Metabolomic search for uremic toxins as indicators of the effect of an oral sorbent AST-120 by liquid chromatography/tandem mass spectrometry

An oral sorbent AST-120 composed of spherical porous carbon particles has superior adsorption ability for certain small-molecular-weight organic compounds known to accumulate in patients with chronic renal failure (CRF). A metabolomic approach was applied to search for uremic toxins as possible indicators of the effect of AST-120. Serum metabolites in normal and CRF rats before and after administration of AST-120 for 3 days were analyzed by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) and principal component analysis. Further, serum and urine levels of the indicators were quantified by selected reaction monitoring of LC/ESI-MS/MS. Indoxyl sulfate was the first principal serum metabolite, which could differentiate CRF from both normal and AST-120-administered CRF rats, followed by hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate. CRF rats showed increased serum levels of indoxyl sulfate, hippuric acid, phenyl sulfate, 4-ethylphenyl sulfate and p-cresyl sulfate. Administration of AST-120 for 3 days to the CRF rats reduced the serum and urine levels of these metabolites. In conclusion, indoxyl sulfate is the best indicator of the effect of AST-120 in CRF rats. Hippuric acid, phenyl sulfate and 4-ethylphenyl sulfate are suggested as the additional indicators. 4-Ethylphenyl sulfate is a newly identified uremic substance.     

Serum metabonomics study of adenine-induced chronic renal failure in rats by ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry

An ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC Q-TOF MS) metabonomics approach was employed to study the serum metabolic profiling of adenine-induced chronic renal failure (CRF) rats. Acquired data were subjected to principal component analysis (PCA) for differentiating the CRF and the normal control groups. Potential biomarkers were screened by using S-plot and were identified by the accurate mass, isotopic pattern and MS/MS fragments information obtained from UPLC Q-TOF MS analysis. Significant differences in the serum level of creatinine, amino acids and LysoPCs were observed, indicating the perturbations of amino acid metabolism and phospholipid metabolism in adenine-induced CRF rats. This research proved that metabonomics is a promising tool for disease research.     

Metabolomic identification of potential phospholipid biomarkers for chronic glomerulonephritis by using high performance liquid chromatography-mass spectrometry

Plasma phospholipids metabolic profile of chronic glomerulonephritis was investigated using high performance liquid chromatography/mass spectrometry (LC/MS) and principal component analysis. The plasma samples of 18 patients with chronic glomerulonephritis, 17 patients with chronic renal failure (CRF) without renal replacement therapy and 18 healthy persons were collected and analyzed. It was found that combination of the LC/MS technique with PCA can be successfully applied to phospholipid profile analysis. The results showed that primary chronic glomerulonephritis and CRF had phospholipids metabolic abnormality. Nineteen phospholipid species were identified as possible biomarkers in plasma samples of chronic glomerulonephritis and chronic renal failure. Moreover, the identification of the molecular structure of the potential phospholipid markers was obtained by ESI-MS/MS experiment. It suggests that phospholipids can be used as potential biomarkers on the progress of primary chronic glomerulonephritis.     

Characterisation of a potential biomarker of phospholipidosis from amiodarone-treated rats

A novel and relatively simple analytical method for the separation, characterisation and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed. This methodology allows PL extracts from cells and tissues to be analysed by liquid chromatography (LC) coupled to electrospray ionisation mass spectrometry (ESI-MS). Complex mixtures of PLs were separated on a high-performance liquid chromatography (HPLC) system using 0.5% ammonium hydroxide in methanol/water/hexane/formate mixture with UV detection at 205 nm. Identification and structural characterisation of molecular species were carried out utilising ESI-MS and MS/MS in the negative ion mode.The abnormal accumulation of PLs (phospholipidosis) was induced in male Sprague-Dawley rats by administration of the cationic amphiphilic drug (CAD), amiodarone. Analysis of the PL profile of liver and lung tissues, lymphocytes and serum from treated rats was carried out using this analytical procedure (LC-ESI/MS/MS). Differences in PL profiles between treated and untreated animals were highlighted by principal component analysis (PCA). This led to the selection of a potential metabolic marker of phospholipidosis (PLD) identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero)phosphate (BMP). This PL was absent in control animals but was present in quantifiable amounts in all samples from amiodarone-treated rats.     

An accurate quantitative LC/ESI-MS/MS method for sirolimus in human whole blood

Sirolimus is a widely used immunosuppressant that requires therapeutic drug monitoring (TDM). We optimized a preanalytical procedure that allows for the accurate quantiation of sirolimus in whole blood by LC/ESI-MS/MS with minimal matrix effects. Sirolimus is highly lipophilic, and solvents containing greater than 50% methanol were required to maintain sirolimus recovery. The final pretreatment procedure developed consists of a zinc sulfate protein precipitation, an extraction using octadecyl silyl-silica gel for eliminating water-soluble and hydrophilic compounds, and HybridSPE cartridge treatment to eliminate phospholipids. Using this procedure prior to LC/ESI-MS/MS led to the accurate and reproducible quantitation of sirolimus in human whole blood. The linear range of detection was 0.5-50 ng/mL, a range appropriate for TDM, and the method demonstrated good repeatability and intermediate precision within this quantitative range. In order to investigate the quantitative performance of this method, we compared it to two commercially available sirolimus immunoassays and our previously reported LC/ESI-MS/MS method. The immunoassays gave consistently greater values for the sirolimus concentration, and this may be related to antibody cross-reactivity with sirolimus metabolites and/or other matrix effects. Although our procedure is too long to support real-time TDM for outpatients, it can serve as reference method to assess the performance of other analytical methods that are currently available or may be developed in the future.     

Effects of parathyroid hormone on plasma zinc concentration in rat with chronic renal failure

Although both secondary hyperparathyroidism (HPT) and hypozincemia are commonly observed in humans and animals with chronic renal failure (CRF), the relationship between secondary HPT and hypozincemia is little delineated. The present study was designed to examine whether the elevated plasma parathyroid hormones (PTH) levels do affect the disposition of extrarenal zinc and decrease plasma zinc level in CRF rats. The experiment was performed in normal and CRF rats with intact parathyroid gland and parathyroidectomized (PTX), using an acute zinc load alone or in combination with PTH infusion in five groups of rats: normal control, CRF control, CRF + PTH, CRF + PTX and CRF + PTX + PTH. Five sixths nephrectomy was used to produce CRF. All rats were infused with 0.05 mg/kg/min ZnSO4 alone or in combination with 10 microg/kg/min PTH through intravenous infusion for 90 min with serial monitoring of plasma zinc levels every 30 min. The alteration of plasma interleukin-6 (IL-6) levels and the effect of zinc levels in red blood cells (RBCs), as well as the output of bile juice zinc and urinary zinc excretion during the 90-min infusion were also examined. After 90-min infusion, liver tissue was harvested to determine its contents of zinc and metallothionein (MT). During zinc sulfate infusion, the responses of plasma zinc concentration in PTH-combined infusion groups markedly decreased as compared with those of the non-PTH-combined infusion groups, especially in the CRF rats with PTX. However, when zinc sulfate alone was infused, the response of plasma zinc concentration was found to increase in CRF rats with PTX as compared with that of the CRF control rats. PTH infusion groups significantly increased the levels of plasma IL-6 (P < 0.05), but it did not alter the levels of RBC zinc and the secretion of bile zinc during the 90-min infusion. After 90-min zinc sulfate infusion, higher liver zinc and MT contents were found in CRF control, CRF + PTH and CRF + PTX + PTH rats, but were [corrected] not found in the CRF + PTX rats. Zinc sulfate infused alone was found to increase the excretion of basal zinc in bile juice and urine, in both normal and CRF rats. The percentage of zinc load translocated out from the plasma during 90-min zinc sulfate infusion significantly rises in CRF rats and CRF rats with PTH-combined infusion as compared with normal control rats. However, in CRF rats with PTX, the percentage of zinc load translocated out from plasma during 90-min zinc sulfate infusion was similar to that in the normal control rats. Therefore, we suggested that in CRF rats, the excessive secretion of PTH may play a role in the pathogenesis of hypozincemia because PTH enhanced extrarenal zinc disposal.     

Application of faecal metabonomics on an experimental model of tubulointerstitial fibrosis by ultra performance liquid chromatography/high-sensitivity mass spectrometry with MSE data collection technique

Chronic renal failure (CRF) is a major challenge for the public healthcare problem. A novel UPLC Q-TOF/MS method with MS^E data collection mode was developed as a very effective biochemical analytical tool for precise identification of important biomarkers in the adenine-induced CRF rats. Nine endogenous metabolites were identified by using metabonomic method combined with multivariate data analysis, the accurate mass, isotopic pattern, MS^E fragments information and MassLynx i-FIT algorithm. The identified metabolites indicated the perturbations of bile acid and phospholipid metabolism are related to CRF rats. This work shows that metabonomics method is a valuable tool in CRF mechanism study.     

Improved method for the quantification of lysophospholipids including enol ether species by liquid chromatography-tandem mass spectrometry

LC/ESI-MS/MS has been previously demonstrated to be a powerful method to detect and quantify molecular species of glycerophospholipids including lysophospholipids. In this study, we provide an improved pre-mass spectrometry lipid extraction procedure that avoids the acid-catalyzed decomposition of plasmenyl phospholipids that is problematic with previously reported methods. We show that the use of lysophospholipid internal standards with perdeuterated fatty acyl chains avoids isobar problems associated with the use of internal standards containing odd carbon number fatty acyl chains. We also show that LC prior to MS is required to avoid numerous problems associated with isobars and with MS in-source decomposition of lysophosphatidylserine. The reported method of using normal phase chromatography/ESI-MS is used to quantify lysophospholipids in serum and to quantify lysophospholipids produced in mammalian cells by human group X secreted phospholipase A₂. The latter shows that group X phospholipase A₂ added exogenously to cells generates a different set of lysophospholipids compared with enzyme produced endogenously in cells, which supports earlier studies showing that this phospholipase A₂ can act on cell membranes prior to externalization from cells.     

Stable-isotope dilution LC–ESI-MS/MS techniques for the quantification of total homocysteine in human plasma

Homocysteine is an endogenous sulphydryl aminoacid irreversibly catabolized by transsulfuration to cysteine or remethylated to methionine. Increased plasma levels of homocysteine are an independent risk factor for atherosclerosis and cardiovascular disease. Accurate and reliable quantification of this amino acid in plasma samples is essential in clinical practice to explore the presence of a hyperhomocysteinemia, for instance after an ischemic event, or to control a possible adjunctive risk factor in patients at higher risk. In this review, LC–ESI-MS/MS methods are discussed and compared with other analytical methods for plasma homocysteine. LC–ESI-MS/MS is a technique combining the physicochemical separation of liquid chromatography with the analysis of mass spectrometry. It is based on stable-isotope dilution and possesses inherent accuracy and precision. Quantitative analysis is achieved by using commercially available homocystine-d₈ as an internal standard. Taking advantage of the high sensitivity and specificity, approaches involving LC–ESI-MS/MS require less laborious sample preparation, no derivatization and produce reliable results.     

UHPLC-Q-Orbitrap-HRMS-based global metabolomics reveal metabolome modifications in plasma of young women after cranberry juice consumption

Plasma metabolome in young women following cranberry juice consumption were investigated using a global UHPLC-Q-Orbitrap-HRMS approach. Seventeen female college students, between 21 and 29 years old, were given either cranberry juice or apple juice for three days using a cross-over design. Plasma samples were collected before and after juice consumption. Plasma metabolomes were analyzed using UHPLC-Q-Orbitrap-HRMS followed by orthogonal partial least squares-discriminant analyses (OPLS-DA). S-plot was used to identify discriminant metabolites. Validated OPLS-DA analyses showed that the plasma metabolome in young women, including both exogenous and endogenous metabolites, were altered following cranberry juice consumption. Cranberry juice caused increases of exogenous metabolites including quinic acid, vanilloloside, catechol sulfate, 3,4-dihydroxyphenyl ethanol sulfate, coumaric acid sulfate, ferulic acid sulfate, 5-(trihydroxphenyl)-gamma-valerolactone, 3-(hydroxyphenyl)proponic acid, hydroxyphenylacetic acid and trihydroxybenzoic acid. In addition, the plasma levels of endogenous metabolites including citramalic acid, aconitic acid, hydroxyoctadecanoic acid, hippuric acid, 2-hydroxyhippuric acid, vanilloylglycine, 4-acetamido-2-aminobutanoic acid, dihydroxyquinoline, and glycerol 3-phosphate were increased in women following cranberry juice consumption. The metabolic differences and discriminant metabolites observed in this study may serve as biomarkers of cranberry juice consumption and explain its health promoting properties in human.     

Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC/ESI-MS/MS) Study for the Identification and Characterization of In Vivo Metabolites of Cisplatin in Rat Kidney Cancer Tissues: Online Hydrogen/Deuterium (H/D) Exchange Study

In vivo rat kidney tissue metabolites of an anticancer drug, cisplatin (cis-diamminedichloroplatinum [II]) (CP) which is used for the treatment of testicular, ovarian, bladder, cervical, esophageal, small cell lung, head and neck cancers, have been identified and characterized by using liquid chromatography positive ion electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with on line hydrogen/deuterium exchange (HDX) experiments. To identify in vivo metabolites, kidney tissues were collected after intravenous administration of CP to adult male Sprague-Dawley rats (n = 3 per group). The tissue samples were homogenized and extracted using newly optimized metabolite extraction procedure which involves liquid extraction with phosphate buffer containing ethyl acetate and protein precipitation with mixed solvents of methanol-water-chloroform followed by solid-phase clean-up procedure on Oasis HLB 3cc cartridges and then subjected to LC/ESI-HRMS analysis. A total of thirty one unknown in vivo metabolites have been identified and the structures of metabolites were elucidated using LC-MS/MS experiments combined with accurate mass measurements. Online HDX experiments have been used to further support the structural characterization of metabolites. The results showed that CP undergoes a series of ligand exchange biotransformation reactions with water and other nucleophiles like thio groups of methionine, cysteine, acetylcysteine, glutathione and thioether. This is the first research approach focused on the structure elucidation of biotransformation products of CP in rats, and the identification of metabolites provides essential information for further pharmacological and clinical studies of CP, and may also be useful to develop various effective new anticancer agents.     

The yeast metabolome addressed by electrospray ionization mass spectrometry: Initiation of a mass spectral library and its applications for metabolic footprinting by direct infusion mass spectrometry

Mass spectrometry (MS) has been a major driver for metabolomics, and gas chromatography (GC)-MS has been one of the primary techniques used for microbial metabolomics. The use of liquid chromatography (LC)-MS has however been limited, but electrospray ionization (ESI) is very well suited for ionization of microbial metabolites without any previous derivatization needed. To address the capabilities of ESI-MS in detecting the metabolome of Saccharomyces cerevisiae, the in silico metabolome of this organism was used as a template to present a theoretical metabolome. This showed that in combination with the specificity of MS up to 84% of the metabolites can be identified in a high mass accuracy ESI-spectrum. A total of 66 metabolites were systematically analyzed by positive and negative ESI-MS/MS with the aim of initiating a spectral library for ESI of microbial metabolites. This systematic analysis gave insight into the ionization and fragmentation characteristics of the different metabolites. With this insight, a small study of metabolic footprinting with ESI-MS demonstrated that biological information can be extracted from footprinting spectra. Statistical analysis of the footprinting data revealed discriminating ions, which could be assigned using the in silico metabolome. By this approach metabolic footprinting can advance from a classification method that is used to derive biological information based on guilt-by-association, to a tool for extraction of metabolic differences, which can guide new targeted biological experiments. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tandem mass spectrometry approach for the investigation of the steroidal metabolism: Structure–fragmentation relationship (SFR) in anabolic steroids and their metabolites by ESI-MS/MS analysis

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used to investigate the effect of different substitutions introduced during metabolism on fragmentation patterns of four anabolic steroids including methyltestosterone, methandrostenolone, cis-androsterone and adrenosterone, along with their metabolites. Collision-induced dissociation (CID) analysis was performed to correlate the major product ions of 19 steroids with structural features. The analysis is done to portray metabolic alteration, such as incorporation or reduction of double bonds, hydroxylations, and/or oxidation of hydroxyl moieties to keto functional group on steroidal skeleton which leads to drastically changed product ion spectra from the respective classes of steroids, therefore, making them difficult to identify. The comparative ESI-MS/MS study also revealed some characteristic peaks to differentiate different steroidal metabolites and can be useful for the unambiguous identification of anabolic steroids in biological fluid. Moreover, LC–ESI-MS/MS analysis of fermented extract of methyltestosterone, obtained by Macrophomina phaseolina was also investigated.     

Metabolomic study reveals a selective accumulation of l-arginine in the d-ornithine treated tobacco cell suspension culture

The non-protein amino acid ornithine (Orn) plays essential roles in regulation of the urea cycle and polyamines biosynthesis in tobacco. Herein, we show that d-enantiomer of Orn, can actively participate in metabolites production in tobacco cells, functioning a positive role in plant cells metabolism, as opposed to the common l-enantiomer. Using a comprehensive amino acids and amines profiling method by liquid chromatography–electrospray ionization triple quadruple mass spectrometry (LC–ESI-QqQ-MS) in combination with chiral LC–ESI-MS, it was shown that d-Orn has a potential advantage in promoting selective and large accumulation of l-arginine (l-Arg) in tobacco cells. Exogenous d-Orn resulted in a selective up-regulation of l-Arg by 80-fold, while l-Orn slightly increased the levels of all amino acids. Changes of all the urea cycle related intermediates, e.g. citrulline, Arg and Orn were also shown to be critical following change of Orn's stereochemistry. GC/MS profiling of the metabolites revealed that high nicotine production was the dominant change driven by l-Orn treatments. From these observations, d-Orn was shown to be a selective regulator of l-Arg biosynthesis and the urea cycle. We propose that d-Orn has a potential function in the tobacco cells which through some previously unidentified mechanism result in l-Arg accumulation.     

Vanadyl Sulfate Administration Protects the Streptozotocin-Induced Oxidative Damage to Brain Tissue in Rats

Diabetes mellitus manifests itself in a wide variety of complications and the symptoms of the disease are multifactorial. The present study was carried out to investigate the effects of vanadyl sulfate on biochemical parameters, enzyme activities and brain lipid peroxidation, glutathione and nonenzymatic glycosylation of normal- and streptozotocin-diabetic rats. Streptozotocin (STZ) was administered as a single dose (65 mg/kg) to induce diabetes. A dose of 100 mg/kg vanadyl sulfate was orally administered daily to STZ-diabetic and normal rats, separately until the end of the experiment, at day 60. In STZ-diabetic group, blood glucose, serum sialic and uric acid levels, serum catalase (CAT) and lactate dehydrogenase (LDH) activities, brain lipid peroxidation (LPO) and nonenzymatic glycosylation (NEG) increased, while brain glutathione (GSH) level and body weight decreased. In the diabetic group given vanadyl sulfate, blood glucose, serum sialic and uric acid levels, serum CAT and LDH activities and brain LPO and NEG levels decreased, but brain GSH and body weight increased.The present study showed that vanadyl sulfate exerted antioxidant effects and consequently may prevent brain damage caused by streptozotocin-induced diabetes.     

Quantification of dinor, dihydro metabolites of F2-isoprostanes in urine by liquid chromatography/tandem mass spectrometry

The F2-isoprostanes (F2-IsoP) are a series of prostaglandin (PG)-F2-like compounds that are produced by free-radical-mediated oxidation of arachidonic acid. One F2-IsoP with potent biological activity is 15-F2t-IsoP and increased levels of 15-F(2t)-IsoP have been measured in several diseases. The major urinary metabolite of 15-F2t-IsoP (8-iso-PGF(2alpha)) is 2,3-dinor-5,6-dihydro-15-F2t-IsoP (15-F2t-IsoP-M). Previously, we developed a stable isotope dilution gas chromatography/negative chemical ionization/mass spectrometry (MS) assay for 15-F2t-IsoP-M, which, while highly sensitive, required time-consuming derivatization and thin-layer chromatography purification. We now report the development of a more rapid high-performance liquid chromatography method coupled to electrospray ionization-tandem mass spectrometry (LC/MS/MS) to analyze all of the dinor,dihydro metabolites of the F2-IsoP isomers (F2-IsoP-M). The precision of this assay was +/-5.0% and the accuracy 80%. The assay remained linear over a range of 1-100 ng injected onto the LC column. Levels of F2-IsoP-M determined by the LC/MS/MS assay method significantly correlated with levels of 15-F2t-IsoP-M determined by the GC/MS assay (R = 0.77y = 67.2x-0.5). The levels of F2-IsoP-M detected in spot urines from 40 normal subjects were 38.1+/-19.1 ng/mg creatinine (mean+/-SD). This method provides an accurate and rapid assay to assess oxidative status in vivo.     

Identification of Ginkgo biloba flavonol metabolites after oral administration to humans

An extract of Ginkgo biloba leaves (EGb) was given to healthy volunteers. Urine samples were collected for 3 days, and blood samples were withdrawn every 30 min for 5 h. The samples were purified through SPE C₁₈ cartridges and analyzed by reversed-phase LC–diode array detection for the presence of EGb metabolites. Only urine samples contained detectable amounts of substituted benzoic acids, i.e., 4-hydroxybenzoic acid conjugate, 4-hydroxyhippuric acid, 3-methoxy-4-hydroxyhippuric acid, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, hippuric acid and 3-methoxy-4-hydroxybenzoic acid (vanillic acid). In contrast to rats no phenylacetic acid or phenylpropionic acid derivatives were found in urine, thus indicating that in humans a more extensive metabolism takes place. As for rats the metabolites found in human urines accounted for less than 30% of the flavonoids given. The same procedure was applied to blood samples, and no metabolites could be detected.     

Utility of electrophoretically derived protein mass estimates as additional constraints in proteome analysis of human serum based on MS/MS analysis

The proteome of a HUPO human serum reference sample was analyzed using multidimensional separation techniques at both the protein and the peptide levels. To eliminate false-positive identifications from the search results, we employed a data filtering method using molecular weight (MW) correlations derived from denaturing 1-DE. First, the six most abundant serum proteins were removed from the sample using immunoaffinity chromatography. 1-DE was then used to fractionate the remaining serum proteins according to the MW. Gel bands were isolated and in-gel digested with trypsin, and the resulting peptides were analyzed by 2-D LC/ESI-MS/MS. A SEQUEST search using the MS/MS results identified 494 proteins. Of these, 202 were excluded formally using protein data filtering as they were single-assignment proteins and their theoretical and electrophoretically-derived MWs did not correlate at high confidence. To evaluate this method, the results were compared with those of 1-D LC/MALDI-TOF/TOF and HUPO Plasma Proteome Project analyses. Our data filtering approach proved valuable in analysis of complex, large-scale proteomes such as human serum.     

Metabolism of DHEA in postmenopausal women following percutaneous administration

The marked decline in serum dehydroepiandrosterone (DHEA) with age is believed to play a role in health problems associated with aging, these health issues being potentially preventable or reversible by the exogenous administration of DHEA. In the present study, liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) and gas chromatrography/mass spectrometry (GC/MS) were used to measure the serum levels of DHEA and 11 of its metabolites in seventy-five 60-65-year-old Caucasian women who received 3g of 0.1%, 0.3%, 1.0% or 2.0% DHEA cream or placebo applied twice daily on the face, upper chest, arms and legs. The serum levels of DHEA increased 574% over control at the 2.0% DHEA dose while the sum of the androgen metabolites androsterone glucuronide (ADT-G), 3alpha-androstenediol-3G (3alpha-diol-3G) and 3alpha-diol-17G increased by only 231%. On the other hand, serum testosterone and dihydrosterone were increased by 192% and 275%, respectively, above basal levels compared to 139% and 158% for estrone and estradiol. Such data show that the transformation of exogenous DHEA in postmenopausal women is preferentially into androgens rather than into estrogens. On the other hand, the present data indicate that serum DHEA measurements following DHEA supplementation in postmenopausal women are an overestimate of the formation of active androgens and estrogens and suggest a decreased efficiency of transformation of DHEA into androgens and estrogens with aging.     

Quantitative determination of cyclic phosphatidic acid in human serum by LC/ESI/MS/MS

An LC/ESI/MS/MS method for cyclic phosphatidic acid (cPA) quantification in serum is established in the present report. The limit of quantitation of the assay reaches low nanomolar level in human serum and the CV% are within 10%. Using this method, we successfully quantify the levels of two cPA species, 16:0 and 18:1, in human serum. We find that the concentrations of 16:0 cPA in the serum of normal subjects and post-surgery ovarian cancer patients are significantly higher than its corresponding concentration in pre-surgery ovarian cancer patients, supporting the observation that cPA has anti-cancer activity. Another discovery is that the addition of strong acids (such as hydrochloric acid) in human serum may lead to the production of artificial cPA. Therefore, strong acids should be avoided in the extraction of cPA present in a complex matrix. Based on this observation, a new lipid extraction method was developed and used to extract cPA. The extraction recovery is close to 80%, guaranteeing an accurate quantification of cPA by LC/ESI/MS/MS can be performed.