Liquid Chromatography-Tandem Mass Spectrometry for Analysis of Intestinal Permeability of Loperamide in Physiological Buffer

Analysis of in vitro samples with high salt concentrations represents a major challenge for fast and specific quantification with liquid chromatography-tandem mass spectrometry (LC-MS/MS). To investigate the intestinal permeability of opioids in vitro employing the Ussing chamber technique, we developed and validated a fast, sensitive and selective method based on LC–MS/MS for the determination of loperamide in HEPES-buffered Ringer''s solution. Chromatographic separation was achieved with an Atlantis dC18 column, 2.1 mm×20 mm, 3 µm particle size and a gradient consisting of methanol/0.1% formic acid and ammonium acetate. The flow rate was 0.7 ml/min, and the total run time was 3 min. For quantification, two mass transitions for loperamide and a deuterated internal standard (methadone-d₃) were used. The lower limit of loperamide quantification was 0.2 ng/ml. This new LC-MS/MS method can be used for the detection of loperamide in any experimental setup using HEPES-buffered Ringer''s solution as a matrix compound.     

Simultaneous determination of tyrosine, phenylalanine and deoxyguanosine oxidation products by liquid chromatography-tandem mass spectrometry as non-invasive biomarkers for oxidative damage

We developed an isotope dilution HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry (HPLC-APCI-MS/MS) method for the simultaneous determination of p-tyrosine, phenylalanine, o,o'-dityrosine, m-tyrosine, o-tyrosine, 3-chlorotyrosine and 3-nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG) that requires no extensive sample pre-treatment. p-[(2)H(4)]Tyrosine and o,o'-[(2)H(6)]dityrosine were used as internal standards. Calibration curves of the method were linear (r(2)=0.990-0.999) over a concentration range of 0.03-10 microM for o-tyrosine; 0.04-10 microM for 3-nitrotyrosine and 3-chlorotyrosine; 0.05-10 microM for o,o'-dityrosine; and for m-tyrosine; 1.0-100 microM for p-tyrosine and for phenylalanine; and 0.01-10 microM for 8-OHdG. The detection limits were from 0.025 to 0.05 microM for the tyrosine derivatives; 0.01 microM for 8-OHdG; and 0.5 microM for p-tyrosine and for phenylalanine, respectively. Within-day coefficients of variation (CV) for spiked human urine samples ranged from 2.7 to 7.0%, except for 8-OHdG (13.7%). Between-day variations ranged from 7.9 to 13.0%, except for o-tyrosine (CV = 18.2%), and for 8-OHdG (CV = 24.7%).The background levels of p-tyrosine, phenylalanine, o,o'-dityrosine, and o-tyrosine in morning urine of eight healthy volunteers were 3890+/-590, 3420+/-730, 5.8+/-0.3, and 9.2+/-1.5 micromol/mol creatinine, respectively. Using the present HPLC-APCI-MS/MS method, the urinary background levels of m-tyrosine, 3-chlorotyrosine, 3-nitrotyrosine and 8-OHdG were below the limit of detection.     

Plasma 3-nitrotyrosine and outcome in neonates with severe bronchopulmonary dysplasia after inhaled nitric oxide

Plasma protein levels of 3-nitrotyrosine and 3-chlorotyrosine were measured by LC-MS/MS at 0 and 72 h after the initiation of inhaled nitric oxide (INO) at 20 ppm in 22 prematurely born infants with clinically documented bronchopulmonary dysplasia. Infants were classified at the time of hospital discharge as either "off mechanical ventilation," "on mechanical ventilation," or "expired/organ failure." These outcomes were tested for association with changes in plasma levels of 3-nitrotyrosine and 3-chlorotyrosine and selected clinical risk factors. Infants whose 3-nitrotyrosine levels decreased over the 72 h period were more likely to wean off of mechanical ventilation (p =.03). There was no significant association between changes in 3-chlorotyrosne levels and outcome. After controlling for other variables, an odds ratio of 8.3 (95% CI: 1.3-54.4) for improved outcomes was observed if the 3-nitrotyrosine levels decreased. These data suggest that nitrative and oxidative stress may be related to the severity of lung disease and, consequentially, the overall outcome in this select group of infants with severe bronchopulmonary dysplasia.     

Antibiotic Toxicity and Absorption in Zebrafish Using Liquid Chromatography-Tandem Mass Spectrometry

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10–1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD₅₀ values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.     

Determination of 3-nitrotyrosine in human urine at the basal state by gas chromatography-tandem mass spectrometry and evaluation of the excretion after oral intake

3-Nitrotyrosine (NO(2)Tyr) is a potential biomarker of reactive-nitrogen species (RNS) including peroxynitrite. 3-Nitrotyrosine occurs in human plasma in its free and protein-associated forms and is excreted in the urine. Measurement of 3-nitrotyrosine in human plasma is invasive and associated with numerous methodological problems. Recently, we have described an accurate method based on gas chromatography (GC)-tandem mass spectrometry (MS) for circulating 3-nitrotyrosine. The present article describes the extension of this method to urinary 3-nitrotyrosine. The method involves separation of urinary 3-nitrotyrosine from nitrite, nitrate and l-tyrosine by HPLC, preparation of the n-propyl-pentafluoropropionyltrimethylsilyl ether derivatives of endogenous 3-nitrotyrosine and the internal standard 3-nitro-l-[(2)H(3)]tyrosine, and GC-tandem MS quantification in the selected-reaction monitoring mode under negative-ion chemical ionization conditions. In urine of ten apparently healthy volunteers (years of age, 36.5+/-7.2) 3-nitrotyrosine levels were determined to be 8.4+/-10.4 nM (range, 1.6-33.2 nM) or 0.46+/-0.49 nmol/mmol creatinine (range, 0.05-1.30 nmol/mmol creatinine). The present GC-tandem MS method provides accurate values of 3-nitrotyrosine in human urine at the basal state. After oral intake of 3-nitro-l-tyrosine by a healthy volunteer (27.6 microg/kg body weight) 3-nitro-l-tyrosine appeared rapidly in the urine and was excreted following a biphasic pharmacokinetic profile. Approximately one third of administered 3-nitro-l-tyrosine was excreted within the first 8 h. The suitability of the non-invasive measurement of urinary 3-nitrotyrosine as a method of assessment of oxidative stress in humans remains to be established.     

Enhancing Proteomic Throughput in Capillary Electrophoresis–Mass Spectrometry by Sequential Sample Injection

In this study we demonstrate the potential of sequential injection of samples in capillary electrophoresis–mass spectrometry for rapid and sensitive proteome characterization of human lymphoblastic T‐cells (line CCRF–CEM). Proteins were extracted, enzymatically digested, and the resulting peptides fractionated by RP–HPLC. Twenty fractions were thereafter analyzed by CE–MS within a single MS analysis. The CE–MS method was designed so that every 10 min a new fraction was injected into the CE system. Without any rinsing or equilibration steps we were able to generate a continuous stream of peptides feeding the mass analyzer. In 250 min, the total analysis time of a single sequential injection experiment, we were able to identify roughly 28 000 peptide sequences counting for 4800 proteins. These numbers could be increased to 62 000 peptides and more than 6100 proteins identified, when performing three experiments analyzing a total of 60 fractions, all within 12.5 h. We found that the electrophoretic mobility of peptides can be used to trace back peptides and assign them to the fraction they originate from.     

A Comparative Study on Structural Elucidation of Sialyl Oligosaccharides by Mass Spectrometry with Fast Atom Bombardment,Electrospray Ionization,and Matrix Assisted Laser Desorption/Ionization

To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS specta being measurable at as low as 200 pmol. Thus, this method consitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.     

Principles and Applications of Liquid Chromatography-Mass Spectrometry in Clinical Biochemistry

Liquid chromatography-mass spectrometry (LC-MS) is now a routine technique with the development of electrospray ionisation (ESI) providing a simple and robust interface. It can be applied to a wide range of biological molecules and the use of tandem MS and stable isotope internal standards allows highly sensitive and accurate assays to be developed although some method optimisation is required to minimise ion suppression effects. Fast scanning speeds allow a high degree of multiplexing and many compounds can be measured in a single analytical run. With the development of more affordable and reliable instruments, LC-MS is starting to play an important role in several areas of clinical biochemistry and compete with conventional liquid chromatography and other techniques such as immunoassay.     

Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein–ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.     

基于质谱的磷酸化蛋白质组学: 富集、检测、鉴定和定量

磷酸化是一种调控生命活动的重要翻译后修饰,调控生物的生长发育、信号转导、以及疾病的发生发展．从上世纪80年代开始,质谱应用于蛋白质磷酸化的检测中,极大地推动了磷酸化蛋白质组学的发展．质谱检测拥有高灵敏度、高通量的特点,更重要的是具有位点分辨率,因此基于质谱的磷酸化蛋白质组检测方法得到不断的发展和推广．常见的磷酸化蛋白质组研究,首先对磷酸化肽段进行富集,然后进行串联质谱分析,最后通过搜索引擎对修饰位点进行鉴定和定量．本文从这个三个基本方面,对磷酸化蛋白质组研究进行综述,并对未来研究发展方向进行讨论．     

Detection and Pharmacokinetics of Alginate Oligosaccharides in Mouse Plasma and Urine after Oral Administration by a Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS) Method

A sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for the detection of alginate oligosaccharides (AOs) in mouse plasma and urine after oral administration. In an AO mixture, dimer, trimer, and tetramer were detected by LC-MS/MS equipped with an anion-exchange column with extremely high sensitivity. By this method, we detected certain levels of AOs in samples prepared from mouse plasma and urine after a single oral administration of the AO mixture. Based on a calibration curve made with an AO trimer peak area as a standard, the maximum plasma and urine concentrations of AOs were estimated to be 24.5 μg/ml at 5 min and 425.5 μg/ml at 30 min, respectively. These results suggest that the LC-MS/MS method is well suited to pharmacokinetic analysis of AOs in an in vivo system, and that some of orally administered AOs, at least from dimer to tetramer, are absorbed by digestive organs promptly, and that unaltered, these oligomers were excreted into an urine after a single oral administration to a mouse.     

Human atherosclerotic intima and blood of patients with established coronary artery disease contain high density lipoprotein damaged by reactive nitrogen species

High density lipoprotein (HDL) is the major carrier of lipid hydroperoxides in plasma, but it is not yet established whether HDL proteins are damaged by reactive nitrogen species in the circulation or artery wall. One pathway that generates such species involves myeloperoxidase (MPO), a major constituent of artery wall macrophages. Another pathway involves peroxynitrite, a potent oxidant generated in the reaction of nitric oxide with superoxide. Both MPO and peroxynitrite produce 3-nitrotyrosine in vitro. To investigate the involvement of reactive nitrogen species in atherogenesis, we quantified 3-nitrotyrosine levels in HDL in vivo. The mean level of 3-nitrotyrosine in HDL isolated from human aortic atherosclerotic intima was 6-fold higher (619 +/- 178 micromol/mol Tyr) than that in circulating HDL (104 +/- 11 micromol/mol Tyr; p < 0.01). Immunohistochemical studies demonstrated striking colocalization of MPO with epitopes reactive with an antibody to 3-nitrotyrosine. However, there was no significant correlation between the levels of 3-chlorotyrosine, a specific product of MPO, and those of 3-nitrotyrosine in lesion HDL. We also detected 3-nitrotyrosine in circulating HDL, and linear regression analysis demonstrated a strong correlation between the levels of 3-chlorotyrosine and levels of 3-nitrotyrosine. These observations suggest that MPO promotes the formation of 3-chlorotyrosine and 3-nitrotyrosine in circulating HDL but that other pathways also produce 3-nitrotyrosine in atherosclerotic tissue. Levels of HDL isolated from plasma of patients with established coronary artery disease contained twice as much 3-nitrotyrosine as HDL from plasma of healthy subjects, suggesting that nitrated HDL might be a marker for clinically significant vascular disease. The detection of 3-nitrotyrosine in HDL raises the possibility that reactive nitrogen species derived from nitric oxide might promote atherogenesis. Thus, nitrated HDL might represent a previously unsuspected link between nitrosative stress, atherosclerosis, and inflammation.     

Global proteome discovery using an online three-dimensional LC-MS/MS

We have developed a proteomics technology featuring on-line three-dimensional liquid chromatography coupled to tandem mass spectrometry (3D LC-MS/MS). Using 3D LC-MS/MS, the yeast-soluble, urea-solubilized peripheral membrane and SDS-solubilized membrane protein samples collectively yielded 3019 unique yeast protein identifications with an average of 5.5 peptides per protein from the 6300-gene Saccharomyces Genome Database searched with SEQUEST. A single run of the urea-solubilized sample yielded 2255 unique protein identifications, suggesting high peak capacity and resolving power of 3D LC-MS/MS. After precipitation of SDS from the digested membrane protein sample, 3D LC-MS/MS allowed the analysis of membrane proteins. Among 1221 proteins containing two or more predicted transmembrane domains, 495 such proteins were identified. The improved yeast proteome data allowed the mapping of many metabolic pathways and functional categories. The 3D LC-MS/MS technology provides a suitable tool for global proteome discovery.     

Myeloperoxidase-catalyzed 3-chlorotyrosine formation in dialysis patients.

Oxidative stress has been implicated in the cardiovascular complications that affect chronic renal failure patients on hemodialysis, though the physiologically relevant pathways mediating oxidative damage are poorly understood. It is known, however, that hemodialysis activates neutrophils, a well-characterized source of hydrogen peroxide and myeloperoxidase. The phagocyte-derived myeloperoxidase-hydrogen peroxide-chloride system generates hypochlorous acid, which reacts with tyrosine residues of proteins to form 3-chlorotyrosine. To explore the role of activated phagocytes in oxidative stress in chronic renal failure, we used 3-chlorotyrosine as a specific marker of myeloperoxidase activity. Utilizing isotope dilution gas chromatography-mass spectrometry, we compared 3-chlorotyrosine levels in plasma proteins of five patients on chronic hemodialysis therapy with those of age- and sex-matched healthy controls. The oxidized amino acid was present in the plasma proteins of 4 of the hemodialysis patients (3.5 +/- 0.8 micromol per mol tyrosine) but was undetectable in the healthy subjects. Therefore, one pathway for oxidative stress in hemodialysis patients appears to involve hypochlorous acid generated by the myeloperoxidase system of activated phagocytes. We also examined intradialytic 3-chlorotyrosine levels using membranes that activate white blood cells and the alternative pathway of complement. Hemodialysis increased plasma myeloperoxidase and the expression of CD11b/CD18 by circulating phagocytes, but failed to demonstrably increase 3-chlorotyrosine levels. 3-chlorotyrosine was detectable in 12 of 19 samples in total, with significant intrasubject variability. Our observations suggest that oxidants generated by myeloperoxidase contribute to the increased oxidative stress observed in renal-failure patients but do not damage plasma proteins during the hemodialysis procedure itself.     

Quantitative Analysis of Cytokinins in Plants by High Performance Liquid Chromatography: Electronspray Ionization Ion Trap Mass Spectrometry

The present paper introduces a highly sensitive and selective method for simultaneous quantification of 12 cytokinins(free form and their conjugates).The method includes a protocol of extraction with methanol/water/formic acid(1514/1,v/v/v)to the micro-scale samples,pre-purification with solid phase extraction(SPE)cartridges of the extracts,separation with a high performance liquid chromatography(HPLC)and detection by an electrospray ionization ion trap mass spectrometry(ESI-Ion trap-MS)system in a consecutive ion monitoring(CRM)mode at the three stage fragmentation of mass spectrometry(MS3).The lowest detection level of the cytokinins of the method reaches 0.1-2.0 pg with a very wide range of linear regression from 1-512 pg,at the coefficient factors of 0.98-0.99.The feasibility of this method has been proven in the application of the method to the analysis of the trace-amount contents of cytokinins in the micro-scale samples of various types of plant materials,such as aerial parts of rice and poplar leaves etc.12 endogenous cytokinins had been identified and quantified in the plant tissues,with an acceptable relatively higher recovery rate from 40% to 70%.     

Mass Spectrometry Based Methods for Analysis of Nucleosides as Antiviral Drugs and Potential Tumor Biomarkers

The intracellular analysis of the phosphorylated metabolites of some anti-HIV nucleosides by liquid chromatography or capillary electrophoresis coupled with tandem mass spectrometry (LC-MS/MS or CE-MS/MS) has been realized on human peripheral blood mononuclear cells (PBMC), with limit of quantitation (LOQ) that allow them to be quantitated intracellularly. We described also the analysis of modified urinary nucleosides as potential tumor biomarkers.     

Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids: development of a clinically translatable biomarker

Measurement of nitrotyrosine levels in biological fluids can serve as a biomarker for oxidative/nitrative damage arising from formation of reactive nitrogen species, including peroxynitrite. Peroxynitrite is formed by the reaction of the superoxide radical (O2.-) with the nitric oxide radical (.NO) that is generated by nitric oxide synthase (NOS). This article describes an immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure 3-nitrotyrosine at very low (picomolar) levels. Incorporation of a pronase digestion step prior to the immunoaffinity LC-MS/MS allowed for measuring not only free amino acid but also protein 3-nitrotyrosine in biological fluids. The use of an in-line antibody column allowed for increased specificity as compared with previously reported assays. The assay is linear over a range of 5 to 500 pg/ml (0.022-2.20 nM, r(2)=0.9987), with the lower detection limit being 5 pg/ml. In addition to its increased sensitivity and specificity, this assay showed great nitrotyrosine recovery from biological fluids when either nitrotyrosine or nitrotyrosine-containing peptides were added exogenously. The utility of this assay for nitrotyrosine as a clinically translatable biomarker was demonstrated by quantifying both free and total nitrotyrosine levels in various biological fluids, including urine, plasma, serum, cerebrospinal fluid (CSF), and synovial fluid (SF) from both preclinical species and human subjects. Thus, whether in an animal model of human disease or in a clinical setting, the quantification of nitrotyrosine levels should provide support for NOS-driven pathology and its blockade following therapeutic intervention.     

Correlation between Serum Levels of 3,3?,5?-Triiodothyronine and Thyroid Hormones Measured by Liquid Chromatography-Tandem Mass Spectrometry and Immunoassay

ObjectiveFor measuring serum 3,3′,5′-triiodothyronine (rT3) levels, radioimmunoassay (RIA) has traditionally been used owing to the lack of other reliable methods; however, it has recently become difficult to perform. Meanwhile, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has recently been attracting attention as a novel alternative method in clinical chemistry. To the best of our knowledge, there are no studies to date comparing results of the quantification of human serum rT3 between LC-MS/MS and RIA. We therefore examined the feasibility of LC-MS/MS as a novel alternative method for measuring serum rT3, thyroxine (T4), and 3,5,3′-triiodothyronine (T3) levels.MethodsAssay validation was performed by LC-MS/MS using quality control samples of rT3, T4, and T3 at 4 various concentrations which were prepared from reference compounds. Serum samples of 50 outpatients in our department were quantified both by LC-MS/MS and conventional immunoassay for rT3, T4, and T3. Correlation coefficients between the 2 measurement methods were statistically analyzed respectively.ResultsMatrix effects were not observed with our method. Intra-day and inter-day precisions were less than 10.8% and 9.6% for each analyte at each quality control level, respectively. Intra-day and inter-day accuracies were between 96.2% and 110%, and between 98.3% and 108.6%, respectively. The lower limit of quantification was 0.05 ng/mL. Strong correlations were observed between the 2 measurement methods (correlation coefficient, T4: 0.976, p < 0.001; T3: 0.912, p < 0.001; rT3: 0.928, p < 0.001).ConclusionsOur LC-MS/MS system requires no manual cleanup operation, and the process after application of a sample is fully automated; furthermore, it was found to be highly sensitive, and superior in both precision and accuracy. The correlation between the 2 methods over a wide range of concentrations was strong. LC-MS/MS is therefore expected to become a useful tool for clinical diagnosis and research.     

A high-throughput method for microbial metabolome analysis using gas chromatography/mass spectrometry

An analytical high-throughput method based on gas chromatography/mass spectrometry (GC/MS) was developed for fast metabolome investigation. By parallelization and partial automation the time needed for the preanalytical steps could be reduced. In addition a strong decrease of the relative standard deviation of metabolite concentrations from independent samples on the same microtiter plate from 25 to 13% was achieved. Between different plates the relative standard deviation is comparable to the one observed in standard experiments with shaking flasks. Using a fast GC the time need for the full GC/MS-based metabolome analysis could be decreased from 60 to 18 min per run, allowing the measurement of 72 single samples per day and GC/MS machine. In samples of the model organism Corynebacterium glutamicum more than 1000 peaks in the total ion current could be detected in a single fast GC/MS run of which 650 were strong enough to be quantified. Approximately 150 compounds of these were identified using our metabolite MS-library. Correlation analysis of the concentration vectors of independent wild-type samples raised under the same conditions show very high correlations of 0.99+/-0.01 (logs). In conclusion this method allows screenings of large mutant libraries for genetically induced metabolic perturbations.     

Mass spectrometry of fatty aldehydes

Fatty aldehydes are important components of the cellular lipidome. Significant interest has been developed towards the analysis of the short chain α,β-unsaturated and hydroxylated aldehydes formed as a result of oxidation of polyunsaturated fatty acids. Multiple gas chromatography-mass spectrometry (GC/MS) and subsequently liquid chromatography-mass spectrometry (LC/MS) approaches have been developed to identify and quantify short-chain as well as long-chain fatty aldehydes. Due to the ability to non-enzymaticaly form Schiff bases with amino groups of proteins, lipids, and with DNA guanidine, free aldehydes are viewed as a marker or metric of fatty acid oxidation and not the part of intracellular signaling pathways which has significantly limited the overall attention this group of molecules have received. This review provides an overview of current GC/MS and LC/MS approaches of fatty aldehyde analysis as well as discusses technical challenges standing in the way of free fatty aldehyde quantitation.