Comprehensive profiling of prostaglandins in human ovarian follicular fluid using mass spectrometry.

Prostaglandins are formed by enzymatic and non-enzymatic mechanisms. They have been detected in human ovarian follicular fluid (HFF), a medium rich in growth factors and nutrients important for oocyte growth and fertility. However, the comprehensive identification of HFF prostaglandins has not been addressed. Here we use hybrid triple quadrupole time-of-flight and triple quadrupole mass spectrometers to comprehensively analyze prostaglandins in HFF. We identified PGE1, PGE2, PGF2α, and other prostaglandins synthesized via prostaglandin-endoperoxide synthase (i.e. Cox) cascades. We also identified specific PGF2α isomers (F2-isoprostanes) and PGF3α analogs whose structures are inconsistent with Cox-dependent formation. A prospective cohort pilot study of infertility patient subtypes revealed two potential associations. F2-isoprostanes are decreased in the diminished ovarian reserve subtype and elevated PGF2α may be associated with decreased live birth. Other than PGF2α, only body mass index >25kg/m2 correlated with poor in vitro fertilization outcome. Our studies suggest that HFF contains prostaglandins formed from at least two mechanisms, which may correlate with distinct clinical parameters.

Comparative Lipidomics of Caenorhabditis elegans Metabolic Disease Models by SWATH Non-Targeted Tandem Mass Spectrometry.

Tandem mass spectrometry (MS/MS) with Sequential Window Acquisition of all Theoretical (SWATH) mass spectra generates a comprehensive archive of lipid species within an extract for retrospective, quantitative MS/MS analysis. Here we apply this new technology in Caenorhabditis elegans (C. elegans) to identify potential lipid mediators and pathways. The DAF-1 type I TGF-ß and DAF-2 insulin receptors transmit endocrine signals that couple metabolic status to fertility and lifespan. Mutations in daf-1 and daf-2 reduce prostaglandin-endoperoxide synthase (i.e., Cox)-independent prostaglandin synthesis, increase triacylglyceride storage, and alter transcription of numerous lipid metabolism genes. However, the extent to which DAF-1 and DAF-2 signaling modulate lipid metabolism and the underlying mechanisms are not well understood. MS/MSALL with SWATH analysis across the groups identified significant changes in numerous lipids, including specific triacylglycerols, diacylglycerols, and phosphatidylinositols. Examples are provided, using retrospective neutral loss and precursor ion scans as well as MS/MS spectra, to help identify annotated lipids and search libraries for lipids of interest. As proof of principle, we used comparative lipidomics to investigate the prostaglandin metabolism pathway. SWATH data support an unanticipated model: Cox-independent prostaglandin synthesis may involve lysophosphatidylcholine and other lyso glycerophospholipids. This study showcases the power of comprehensive, retrospectively searchable lipid archives as a systems approach for biological discovery in genetic animal models.

Cholesterol-Independent Suppression of Lymphocyte Activation, Autoimmunity, and Glomerulonephritis by Apolipoprotein A-I in Normocholesterolemic Lupus-Prone Mice.

Apolipoprotein (Apo)A-I, the major lipid-binding protein of high-density lipoprotein, can prevent autoimmunity and suppress inflammation in hypercholesterolemic mice by attenuating lymphocyte cholesterol accumulation and removing tissue-oxidized lipids. However, whether ApoA-I mediates immune-suppressive or anti-inflammatory effects under normocholesterolemic conditions and the mechanisms involved remain unresolved. We transferred bone marrow from systemic lupus erythematosus (SLE)-prone Sle123 mice into normal, ApoA-I-knockout (ApoA-I(-/-)) and ApoA-I-transgenic (ApoA-I(tg)) mice. Increased ApoA-I in ApoA-I(tg) mice suppressed CD4(+) T and B cell activation without changing lymphocyte cholesterol levels or reducing major ApoA-I-binding oxidized fatty acids. Unexpectedly, oxidized fatty acid peroxisome proliferator-activated receptor γ ligands 13- and 9-hydroxyoctadecadienoic acid were increased in lymphocytes of autoimmune ApoA-I(tg) mice. ApoA-I reduced Th1 cells independently of changes in CD4(+)Foxp3(+) regulatory T cells or CD11c(+) dendritic cell activation and migration. Follicular helper T cells, germinal center B cells, and autoantibodies were also lower in ApoA-I(tg) mice. Transgenic ApoA-I also improved SLE-mediated glomerulonephritis. However, ApoA-I deficiency did not have the opposite effects on autoimmunity or glomerulonephritis, possibly as the result of compensatory increases in ApoE on high-density lipoprotein. We conclude that, although compensatory mechanisms prevent the proinflammatory effects of ApoA-I deficiency in normocholesterolemic mice, increasing ApoA-I can attenuate lymphocyte activation and autoimmunity in SLE independently of cholesterol transport, possibly through oxidized fatty acid peroxisome proliferator-activated receptor γ ligands, and it can reduce renal inflammation in glomerulonephritis.

Neurosensory perception of environmental cues modulates sperm motility critical for fertilization.

Environmental exposures affect gamete function and fertility, but the mechanisms are poorly understood. Here, we show that pheromones sensed by ciliated neurons in the Caenorhabditis elegans nose alter the lipid microenvironment within the oviduct, thereby affecting sperm motility. In favorable environments, pheromone-responsive sensory neurons secrete a transforming growth factor-ß ligand called DAF-7, which acts as a neuroendocrine factor that stimulates prostaglandin-endoperoxide synthase [cyclooxygenase (Cox)]-independent prostaglandin synthesis in the ovary. Oocytes secrete F-class prostaglandins that guide sperm toward them. These prostaglandins are also synthesized in Cox knockout mice, raising the possibility that similar mechanisms exist in other animals. Our data indicate that environmental cues perceived by the female nervous system affect sperm function.

Mitochondria-targeted heme oxygenase-1 decreases oxidative stress in renal epithelial cells.

Mitochondria are both a source and target of the actions of reactive oxygen species and possess a complex system of inter-related antioxidants that control redox signaling and protect against oxidative stress. Interestingly, the antioxidant enzyme heme oxygenase-1 (HO-1) is not present in the mitochondria despite the fact that the organelle is the site of heme synthesis and contains multiple heme proteins. Detoxification of heme is an important protective mechanism since the reaction of heme with hydrogen peroxide generates pro-oxidant ferryl species capable of propagating oxidative stress and ultimately cell death. We therefore hypothesized that a mitochondrially localized HO-1 would be cytoprotective. To test this, we generated a mitochondria-targeted HO-1 cell line by transfecting HEK293 cells with a plasmid construct containing the manganese superoxide dismutase mitochondria leader sequence fused to HO-1 cDNA (Mito-HO-1). Nontargeted HO-1-overexpressing cells were generated by transfecting HO-1 cDNA (HO-1) or empty vector (Vector). Mitochondrial localization of HO-1 with increased HO activity in the mitochondrial fraction of Mito-HO-1 cells was observed, but a significant decrease in the expression of heme-containing proteins occurred in these cells. Both cytosolic HO-1- and Mito-HO-1-expressing cells were protected against hypoxia-dependent cell death and loss of mitochondrial membrane potential, but these effects were more pronounced with Mito-HO-1. Furthermore, decrement in production of tricarboxylic acid cycle intermediates following hypoxia was significantly mitigated in Mito-HO-1 cells. These data suggest that specific mitochondrially targeted HO-1 under acute pathological conditions may have beneficial effects, but the selective advantage of long-term expression is constrained by a negative impact on the synthesis of heme-containing mitochondrial proteins.

Simultaneous quantification of F2-isoprostanes and prostaglandins in human urine by liquid chromatography tandem-mass spectrometry.

A specific and sensitive LC-MS/MS method for analysis of F(2)-isoprostanes (F(2)-IsoPs) and prostaglandins (PGs) in urine was developed and validated to examine the levels of F(2)-IsoPs and prostaglandin F(2α) (PGF(2α)), in human urine in patients undergoing cardiac surgery. The rapid extraction for F(2)-IsoPs and PGs from urine was achieved using a polymeric weak anion solid phase extraction cartridge. The base-line separation of 8-iso-PGF(2α), 8-iso-15(R)-PGF(2α), PGF(2α), and 15(R)-PGF(2α) was carried out on a Hydro-RP column (250mm×2.0mm i.d., Phenomenex, CA) using a linear gradient of methanol:acetonitrile (1:1, v/v) in 0.1% formic acid at a flow rate of 0.2mL/min. The method was proved to be accurate and precise for simultaneous quantification of each analyte over a linear dynamic range of 0.05-50ng/mL with correlation coefficients greater than 0.99. The intra-day and inter-day assay precision at the lowest quality control (0.07ng/mL) level were less than 17%. The mean extraction recoveries of F(2)-IsoPs and PGs were in a range of 79-100%. In applications of this method to patients undergoing cardiac surgery, post-surgery urinary concentrations of 8-iso-PGF(2α) increased significantly in patients (n=14) who did not develop acute kidney (AKI) (pre-surgery 0.344±0.039 vs. post-surgery 0.682±0.094ng/mg creatinine, p<0.01), whereas there was no significant change in this isoprostane in the patients (n=4) who developed AKI (pre-surgery 0.298±0.062 vs. post-surgery 0.383±0.117ng/mg creatinine, NS). Therefore, the method is suitable for the analysis of individual F(2)-IsoPs and PGF(2α)'s in both clinical and research studies.

Determination of cranberry phenolic metabolites in rats by liquid chromatography-tandem mass spectrometry.

The glycosides of flavonoid, anthocyanins and A type proanthocyanidins in cranberry concentrate were characterized and quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cranberry concentrate (1 g/body weight) was orally gavaged to Fischer-344 rats (n = 6), and blood and urine samples were collected over 24 h periods. Quercetin, 3'-O-methylquercetin (isorhamnetin), myricetin, kaempferol, and proanthocyanidin dimer A2, together with thirteen conjugated metabolites of quercetin and methylquercetin and intact peonidin 3-O-galactoside and cyanidin 3-O-galactoside were identified in the rat urine after cranberry treatment. Very low levels of isorhamnetin (0.48 ± 0.09 ng/mL) and proanthocyanidin dimer A2 (0.541 ± 0.10 ng/mL) were found in plasma samples after 1 h of cranberry administration. Although no quercetin was detected in plasma, MRM analysis of the methanolic extract of urinary bladder showed that chronic administration of cranberry concentrate to rats resulted in accumulation of quercetin and isorhamnetin in the bladder. These results demonstrate that cranberry components undergo rapid metabolism and elimination into the urine of rats and are present in the urinary bladder tissue potentially allowing them to inhibit urinary bladder carcinogenesis.

Chronic dietary kudzu isoflavones improve components of metabolic syndrome in stroke-prone spontaneously hypertensive rats.

The present study tested the long-term effects of dietary kudzu root extract supplementation on the regulation of arterial pressure, plasma glucose, and circulating cholesterol in stroke-prone spontaneously hypertensive rats (SP-SHR). Female SP-SHR were maintained for 2 months on a polyphenol-free diet, with or without the addition of 0.2% kudzu root extract. Half of the rats in each diet group were ovariectomized, whereas the other half remained intact. Following 2 months on the diets, the 0.2% kudzu root extract supplementation (compared to control diet) significantly lowered arterial pressure (11-15 mmHg), plasma cholesterol, fasting blood glucose (20-30%), and fasting plasma insulin in both the ovariectomized and intact SP-SHR. These results indicate that long-term dietary kudzu root extract supplementation can improve glucose, lipid, and blood pressure control in intact and ovariectomized SP-SHR.

Liquid chromatography tandem mass spectrometry identification of proanthocyanidins in rat plasma after oral administration of grape seed extract.

Proanthocyanidin rich plant extracts derived from grape seed extract (GSE), hawthorn and cranberry are on markets for their preventive effects against cardiovascular diseases and uroinfections in woman. However, the importance of these health beneficial effects of these botanicals remains elusive due to incomplete understanding of uptake, metabolism and bioavailability of proanthocyanidins in vivo. In the present study rats were given GSE orally (300 mg/kg, twice a day) and blood and urine were collected over a 24 h period. Monomeric catechins and their methylated metabolites, and proanthocyanidins up to trimers were detected in blood samples treated with GSE using LC-MS/MS operating in the multiple reaction monitoring (MRM) mode. A new tetramethylated metabolite of dimeric proanthocyanidin (m/z 633) in GSE-treated urine was tentatively identified. Using LC-MS/MS, (+)-catechin and (-)-epicatechin were identified in the brain conclusively. These data suggested that GSE catechins cross the blood brain barrier and may be responsible for the neuroprotective effects of GSE.

Tissue distribution of puerarin and its conjugated metabolites in rats assessed by liquid chromatography-tandem mass spectrometry.

Puerarin (an isoflavone C-glucoside from kudzu root) has been the focus of several studies investigating its potential effects on health benefits. In this study, we determined single dose tissue distribution of puerarin and its metabolites in order to examine whether they undergo selective uptake by specific organs. Puerarin was administered orally (50 mg/kg) to rats and the concentration of puerarin in tissue compartments was determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Puerarin was widely distributed in rat tissues with highest concentrations in lungs (799+/-411.6 ng/g wet tissues). In addition, we examined the excretion of puerarin into the bile. LC-MS/MS analysis of bile samples collected after infusing puerarin directly into the portal vein indicated that puerarin was excreted into the bile predominantly in the form of unconjugated puerarin. This report identifying puerarin in several organs including kidney and pancreas may explain its beneficial effects in diabetes.

A liquid chromatography mass spectrometry assay for determination of PD168393, a specific and irreversible inhibitor of erbB membrane tyrosine kinases, in rat serum.

For the first time, a rapid, sensitive and simple liquid chromatography/tandem mass spectrometry (LC-MS/MS) method using an atmospheric pressure chemical ionization (APCI) source for the quantification of PD168393 in rat serum was developed and validated. Serum samples were pretreated with methanol for protein precipitation. The chromatographic separation was performed on a Jupiter-C5 column (250 mm x 2.0 mm i.d.) pre-equilibrated with 0.1% formic acid. The tandem mass spectrometer was tuned in the multiple reaction monitoring mode to monitor the m/z transitions 369/313 for PD168393 and m/z 343/308 for the internal standard triazolam, using positive ion mode. The MS/MS response was linear over the concentration range from 2 ng/mL to 5000 ng/mL, with a lower limit of quantification (LLQ) of 2 ng/mL. At the lowest quality control (4 ng/mL), the intra- and inter-day precisions (CV%) for PD168393 were less than 10% and the accuracies were between 92% and 111%. The validated method can be used in most or all stages of the screening and optimizing process for future method validation of pharmacokinetic studies.

Effect of cranberry juice concentrate on chemically-induced urinary bladder cancers.

The chemopreventive efficacy of cranberry juice concentrate in an experimental model of urinary bladder cancer was evaluated using female Fischer-344 rats. The animals received N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN) for a period of eight weeks. Cranberry juice concentrate was administered at doses of 1.0 or 0.5 ml/rat/day beginning one week after the final OH-BBN treatment and continuing until the end of the study. The urinary bladders of all the rats were weighed and examined grossly for lesions, and all masses were submitted for pathological evaluation. A dose-dependent preventive effect of cranberry treatment was observed, with a reduced number of urinary bladder cancers (38%) in the 1.0 ml/rat/day group versus the control group. The cranberry extract neither affected body weight gain nor caused other signs of toxicity. For the metabolic studies, serum and urine were collected at 4 and 12 h after the administration of the cranberry juice concentrate and were analyzed by LC-MS/MS. Quercetin and its methylated derivative were detected in the urine samples. However, no quercetin was detected in the serum samples, indicating its poor bioavailability. These data suggest that components of cranberries may be effective in preventing urinary bladder carcinogenesis.

Naturally occurring progesterone in loblolly pine (Pinus taeda L.): a major steroid precursor of environmental androgens.

Progesterone, androstenedione, and androstadienedione were previously identified in the water and sediment of the Fenholloway River (Taylor County, FL, USA), a river that contains populations of masculinized female mosquitofish downstream of a paper mill, at levels higher than those in the nearby Spring Creek. Plant sterols, such as beta-sitosterol in mill effluent derived from pine tree pulp, were suggested to be metabolized by bacteria to progesterone and androgens to account for the masculinization phenomenon. The current study made use of standard solid-phase methanol extraction procedures, high-performance liquid chromatography, liquid chromatography-mass spectrometry, and a cell-based, androgen-receptor transcription assay to determine naturally occurring progesterone levels in mature pine trees. Progesterone concentrations in the loblolly pine (Pinus taeda L.) were 49.34 +/- 4.1 nmol/g dry mature wood (15.5 +/- 1.29 microg/g), 12.26 +/- 1.78 nmol/g pine needles (3.85 +/- 0.56 microg/g), and 3.81 +/- 0.36 nmol/g pine bark (1.19 +/- 0.11 mug/g). The results suggest that naturally occurring progesterone from pine wood pulp contributes to increased progesterone levels downstream of paper mill effluent discharges and may serve as the natural steroid precursor for environmental androgen production that causes masculinization of female mosquitofish.

Sex, pain and cranberries - ideas from the 2006 Registrar Research Workshop.

The Registrar Research Workshop has been a feature of Australian general practice training since 1994. Twenty five general practice registrars attend the annual 3 day event, which aims to develop registrars' understanding of the research process. Presenters and facilitators are drawn from the academic general practitioner and primary health care research community. Presentations alternate with small group sessions, where groups of five registrars are guided through the process of developing a research question, identifying appropriate research methods, and addressing ethical and funding concerns, before preparing a presentation about their research proposal for their peers. Research questions are developed from unanswered questions that have arisen in registrars' clinical practice.

Pharmacokinetic study of puerarin in rat serum by liquid chromatography tandem mass spectrometry.

A highly sensitive and specific atmospheric pressure chemical ionization liquid chromatography-tandem mass spectrometry method was developed for serum pharmacokinetic studies of puerarin in rats. Chromatography was carried out on a reversed-phase Phenomenex Synergi 4 microm Fusion-RP80 column (150 x 2.0 mm i.d.) using a mobile phase consisting of acetonitrile-water (10:90, v/v) in 10 mm NH(4)OAc with a flow rate of 0.2 mL/min. Puerarin was analyzed in the multiple reaction monitoring mode with a precursor/product ion transition of m/z 415/267. The method was demonstrated to be specific and sensitive, and a linear response was observed over a range of 2-5000 ng/mL in rat serum. The validated method was successfully applied to the characterization of the pharmacokinetics of puerarin in rat serum after oral administration to spontaneously hypertensive rats. The blood concentration-time profile of puerarin showed a rapid initial increase, reaching a maximum and then declining within 1 h. Puerarin could not be detected after 24 h. The main pharmacokinetic parameters for puerarin after oral administration were as follows: C(max) (3.54 +/- 2.03 mg/L), T(max) (0.68 +/- 0.37 h), AUC(0-t) (7.29 +/- 3.79 mg h/L), AUC(0-infinity) (9.17 +/- 4.87 mg h/L), T(1/2) (1.7 +/- 0.6 h), CL/F (7.24 +/- 4.27 L/h/kg) and V/F (17.88 +/- 13.55 L/h/kg).

Efficacy of benfluorex in combination with sulfonylurea in type 2 diabetic patients: an 18-week, randomized, double-blind study.

OBJECTIVE: The aim of this study was to demonstrate the superiority of benfluorex over placebo as an add-on therapy in type 2 diabetic patients in whom diabetes is insufficiently controlled by sulfonylurea monotherapy and who have a limitation for the use of metformin. RESEARCH DESIGN AND METHODS: Type 2 diabetic patients with HbA(1c) (A1C) (7-10%) who were receiving the maximum tolerated sulfonylurea dose and had a contraindication to or poor tolerance of metformin were randomly assigned (double blind) to receive benfluorex 450 mg/day (n = 165) or placebo (n = 160) for 18 weeks. The main efficacy criterion was A1C, analyzed as the change from baseline to the end of treatment using ANCOVA with baseline and country as covariates. Secondary criteria were fasting plasma glucose (FPG), insulin resistance, and plasma lipid level. RESULTS: Both groups were similar at baseline in the intention-to-treat population. A1C significantly decreased with benfluorex from 8.34 +/- 0.83 to 7.52 +/- 1.04% (P < 0.001) and tended to increase with placebo from 8.33 +/- 0.87 to 8.52 +/- 1.36% (NS), resulting in a mean adjusted difference between groups of -1.01% (95% CI -1.26 to -0.76; P < 0.001). The target A1C (< or =7%) was achieved in 34% of patients receiving benfluorex versus 12% of patients receiving placebo. Significant between-group differences in favor of benfluorex were observed for mean FPG (-1.65 mmol/l) (P < 0.001) and for homeostasis model assessment of insulin resistance. Overall tolerance was similar in both groups. Serious adverse events were more frequent in the benfluorex group, without evidence of causality relationship. CONCLUSIONS: Benfluorex as an add-on therapy was superior to placebo in lowering A1C with a between-group difference of 1% in type 2 diabetic patients whose disease was insufficiently controlled with sulfonylurea alone and in whom metformin was contraindicated or not tolerated.

Contrasting effects of puerarin and daidzin on glucose homeostasis in mice.

Puerarin and daidzin are the major isoflavone glucosides found in kudzu dietary supplements. In this study, we demonstrated that puerarin significantly improves glucose tolerance in C57BL/6J-ob/ob mice, an animal model of type 2 diabetes mellitus, blunting the rise in blood glucose levels after i.p. administration of glucose. In contrast, daidzin, the O-glucoside, had a significant but opposite effect, impairing glucose tolerance as compared to saline-treated controls. When they were administered i.p. with (14)C-glucose to C57BL/6J lean mice, puerarin inhibited glucose uptake into tissues and incorporation into glycogen, while daidzin stimulated glucose uptake, showing an opposite effect to puerarin. Puerarin also antagonized the stimulatory effect of decyl-beta-D-thiomaltoside, an artificial primer of glycogen synthesis, which increases (14)C-glucose uptake and incorporation into glycogen in mouse liver and heart. A liquid chromatography-tandem mass spectrometry procedure was used to investigate the metabolism and bioavailability of puerarin and daidzin. The blood puerarin concentration-time curve by i.p. and oral administration indicated that puerarin was four times more bioavailable via i.p. injection than via the oral route of administration. This may account for the increased hypoglycemic effect seen in the i.p. glucose tolerance test vs that seen orally. Our results suggest that puerarin is rapidly absorbed from the intestine without metabolism, while daidzin is hydrolyzed to the aglycone daidzein. The opposing effects of puerarin and daidzin on glucose homeostasis may have implications for the activity of dietary supplements that contain both of these isoflavonoids.

Long-term dietary habits affect soy isoflavone metabolism and accumulation in prostatic fluid in caucasian men.

The soy isoflavones daidzein and genistein are believed to reduce prostate cancer risk in soy consumers. However, daidzein can be metabolized by the intestinal flora to form a variety of compounds with different bioactivities. In the current study, we investigated the influence of long-term dietary habits on daidzein metabolism in healthy Caucasian men (19-65 y old). A secondary goal was to compare plasma and prostatic fluid concentrations of 5 isoflavonoids: genistein, daidzein, equol, dihydrodaidzein, and O-desmethylangolensin. Baseline plasma levels of isoflavonoids were quantitated in 45 men by HPLC-electrospray ionization-MS. Participants then consumed a soy beverage daily for 1 wk, and post-soy isoflavonoid levels were quantitated in plasma and prostatic fluid. Equol was the only metabolite that appeared to be influenced by routine dietary habits. Stratified analyses revealed that men who had consumed > or =30 mg soy isoflavones/d for at least 2 y had 5.3-times the probability of producing equol than men who had consumed < or =5 mg/d (P = 0.014). Additionally, those men who consumed animal meat regularly had 4.7-times the probability of producing equol than men who did not consume meat (P = 0.023). Equol production was not linked to age, BMI, or the consumption of yogurt, dairy, fruit, or American-style fast food. Daidzein and its metabolites (but not genistein) were typically present at higher levels in prostate fluid than plasma (median = 4-13 times that in plasma). In conclusion, our data suggest that the ability of Caucasian men to produce equol is favorably influenced by the long-term consumption of high amounts of soy and the consumption of meat. Last, the high concentrations of isoflavonoids in prostatic fluid increases the potential for these compounds to have direct effects in the prostate.

Electrospray tandem mass spectrometric analysis of zeaxanthin and its oxidation products.

Carotenoids have been implicated in protection of the eye from light-mediated photo-toxicity caused by free radicals. Under conditions of normal oxidative stress the carotenoids serve as protective antioxidants; however, when the oxidative stress exceeds the antioxidant capacity, carotenoids can be oxidized into numerous cleavage products. The determination and identification of oxidized carotenoids in biological samples remains a major challenge due to the small sample size and low stability of these compounds. We investigated the reaction of various zeaxanthin cleavage products with O-ethyl hydroxylamine to evaluate their levels in a biological sample. For this, a sensitive and specific electrospray tandem mass spectrometry (ESI-MS/MS) was developed, avoiding the classical lower sensitive and specific HPLC-UV and fluorescence absorption methods. Protonated molecules [M + H](+) of carotenoids upon collision-induced dissociation produced a number of structurally characteristic product ions. A series of complicated clusters of product ions differing in 14 (CH(2))and 26 (C(2)H(2))Da was characteristic of the polyene chain of intact carotenoids. All carotenoid ethyl oximes of zeaxanthin cleavage products were characterized by the losses of 60 and 61 Da in their MS/MS spectra. Through the application of the LC/MS/MS method, we identified two oxime derivatives of 3-hydroxy-beta-ionone and 3-hydroxy-14'-apocarotenal with protonated molecules at m/z 252 and m/z 370 respectively, in a human eye sample.