborns and SLOS patients under therapy in serum as well as dried blood spot samples.
Keywords: 7-Dehydrocholesterol; Dried blood spots; Smith–Lemli–Opitz syndrome; Tandem mass spectrometry

Revised sample preparation for the analysis of oxysterols by enzyme-assisted derivatisation for sterol analysis (EADSA) by Peter J. Crick; T. William Bentley; Yuqin Wang; William J. Griffiths (5235-5239).
Sterols, and specifically oxysterols, play important roles in the biosynthesis of bile acids and steroid hormones as well as possessing biological activities in their own right. Analysis of oxysterols is complicated due to their low abundance in biological systems and poor ionisation characteristics in mass spectrometry. Over the past decade, we have developed a liquid chromatography–mass spectrometry method termed enzyme-assisted derivatisation for sterol analysis (EADSA). Our derivatisation procedure relies on two solid-phase extraction steps to (i) separate cholesterol from oxysterols and (ii) remove excess derivatisation reagents. Recent inter-batch variation in C18 reversed-phase cartridges has led us to experiment with alternative columns. Here, we present our findings and report an improved sample preparation procedure using polymeric hydrophilic–lipophilic balanced reversed-phase cartridges.
Keywords: Sample preparation; Solid-phase extraction; Lipidomics; Cholesterol; Oxysterol; Derivatisation; Liquid chromatography; Mass spectrometry