Journal of Chromatography B (v.964, #C)
Analysis of eicosanoids, amino acids, organic acids, and microRNAs by Dimitrios Tsikas; Alexander A. Zoerner (vii-viii).
Editorial Board (i).
Liquid chromatography–tandem mass spectrometry for the analysis of eicosanoids and related lipids in human biological matrices: A review by Linda Kortz; Juliane Dorow; Uta Ceglarek (1-11).
Today, there is an increasing number of liquid chromatography tandem-mass spectrometric (LC–MS/MS) methods for the analysis of eicosanoids and related lipids in biological matrices. An overview of currently applied LC–MS/MS methods is given with attention to sample preparation strategies, chromatographic separation including ultra high performance liquid chromatography (UHPLC) and chiral separation, as well as to mass spectrometric detection using multiple reacting monitoring (MRM). Further, the application in recent clinical research is reviewed with focus on preanalytical aspects prior to LC–MS/MS analysis as well as applications in major diseases of Western civilization including respiratory diseases, diabetes, cancer, liver diseases, atherosclerosis, and neurovascular diseases.
Keywords: Tandem mass spectrometry; Liquid chromatography; Eicosanoids; UHPLC;
Liquid chromatography–mass spectrometry measurement of leukotrienes in asthma and other respiratory diseases by Paolo Montuschi; Giuseppe Santini; Salvatore Valente; Chiara Mondino; Francesco Macagno; Paola Cattani; Gina Zini; Nadia Mores (12-25).
Leukotrienes (LTs), including cysteinyl-LTs (LTC4, LTD4 and LTE4) and LTB4, are potent inflammatory lipid mediators which have been involved in the pathophysiology of respiratory diseases. LC–MS/MS techniques for measuring LT concentrations in sputum supernatants, serum, urine and exhaled breath condensate (EBC) have been developed. In asthmatic adults, reported LTB4 and LTE4 concentrations in sputum range from 79 to 7220 pg/ml and from 11.9 to 891 pg/ml, respectively. Data on sputum LT concentrations in healthy subjects are not available. In EBC, reported LTE4 concentrations range from 38 to 126 pg/ml (95% CI) in adult asthma patients and from 34 to 48 pg/ml in healthy subjects. LTB4 concentrations in EBC range from 175 to 315 pg/ml (interquartile range) in asthmatic children, and from 25 to 245 pg/ml in healthy children. Enabling an accurate quantitative assessment of LTs in biological fluids, LC–MS/MS techniques provide a valuable tool for exploring the pathophysiological role of LTs in respiratory disease and might be useful for assessing the effects of therapeutic intervention. This review presents the analytical aspects of the LC–MS/MS techniques for measuring LT concentrations in biological fluids and discusses their potential utility for the assessment of airway inflammation and monitoring of pharmacological treatment in patients with asthma phenotypes and other respiratory diseases.
Keywords: Airway inflammation; Asthma; Biomarkers; Exhaled breath condensate; Leukotrienes; Liquid chromatography–mass spectrometry; Respiratory medicine;
Analysis, physiological and clinical significance of 12-HETE: A neglected platelet-derived 12-lipoxygenase product by Benedetta Porro; Paola Songia; Isabella Squellerio; Elena Tremoli; Viviana Cavalca (26-40).
While the importance of cyclooxygenase (COX) in platelet function has been amply elucidated, the identification of the role of 12-lipoxygenase (12-LOX) and of its stable metabolite, 12-hydroxyeicosatretraenoic acid (12-HETE), has not been clarified as yet. Many studies have analysed the implications of 12-LOX products in different pathological disorders but the information obtained from these works is controversial. Several analytical methods have been developed over the years to simultaneously detect eicosanoids, and specifically 12-HETE, in different biological matrices, essentially enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), high performance liquid chromatography (HPLC) and mass spectrometry coupled with both gas and liquid chromatography methods (GC– and LC–MS). This review is aimed at summarizing the up to now known physiological and clinical features of 12-HETE together with the analytical methods used for its determination, focusing on the critical issues regarding its measurement.
Keywords: Arachidonic acid metabolism; 12-HETE; Platelet; Analytical method; 12-LOX clinical implication;
Liquid chromatography-tandem mass spectrometry analysis of eicosanoids and related compounds in cell models by Raquel Martin-Venegas; Olga Jáuregui; Juan Jose Moreno (41-49).
Enzyme- and free radical-catalyzed oxidation of polyunsaturated fatty acids (PUFAs) produces the eicosanoids, docosanoids and octadecanoids. This large family of potent bioactive lipids is involved in many biochemical and signaling pathways which are implicated in physiological and pathophysiological processes and can be viable therapeutic targets. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers selectivity, sensitivity, robustness and high resolution and is able to analyze a large number of eicosanoids in biological samples in a short time. The present article reviews and discusses reported LC-MS/MS methods and the results obtained from their application in cell models. Reliable analytical outcomes are critically important for understanding physiological and pathophysiological cellular processes, such as inflammation, diseases with inflammatory components (e.g., cardiovascular disease, diabetes, metabolic syndrome), as well as cancer. Reported findings obtained by using the LC-MS/MS methodology in cell systems may have important predictive as well as nutritional and pharmacological implications. We conclude that the LC-MS/MS methodology is a versatile and reliable analytical tool for the simultaneous analysis of multiple PUFA-derived metabolites including the eicosanoids in cell culture samples at concentrations on the pM/nM threshold, i.e. at baseline and after stimulation.
Keywords: LC-MS/MS; Prostaglandins; Leukotrienes; Hydroxyeicosatetraenoic acids; Epoxyeicosatrienoic acids; Cell culture;
A review of analytical methods for eicosanoids in brain tissue by Michael Puppolo; Deepti Varma; Susan A. Jansen (50-64).
Eicosanoids are potent lipid mediators of inflammation and are known to play an important role in numerous pathophysiological processes. Furthermore, inflammation has been proven to be a mediator of diseases such as hypertension, atherosclerosis, Alzheimer's disease, cancer and rheumatoid arthritis. Hence, these lipid mediators have gained significant attention in recent years. This review focuses on chromatographic and mass spectrometric methods that have been used to analyze arachidonic acid and its metabolites in brain tissue. Recently published analytical methods such as LC–MS/MS and GC–MS/MS are discussed and compared in terms of limit of quantitation and sample preparation procedures, including solid phase extraction and derivatization. Analytical challenges are also highlighted.
Keywords: Eicosanoids; Brain tissue; Ultra high pressure liquid chromatography–mass spectrometry; High pressure liquid chromatography–mass spectrometry; Gas chromatography–mass spectrometry; Tandem mass spectrometry;
Non-enzymatic lipid oxidation products in biological systems: Assessment of the metabolites from polyunsaturated fatty acids by Claire Vigor; Justine Bertrand-Michel; Edith Pinot; Camille Oger; Joseph Vercauteren; Pauline Le Faouder; Jean-Marie Galano; Jetty Chung-Yung Lee; Thierry Durand (65-78).
Metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acids notably omega-3 and omega-6 fatty acids have become important biomarkers of lipid products. Especially the arachidonic acid-derived F2-isoprostanes are the classic in vivo biomarker for oxidative stress in biological systems. In recent years other isoprostanes from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic acids have been evaluated, namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively. These have been gaining interest as complementary specific biomarkers in human diseases. Refined extraction methods, robust analysis and elucidation of chemical structures have improved the sensitivity of detection in biological tissues and fluids. Previously the main reliable instrumentation for measurement was gas chromatography–mass spectrometry (GC–MS), but now the use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) and immunological techniques is gaining much attention. In this review, the types of prostanoids generated from non-enzymatic lipid peroxidation of some important omega-3 and omega-6 fatty acids and biological samples that have been determined by GC–MS and LC–MS/MS are discussed.
Keywords: Oxidative stress; Lipid peroxidation; Isoprostanes; Fatty acid; GC–MS; LC–MS/MS;
Analysis of eicosanoids by LC-MS/MS and GC-MS/MS: A historical retrospect and a discussion by Dimitrios Tsikas; Alexander A. Zoerner (79-88).
Eicosanoids are a large family that derives from arachidonic acid, i.e., eicosatetraenoic acid. Prominent members include prostaglandins, thromboxane and leukotrienes. They are biologically highly active lipid mediators and play multiple physiological roles. GC-MS/MS has played a pivotal role in the identification and quantification of eicosanoids in biological samples. This technology generated a solid knowledge of their analytical chemistry, biochemistry, physiology and pharmacology. Since about a decade, GC-MS and GC-MS/MS are increasingly displaced by the seemingly more simple, rapid and powerful LC-MS/MS in the area of instrumental analysis of physiological substances, drugs and their metabolites. In this article, we review and discuss LC-MS/MS methods published over the last decade from the perspective of the GC-MS/MS user. Our analysis revealed that the shift from the adult GC-MS/MS to the youthful emerging LC-MS/MS technology in eicosanoid analysis is associated with several important challenges. Known pitfalls and problematic issues discovered by eicosanoid pioneers by using GC-MS/MS are often ignored by LC-MS/MS users. Established reference values and intervals provided by GC-MS-based methods are not considered properly in developing and validating LC-MS/MS methods. Virtually, there is a belief in the unlimited capability of the LC-MS/MS technique in eicosanoid analysis, a thought that simulates analytical certainty. LC-MS/MS users should profit from the plethora of solid knowledge acquired from the use of GC-MS/MS in eicosanoid analysis in basic and clinical research.
Keywords: Artefactual formation; Blood; Plasma; Tandem mass spectrometry; Urine; Validation;
Mass spectrometric quantification of L-arginine and its pathway related substances in biofluids: The road to maturity by Jens Martens-Lobenhoffer; Stefanie M. Bode-Böger (89-102).
The amino acid L-arginine together with its metabolites and related substances is in the center of many biologically important pathways, especially the urea cycle and the nitric oxide (NO) synthesis. Therefore, the concentrations of these substances in various biological fluids are of great interest as predictive markers for health and disease. Yet, they provide major analytical difficulties as they are very polar in nature and therefore not easily to be separated on standard reversed phase HPLC stationary phases. Furthermore, as endogenous substances, no analyte-free matrix is available, a fact that results in complicated calibration procedures. This review evaluates the analytical literature for the determination of L-arginine, symmetric dimethylarginine, asymmetric dimethylarginine, monomethylarginine, L-citrulline, L-ornithine, L-homoarginine, agmatine and dimethylguanidinovaleric acid in biological fluids. Papers are discussed, which were published since 2007 and describe methods applying capillary electrophoresis (CE), gas chromatography (GC), reversed phase HPLC or polar phase HPLC, coupled to mass spectrometric quantification. Nowadays, many carefully developed and validated methods for L-arginine and its related substances are available to the scientific community. The use of stable isotope labeled internal standards enables high precision and accuracy in mass spectrometry-based quantitative analysis
Keywords: Review; L-arginine; Metabolites; Mass spectrometry; HPLC; GC; CE;
Analytical methods involving separation techniques for determination of low-molecular-weight biothiols in human plasma and blood by Muneki Isokawa; Takahiro Kanamori; Takashi Funatsu; Makoto Tsunoda (103-115).
Low-molecular-weight biothiols such as homocysteine, cysteine, and glutathione are metabolites of the sulfur cycle and play important roles in biological processes such as the antioxidant defense network, methionine cycle, and protein synthesis. Thiol concentrations in human plasma and blood are related to diseases such as cardiovascular disease, neurodegenerative disease, and cancer. The concentrations of homocysteine, cysteine, and glutathione in plasma samples from healthy human subjects are approximately in the range of 5–15, 200–300, and 1–5 μM, respectively. Glutathione concentration in the whole blood is in the millimolar range. Measurement of biothiol levels in plasma and blood is thought to be important for understanding the physiological roles and biomarkers for certain diseases. This review summarizes the relationship of biothiols with certain disease as well as pre-analytical treatment and analytical methods for determination of biothiols in human plasma and blood by using high-performance liquid chromatography and capillary electrophoresis coupled with ultraviolet, fluorescence, or chemiluminescence detection; or mass spectrometry.
Keywords: HPLC; Fluorescence; Chemiluminescence; Derivatization;
Analysis of amino acid composition in proteins of animal tissues and foods as pre-column o-phthaldialdehyde derivatives by HPLC with fluorescence detection by Zhaolai Dai; Zhenlong Wu; Sichao Jia; Guoyao Wu (116-127).
Studies of protein nutrition and biochemistry require reliable methods for analysis of amino acid (AA) composition in polypeptides of animal tissues and foods. Proteins are hydrolyzed by 6 M HCl (110 °C for 24 h), 4.2 M NaOH (105 °C for 20 h), or proteases. Analytical techniques that require high-performance liquid chromatography (HPLC) include pre-column derivatization with 4-chloro-7-nitrobenzofurazan, 9-fluorenyl methylchloroformate, phenylisothiocyanate, naphthalene-2,3-dicarboxaldehyde, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, and o-phthaldialdehyde (OPA). OPA reacts with primary AA (except cysteine or cystine) in the presence of 2-mercaptoethanol or 3-mercaptopropionic acid to form a highly fluorescent adduct. OPA also reacts with 4-amino-1-butanol and 4-aminobutane-1,3-diol produced from oxidation of proline and 4-hydroxyproline, respectively, in the presence of chloramine-T plus sodium borohydride at 60 °C, or with S-carboxymethyl-cysteine formed from cysteine and iodoacetic acid at 25 °C. Fluorescence of OPA derivatives is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 50 fmol for AA. This technique offers the following advantages: simple procedures for preparation of samples, reagents, and mobile-phase solutions; rapid pre-column formation of OPA-AA derivatives and their efficient separation at room temperature (e.g., 20–25 °C); high sensitivity of detection; easy automation on the HPLC apparatus; few interfering side reactions; a stable chromatography baseline for accurate integration of peak areas; and rapid regeneration of guard and analytical columns. Thus, the OPA method provides a useful tool to determine AA composition in proteins of animal tissues (e.g., skeletal muscle, liver, intestine, placenta, brain, and body homogenates) and foods (e.g., milk, corn grain, meat, and soybean meal).
Keywords: Amino acids; HPLC; Hydrolysates; OPA (o-phthaldialdehyde); Proteins;
Chromatographic techniques coupled with mass spectrometry for the determination of organic acids in the study of autism by Joanna Kałużna-Czaplińska; Ewa Żurawicz; Jagoda Jóźwik (128-135).
Chromatographic methods find application in the diagnostics and prognosis of diseases. They are used in finding new biomarkers, which may result in early medical intervention. Early diagnosis and intervention are especially important in the case of diseases of unknown etiology. One of these is autism. Autism is a neurodevelopmental disorder characterized by severe impairment in reciprocal social interaction and communication and a pattern of repetitive or stereotyped behavior. Organic acids are intermediate metabolites of all major groups of organic cellular components and can play a role in the pathogenesis of autism. This review presents information about abnormal levels of some organic acids observed in the urine of children with autism and determination of acids with the use of chromatographic techniques. 342 literature sources on frequency (2005–2012) of the use of chromatographic methods in the determination of organic compounds in various body fluids were searched.
Keywords: Chromatographic methods; Mass spectrometry; Organic acids; Autism;
A survey of liquid chromatographic–mass spectrometric analysis of mercapturic acid biomarkers in occupational and environmental exposure monitoring by Patricia I. Mathias; Clayton B’Hymer (136-145).
High-performance liquid chromatography/mass spectrometry (HPLC/MS) is sensitive and specific for targeted quantitative analysis and is readily utilized for small molecules from biological matrices. This brief review describes recent selected HPLC/MS methods for the determination of urinary mercapturic acids (mercapturates) which are useful as biomarkers in characterizing human exposure to electrophilic industrial chemicals in occupational and environmental studies. Electrophilic compounds owing to their reactivity are used in chemical and industrial processes. They are present in industrial emissions, are combustion products of fossil fuels, and are components in tobacco smoke. Their presence in both the industrial and general environments are of concern for human and environmental health. Urinary mercapturates which are the products of metabolic detoxification of reactive chemicals provide a non-invasive tool to investigate human exposure to electrophilic toxicants. Selected recent mercapturate quantification methods are summarized and specific cases are presented. The biological formation of mercapturates is introduced and their use as biomarkers of metabolic processing of electrophilic compounds is discussed. Also, the use of liquid chromatography/tandem mass spectrometry in simultaneous determinations of the mercapturates of multiple parent compounds in a single determination is considered, as well as future trends and limitations in this area of research.
Keywords: Liquid chromatography; Mass spectrometry; Occupational exposure; Industrial chemicals; Urinary biomarker; Mercapturic acid;
Analytical approaches in microRNA therapeutics by Sandor Batkai; Thomas Thum (146-152).
MicroRNAs are non-coding oligonucleotides with regulatory roles in virtually all biological processes. Deregulation of microRNAs lead to impaired cellular function and disease development. Thus, microRNAs are of potential diagnostic and therapeutic relevance. Several technology platforms are currently available for quantitative microRNA analysis and profiling, including the most extensively used PCR-based methods. Each of these technologies has its own advantages and limitations. Mass spectrometry combines low-level detectability with high selectivity and has been used for oligonucleotide sequence analysis. Its use for native microRNA analysis has been limited due to the very low abundance and chemical similarity of microRNAs. However, with the advancement of technology, this analytical method has become a powerful complementary tool for comprehensive analysis of native and synthetic microRNAs. This brief review highlights current developments in the field of microRNA analytics, detection techniques for extracellular microRNAs, their synthetic inhibitors, and the dynamics of their interactions.
Keywords: MicroRNA; Oligonucleotide; Purification; Functional assay; Biomarker; Liquid chromatography–mass spectrometry;
UHPLC–MS/MS analysis of arachidonic acid and 10 of its major cytochrome P450 metabolites as free acids in rat livers: Effects of hepatic ischemia by Vindhya Edpuganti; Reza Mehvar (153-163).
The cytochrome P450 metabolites of arachidonic acid (AA) are mostly present in tissues, such as the liver, as bound to phospholipids, with only a small fraction available as free acids. The purpose of this study was to develop and validate a UHPLC–MS/MS method for quantitation of free liver concentrations of AA and four epoxygenated (5,6-, 8,9-, 11,12-, and 14,15-EET), four dihydroxylated (5,6-, 8,9-, 11,12-, and 14,15-DHET), and two ω/(ω − 1) hydroxylated (19- and 20-HETEs) metabolites of AA in rat livers using deuterated internal standards. The analytes were rapidly and efficiently (79–92%) recovered from 100 mg of fresh liver into methanol. After evaporation, the reconstituted samples were injected either undiluted (for the simultaneous analysis of the metabolites) into a gradient or diluted (for AA analysis) into an isocratic UHPLC system with run times of 5 and 2 min, respectively. Mass spectrometry was conducted using multiple reaction monitoring in negative mode. The method was linear (r 2 ≥ 0.98) in the concentration ranges tested for metabolites (0.19–120 ng/g liver) and AA (7.8–500 μg/g liver). The lower limit of quantitation of the assay was between 0.57 and 5.6 pg injected on column for different AA metabolites. The assay was validated (n = 5) based on acceptable intra- and inter-run accuracy and precision values. Additionally, matrix effect was minimal for most analytes. Freeze-thaw of samples drastically increased the free liver concentrations of analytes, presumably due to their release from the membrane storage sites. Therefore, fresh liver samples should be used for analysis. However, the methanolic extracts may be stored at −80 °C for at least two weeks without any compromise. The method was successfully used in the measurement of all the analytes in the rats subjected to 60 min of hepatic ischemia (n = 6) or sham operation (n = 6). Ischemia resulted in significantly higher free concentrations of AA and most of its studied metabolites. The method is precise, accurate, and sensitive for measurement of free liver concentrations of AA and its P450 metabolites in the rat liver.
Keywords: Arachidonic acid; Cytochrome P450 metabolites; UHPLC–MS/MS; Liver; Ischemia;
The impact of exogenous ω-6 and ω-3 polyunsaturated fatty acids on the induced production of pro- and anti-inflammatory prostaglandins and leukotrienes in Atlantic salmon head kidney cells using a full factorial design and LC–MS/MS by Pedro Araujo; Eva Lucena; Yang Yang; Bupesh Ceemala; Zebasil Mengesha; Elisabeth Holen (164-171).
The production of prostaglandins (PGE2, PGE3) and leukotrienes (LTB4, LTB5) in salmon head kidney cell cultures, exposed to different combinations of 20:4ω-6, 20:5ω-3 and 22:6ω-3 polyunsaturated fatty acids (PUFAs), was evaluated by means of a two level factorial design and LC–MS/MS. The method was selective for the pro- and anti-inflammatory analytes and their corresponding stable-isotope labelled internal standards. The regression models were linear over the concentration range 0.5–150 ng/ml with limits of detection of 0.25 ng/ml and quantification of 0.40 ng/ml for the analysed metabolites. The recovery ranged from 78 to 107% for prostaglandins and 73 to 115% for leukotrienes. The analysis of the samples exposed to different combinations of PUFAs revealed that the presence of single ω-3 PUFAs brought an enhancement of the metabolites from the lipooxygenase pathway, specially LTB4, and a reduction of the metabolites from the cyclooxygenase pathway (PGE2 and PGE3), while the two-term interactions generated the opposite effect (high concentration of prostaglandins and low concentrations of leukotrienes). To our knowledge, this is the first implementation of a fully crossed design for investigating the impact of ω-6 and ω-3 PUFAs on the production of eicosanoids not only through their individual but also through their combined effects on Atlantic salmon head kidney cells.
Keywords: Eicosanoids; Prostaglandins; Leukotrienes; Liquid Chromatography Tandem Mass Spectrometry; Polyunsaturated fatty acids; Experimental design; Salmon head kidney cell cultures;
GC–MS/MS and LC–MS/MS studies on unlabelled and deuterium-labelled oleic acid (C18:1) reactions with peroxynitrite (O＝N―O―O−) in buffer and hemolysate support the pM/nM-range of nitro-oleic acids in human plasma by Arne Trettin; Anke Böhmer; Alexander A. Zoerner; Frank-Mathias Gutzki; Jens Jordan; Dimitrios Tsikas (172-179).
Oleic acid (cis-9,10-octadecenoic acid) is the most abundant monounsaturated fatty acid in human blood. Peroxynitrite (ONOO−) is a short-lived species formed from the reaction of nitric oxide (•NO) and superoxide (O2 • −). Peroxynitrite is a potent oxidizing and moderate nitrating agent. We investigated reactions of unlabelled and deuterium labelled oleic acid in phosphate buffered saline (PBS) and lysed human erythrocytes with commercially available sodium peroxynitrite (Na+ONOO−). Non-derivatized reaction products were analyzed by spectrophotometry, HPLC with UV absorbance detection, and LC–MS/MS electrospray ionization in the negative-ion mode. Reaction products were also analyzed by GC–MS/MS in the electron capture negative-ion chemical ionization mode after derivatization first with pentafluorobenzyl (PFB) bromide and then with N,O-bis(trimethylsilyl)trifluoroacetamide. Identified oleic acid reaction products in PBS and hemolysate include cis-9,10-epoxystearic acid and trans-9,10-epoxystearic acid (about 0.1% with respect to oleic acid), threo- and erythro-9,10-dihydroxy-stearic acids. Vinyl nitro-oleic acids, 9-nitro-oleic acid (9-NO2OA) and 10-nitro-oleic acid (10-NO2OA), or other nitro-oleic acids were not found to be formed from the reaction of oleic acid with peroxynitrite in PBS or hemolysate. Our in vitro study suggests that peroxynitrite oxidizes but does not nitrate oleic acid in biological samples. Unlike thiols and tyrosine, oleic acid is not susceptible to peroxynitrite. GC–MS/MS analysis of PFB esters is by far more efficient than LC–MS/MS analysis of non-derivatized oleic acid and its derivates. Our in vitro results support our previous in vivo findings that nitro-oleic acid plasma concentrations of healthy and diseased subjects are in the pM/nM-range.
Keywords: Epoxidation; Erythrocytes; Nitration; Oleic acid; Peroxynitrite; Tandem mass spectrometry;
High-throughput quantitation of amino acids in rat and mouse biological matrices using stable isotope labeling and UPLC–MS/MS analysis by Edward Takach; Thomas O'Shea; Hanlan Liu (180-190).
Quantifying amino acids in biological matrices is typically performed using liquid chromatography (LC) coupled with fluorescent detection (FLD), requiring both derivatization and complete baseline separation of all amino acids. Due to its high specificity and sensitivity, the use of UPLC–MS/MS eliminates the derivatization step and allows for overlapping amino acid retention times thereby shortening the analysis time. Furthermore, combining UPLC–MS/MS with stable isotope labeling (e.g., isobaric tag for relative and absolute quantitation, i.e., iTRAQ) of amino acids enables quantitation while maintaining sensitivity, selectivity and speed of analysis. In this study, we report combining UPLC–MS/MS analysis with iTRAQ labeling of amino acids resulting in the elution and quantitation of 44 amino acids within 5 min demonstrating the speed and convenience of this assay over established approaches. This chromatographic analysis time represented a 5-fold improvement over the conventional HPLC–MS/MS method developed in our laboratory. In addition, the UPLC–MS/MS method demonstrated improvements in both specificity and sensitivity without loss of precision. In comparing UPLC–MS/MS and HPLC–MS/MS results of 32 detected amino acids, only 2 amino acids exhibited imprecision (RSD) >15% using UPLC–MS/MS, while 9 amino acids exhibited RSD >15% using HPLC–MS/MS. Evaluating intra- and inter-assay precision over 3 days, the quantitation range for 32 detected amino acids in rat plasma was 0.90–497 μM, with overall mean intra-day precision of less than 15% and mean inter-day precision of 12%. This UPLC–MS/MS assay was successfully implemented for the quantitative analysis of amino acids in rat and mouse plasma, along with mouse urine and tissue samples, resulting in the following concentration ranges: 0.98–431 μM in mouse plasma for 32 detected amino acids; 0.62–443 μM in rat plasma for 32 detected amino acids; 0.44–8590 μM in mouse liver for 33 detected amino acids; 0.61–1241 μM in mouse kidney for 37 detected amino acids; and 1.39–1681 μM in rat urine for 34 detected amino acids. The utility of the assay was further demonstrated by measuring and comparing plasma amino acid levels between pre-diabetic Zucker diabetic fatty rats (ZDF/Gmi fa/fa) and their lean littermates (ZDF/Gmi fa/?). Significant differences (P < 0.001) in 9 amino acid concentrations were observed, with the majority ranging from a 2- to 5-fold increase in pre-diabetic ZDF rats on comparison with ZDF lean rats, consistent with previous literature reports.
Keywords: Amino acids; Quantitation; UPLC; MS/MS; iTRAQ; Isotope labeling;
Micro-method for the determination of glutathione in human blood by Daniela Giustarini; Paolo Fanti; Elena Matteucci; Ranieri Rossi (191-194).
A new procedure is described for the visible-range spectrophotometric analysis of glutathione (GSH) in microvolumes of blood (as low as 0.5 μL) collected by fingerstick. Samples are diluted 1 to 300 (v/v) in a stabilizing solution, followed by determination of haemoglobin concentration and by acid deproteination. GSH is then measured in the clear supernatant by colorimetry using DTNB, i.e., 5,5′-dithio-bis(2-nitrobenzoic acid), in aqueous solution at pH 7.8. The DTNB reagent is prepared and kept at pH 6.2 until just prior its addition, thus avoiding spontaneous decomposition of the reagent. The assay is rapid, easy to adapt to large-scale studies and it avoids artefactual oxidation of GSH, a common methodological shortcoming. The method is precise with 1.7 to 3.4% intra-day relative standard deviation (RSD) and 2.2 to 4.2% inter-day RSD, and accurate with −1.4% to 2.3% intra-day relative error (RE) and −2.8% to 1.6% inter-day RE. GSH is recovered by 97.5 to 100% at all tested concentrations. The new colorimetric micro-method was validated by a reliable previously reported HPLC method. The procedure is suitable for minimally invasive investigation of oxidative stress in peripheral blood.
Keywords: Blood; DTNB; Fingerstick; Glutathione; Oxidative stress;
GC-MS analysis of organic acids in human urine in clinical settings: A study of derivatization and other analytical parameters by Chrysoula Christou; Helen. G. Gika; Nikolaos Raikos; Georgios Theodoridis (195-201).
In the current paper the analytical conditions for the determination of ten free organic acids by GC-MS are studied with the aim to establish a method for organic acid profiling in human urine to be used as a tool for the detection of metabolic or other health disorders. Studies included the GC-MS method development, the derivatization (trimethylsilylation) reaction conditions, the stability of the standard solutions during storage in the freezer, and the stability of the formed trimethylsilyl derivatives. Best results were obtained at a derivatization temperature of 50 °C, and a reaction time of 30 min. Standard solutions were stable for 22 days, derivatized samples were stable at least for 30 h when stored at −24 °C. GC-MS analysis achieved sensitive determination of the organic acids under study with limits of detection ranging from 0.03 mmol/mol creatinine for glutaric acid, to 0.34 mmol/mol creatinine for glycolic acid. Within-day and day-to-day assay imprecision was found satisfactory with relative standard deviations being below 10%. The developed method was successfully applied to the quantitative analysis of free organic acids in urine samples obtained from hospitalized children. Creatinine-corrected excretion rates of all analyzed organic acids were within reference intervals.
Keywords: Organic acids analysis; GC-MS; Inborn errors of metabolism; Acidurias;
A sensitive LC–MS/MS method for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells by Xiaowei Tu; Youming Lu; Dafang Zhong; Yifan Zhang; Xiaoyan Chen (202-207).
Clofarabine triphosphate is an intracellular active metabolite of clofarabine. In the present study, we developed and validated a rapid, sensitive, and selective liquid chromatography–tandem mass spectrometry method (LC–MS/MS) for quantifying clofarabine triphosphate concentrations in human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood using the Ficoll gradient centrifugation method. Chromatographic separation was performed on a CN column using an isocratic mobile phase comprising acetonitrile/5 mM ammonium acetate with 0.001% ammonium hydroxide (20/80, v/v) at a flow rate of 0.60 mL/min. Detection was carried out by MS/MS in the multiple reaction monitoring mode using a negative electrospray ionization interface. The method was validated in concentration ranges of 1.25–100 ng/107 cells with acceptable accuracy and precision using 50 μL of cell extract. Clofarabine triphosphate was stable in a series of stability studies with bench-top, auto-sampler, and repeated freeze–thaw cycles. The validated method was successfully used to measure the concentrations of clofarabine triphosphate in PBMCs from cancer patients treated with clofarabine
Keywords: Clofarabine; Clofarabine triphosphate; Liquid chromatography–tandem mass spectrometry; Peripheral blood mononuclear cells;
Measurement of 2′,3′-cyclic nucleotides by liquid chromatography–tandem mass spectrometry in cells by Heike Bähre; Volkhard Kaever (208-211).
Recently, the occurrence of 2′,3′-cyclic nucleoside monophosphates (2′,3′-cNMPs) in addition to 3′,5′-cNMPs in mammalian tissues was reported. We developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the measurement of four 2′,3′-cyclic nucleotides, i.e., 2′,3′-cAMP, 2′,3′-cCMP, 2′,3′-cGMP, 2′,3′-cUMP, in cell samples. Chromatographic separation was achieved using a Zorbax eclipse XCB-C18 (50 mm × 4.6 mm; 1.8 μm column; Agilent) connected to a QTRAP5500 system (AB Sciex) operating in positive ionization mode. Calibration curves were constructed in the range 0.41 fmol/μL to 1666.6 fmol/μL for 2′,3′-cAMP, 2′,3′-cCMP, and 2′,3′-cGMP, and 3.3–1666.6 fmol/μL for 2′,3′-cUMP, respectively, showing squared correlation coefficients >0.9992. Accuracy and inter- and intra-day precision lay within the required ranges of <20% for LLOQ and <15% for higher concentration levels. The method was applied to the analysis of nucleotides in two different cell lines (Hek293T and HuT-78).
Keywords: Cells; Liquid chromatography–tandem mass spectrometry; 2′,3′-Cyclic nucleotide monophosphates;
Quantification of vorinostat and its main metabolites in plasma and intracellular vorinostat in PBMCs by liquid chromatography coupled to tandem mass spectrometry and its relation to histone deacetylase activity in human blood by Lu Liu; Jan-Christoph Detering; Till Milde; Walter Emil Haefeli; Olaf Witt; Jürgen Burhenne (212-221).
Vorinostat (suberoylanilide hydroxamic acid) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. Intracellular access of vorinostat is essential to exert its epigenetic effects. Therefore, we studied the relationship between vorinostat extracellular (plasma) and intracellular (peripheral blood mononuclear cells, PBMCs) concentration and assessed its concentration-effect relationship by HDAC activity testing. Assays were developed and validated for the low nanomolar quantification of vorinostat and two inactive metabolites in human plasma and PBMCs. For the vorinostat extraction from plasma and PBMCs solid-phase extraction and liquid–liquid extraction methods were applied. Extraction recoveries ranged from 88.6% to 114.4% for all analytes and extraction methods. Extracts were chromatographed on a Phenomenex Luna column isocratically (plasma) or by gradient (PBMCs) consisting of acidic ammonium acetate, acetonitrile, and methanol. The analytes were quantified using deuterated internal standards and positive electrospray tandem mass spectrometry (multiple reaction monitoring) with lower limits of quantification of 11.0 ng/mL (plasma) and 0.1 ng/3 × 106 cells (PBMCs). The calibrated ranges were linear for vorinostat in plasma 11.0–1100 (11,000) ng/mL (metabolites) and PBMCs 0.1–10.0 ng/3 × 106 cells with correlation coefficients >0.99, an overall accuracy varying between −6.7% and +3.8% in plasma, −8.1% and −1.5% in PBMCs, and an overall precision ranging from 3.2% to 6.1% in plasma and 0.8% to 4.0% in PBMCs (SD batch-to-batch). The application to blood samples from healthy volunteers incubated with vorinostat revealed accumulation of vorinostat in PBMCs, effective intracellular HDAC inhibition at therapeutic vorinostat concentrations and a direct vorinostat concentration dependency to HDAC inhibition.
Keywords: Vorinostat; Tandem mass spectrometry; Plasma; Peripheral blood mononuclear cells; Histone deacetylase activity;