Profiling of urinary bile acids in piglets by a combination of enzymatic deconjugation and targeted LC-MRM-MS.

We present a method using a combination of enzymatic deconjugation and targeted LC-multiple reaction monitoring (MRM)-MS analysis for analyzing all common bile acids (BAs) in piglet urine, and in particular, for detecting conjugated BAs either in the absence of their standards, or when present in low concentrations. Initially, before enzymatic deconjugation, 19 unconjugated BAs (FBAs) were detected where the total concentration of the detected FBAs was 9.90 µmol/l. Sixty-seven conjugated BAs were identified by LC-MRM-MS analysis before and after enzymatic deconjugation. Four enzymatic assays were used to deconjugate the BA conjugates. FBAs in urine after cholylglycine hydrolase/sulfatase treatment were 33.40 µmol/l, indicating the urinary BAs were comprised of 29.75% FBAs and 70.25% conjugated BAs in single and multiple conjugated forms. For the conjugates in single form, released FBAs from cholylglycine hydrolase deconjugation indicated that the conjugates with amino acids were 14.54% of urinary BAs, 16.27% glycosidic conjugates were found by ß-glucuronidase treatment, and sulfatase with glucuronidase inhibitor treatment liberated FBAs that constituted 16.67% of urinary BAs. Notably, chenodeoxycholic acid (CDCA) was initially detected only in trace amounts in urine, but was found at significant levels after the enzymatic assays above. These results support that CDCA is a precursor of γ-muricholic acid in BA biosynthesis in piglets.

Diets Containing Shiitake Mushroom Reduce Serum Lipids and Serum Lipophilic Antioxidant Capacity in Rats.

BACKGROUND: We previously reported that dietary intake of shiitake mushroom (SM; Lentinus edodes) decreased serum concentrations of polar lipids in male rats. OBJECTIVE: This study evaluated the dietary effects of SM on serum cholesterol-related and serum antioxidant indexes in rats of both sexes. METHODS: Sprague-Dawley rats [38 dams and their offspring (20 males and 20 females/diet)] were fed diets containing 0 (control), 1%, 4%, or 10% (wt:wt) SM powder from gestation day 4 through to postnatal day (PND) 126. Biochemical indexes were monitored during the midgrowth phase (PNDs 50-66). RESULTS: The food consumption by offspring fed the control diet and diets supplemented with SM was not different when measured on PND 65. However, the 4% and 10% SM diets resulted in male rats with 7% lower body weights than those of the other 2 groups on PND 66. SM consumption dose-dependently decreased the concentrations of lipidemia-related factors in sera, irrespective of sex. At PND 50, serum concentrations of total cholesterol, HDL cholesterol, and non-HDL cholesterol in SM-fed male and female rats were generally lower (3-27%) than those in the corresponding control groups. Consumption of the 10% SM diet resulted in significantly decreased (55%) serum triglyceride concentrations relative to the control groups for both sexes. The 10% SM diet elicited a 62% reduction of serum leptin concentrations in females but not in males, and this same diet increased serum insulin (137%) and decreased serum glucose (15%) in males compared with controls. Serum lipophilic antioxidant capacity in males and females fed SM diets was generally lower (31-86%) than that in the control groups. CONCLUSION: SM decreased the concentrations of lipidemia-related factors in rat sera irrespective of sex. The SM-elicited reduction of lipophilic antioxidant capacity irrespective of sex may reflect a lower pro-oxidative state and, hence, improved metabolic profile.

Moutan cortex extract exerts protective effects in a rat model of cardiac ischemia/reperfusion.

Moutan cortex (MC) is a traditional Chinese medicine with diverse biological effects. The present study was performed to investigate the effects of MC on myocardial ischemia/reperfusion (I/R) in rats and to explore its possible mechanisms. Sprague-Dawley rats were administered MC extract (1.98 g/kg, i.g.) for 14 days and underwent a subsequent open-chest procedure involving 30 min of myocardial ischemia and 60 min of reperfusion. The cardioprotective effect of MC was demonstrated by reduced infarct size and marked improvement in the histopathological examination. The increase in the activity of superoxide dismutase (SOD) and glutathione (GSH) as well as the reduction of malondialdehyde (MDA) indicated that MC effectively promoted the anti-oxidative defense system. Increased anti-oxidative defense was accompanied by decreased release of lactate dehydrogenase (LDH) and creatine kinase (CK). The reduction in TUNEL-positive myocytes demonstrated that MC decreased myocardial apoptosis. The mRNA expression of B cell leukemia-2 (Bcl-2) was upregulated by MC and the ratio of Bcl-2/Bcl-2-associated X protein (Bax) mRNA expression was increased. MC pretreatment decreased the mRNA expression of inducible nitric oxide synthase (iNOS). The data from this study suggest that MC exerted protective effects on acute myocardial I/R injury via anti-oxidative and anti-apoptotic activities.

Antidiabetic Effect of an Active Components Group from Ilex kudingcha and Its Chemical Composition.

The leaves of Ilex kudingcha are used as an ethnomedicine in the treatment of symptoms related with diabetes mellitus and obesity throughout the centuries in China. The present study investigated the antidiabetic activities of an active components group (ACG) obtained from Ilex kudingcha in alloxan-induced type 2 diabetic mice. ACG significantly reduced the elevated levels of serum glycaemic and lipids in type 2 diabetic mice. 3-Hydroxy-3-methylglutaryl coenzyme A reductase and glucokinase were upregulated significantly, while fatty acid synthetase, glucose-6-phosphatase catalytic enzyme was downregulated in diabetic mice after treatment of ACG. These findings clearly provided evidences regarding the antidiabetic potentials of ACG from Ilex kudingcha. Using LC-DAD/HR-ESI-TOF-MS, six major components were identified in ACG. They are three dicaffeoylquinic acids that have been reported previously, and three new triterpenoid saponins, which were the first time to be identified in Ilex kudingcha. It is reasonable to assume that antidiabetic activity of Ilex kudingcha against hyperglycemia resulted from these six major components. Also, synergistic effects among their compounds may exist in the antidiabetic activity of Ilex kudingcha.

Enhanced expression and glucocorticoid-inducibility of hepatic cytochrome P450 3A involve recruitment of the pregnane-X-receptor to promoter elements in rats fed soy protein isolate.

Previous studies and Expt. 1 of the current study demonstrate that diets made with soy protein isolate (SPI) enhance the glucocorticoid-inducibility of hepatic cytochrome P450 (CYP)3A-dependent monooxygenase activities (P < 0.05) compared with diets made with casein (CAS). To determine the underlying molecular mechanism, in a second experiment, we analyzed the time course of dexamethasone (DEX)-induction of hepatic CYP3A mRNA expression on postnatal d (PND) 25 and PND60 in male and female rats fed SPI- or CAS-based diets. After 50 mg(/)kg DEX, CYP3A1 mRNA expression increased >200-fold in SPI-fed males and females at PND25 compared with a 100-fold increase in CAS-fed rats (P < 0.05). The DEX-induced increase in CYP3A1 mRNA in SPI-fed rats on PND60 was also greater than that in CAS-fed rats. The induction by DEX of CYP3A2 mRNA was 1- to 3-fold greater in rats fed SPI compared with those fed CAS on PND25 (P < 0.05). Quantitation of newly synthesized CYP3A1 RNA transcripts by nuclear run-on analysis demonstrated a greater rate of basal transcription in SPI-fed compared with CAS-fed rats on PND60 accompanied by greater binding of the pregnane X receptor (PXR) to a response element on the CYP3A1 promoter in SPI-fed compared with CAS-fed rats (P < 0.05). These data suggest that increased hepatic CYP3A expression and inducibility following SPI feeding involves recruitment of PXR to its response element and suggests that soy consumption has potential effects on metabolism and transport of a wide variety of drugs and on bile acid homeostasis via proteins regulated by this transcription factor.

Gambogenic acid inhibits proliferation of A549 cells through apoptosis-inducing and cell cycle arresting.

Although anticancer effect of gambogic acid (GA) and its potential mechanisms were well documented in past decades, limited information is available on the anticancer effect of gambogenic acid (GNA), another major active component of Gamboge. Here we performed a study to determine whether GNA possesses anticancer effect and find its potential mechanisms. The results suggested that GNA significantly inhibited the proliferation of several tumor cell lines in vitro and in vivo. Treatment with GNA dose and time dependently induced A549 cells apoptosis, arrested the cells to G0/G1 phase in vitro and down-regulated the expression of cyclin D1 and cyclooxygenase (COX)-2 in mRNA level. In addition, anticancer effect was further demonstrated by applying xenografts in nude mice coupled with the characteristic of apoptosis in the GNA treated group. Taken together, these observations might suggest that GNA inhibits tumor cell proliferation via apoptosis-induction and cell cycle arrest.

Involvement of ubiquitin proteasome system in protective mechanisms of Puerarin to MPP(+)-elicited apoptosis.

It has been well documented that dysfunction of ubiquitin proteasome system (UPS) in the neuron exacerbated the Parkinson's disease (PD). However, whether or not UPS is involved in the protective effect of Puerarin on 1-Methyl-4-Phenyl-1, 2, 3, 6-Tetrahydropyridine (MPP(+))-elicited cell death is yet to be elucidated. In this study, treatment of SH-SY5Y cells with 1mM MPP(+)-elicited a characteristic apoptotic cell death and pretreatment with Puerarin protected cells against MPP(+)-induced apoptosis as evidenced by promoting cell viability, improving morphological changes and reducing apoptotic rate. To further explore the potential protective mechanism of Puerarin in MPP(+)-induced SH-SY5Y cell death, UPS activity, mitochondria-dependent apoptosis and caspase-3 activity were measured. Puerarin pretreatment attenuated MPP(+)-induced dysfunction of protease activity, thereby reducing accumulation of ubiquitin-conjugated proteins. Meanwhile, caspase-3 activity was remarkably attenuated by Puerarin. In addition, the ratio of bcl-2/bax was increased by Puerarin in comparison with MPP(+)-treated group. Taken together, these results suggest that Puerarin could protect MPP(+)-induced SH-SY5Y cells from apoptosis by regulating the function of UPS.

Quantitative analysis of polar lipids in the nanoliter level of rat serum by liquid chromatography/mass spectrometry/mass spectrometry.

Polar lipids in serum, including lysophospholipids (LPLs) and free fatty acids (FFAs), have a broad range of biological activities and require a suitable method for their quantitative analysis. Conventional methods use multistep procedures to simultaneously purify and analyze polar lipids and non-polar lipids in serum. However, the methods could result in inaccurate quantifications of polar and/or non-polar lipids because compounds with different polarities have different behaviors in solvent extraction and mass spectrometric ionization. In this study, a method was designed to analyze polar lipids in serum based on the polarities of LPLs and FFAs. The method consisted of extraction without filtration and analysis of the crude extract without multistep purification. Fifty LPLs and 32 FFAs were detected in rat serum. The concentrations of LPLs (1272.1 micromole/L in female and 999.8 micromole/L in male) and FFAs (1910.9 micromole/L in female and 1651.4 micromole/L in male) were determined. Peak areas of MS ion in Extract Ion Chromatogram (EIC) were used for the quantification in this study. The approach of quantification should be perfectly suitable for precise quantification of a specific serum component by adding its isotope standard to the serum before extraction.

Triterpenoid saponins from Anemone flaccida induce apoptosis activity in HeLa cells.

Five triterpenoid saponins were isolated from Anemone flaccida Fr. Schmidt. Their structures were identified as glycoside St-I4a (1), glycoside St-J (2), anhuienoside E (3), hederasaponin B (4), and flaccidoside II (5). Compounds 1-2 were isolated from Anemone family for the first time, and compounds 3-4 were isolated from this plant for the first time. The inhibitory effects of saponins on proliferation of HeLa cells were studied by MTT assay, the apoptosis-induction activity was observed by cell-cycle analysis and caspase-3 expression assay. The antitumor activities of the saponins were ranked in the following order: 5 > 3 > 4 > 1 > 2. The data presented here indicated that naturally occurring triterpenoid saponins can be regarded as excellent structures for the potential development of new anticancer agents.

Quantification of vitamin E and gamma-oryzanol components in rice germ and bran.

Rice bran is a rich natural source of vitamin E and gamma-oryzanol, which have been extensively studied and reported to possess important health-promoting properties. However, commercial rice bran is a mixture of rice bran and germ, and profiles of vitamin E and gamma-oryzanol components in these two different materials are less well-studied. In the current study, vitamin E and gamma-oryzanol components in rice bran and germ were analyzed by liquid chromatography/mass spectrometry/mass spectrometry. The components were identified by electrospray ionization mass spectrometry (ESI-MS) with both positive- and negative-ion modes. Both deprotonated molecular ion [M - H](-) and protonated molecular ion [M + H](+) found as the base peaks in spectra of vitamin E components made ESI-MS a valuable analytic method in detecting vitamin E compounds, especially when they were at very low levels in samples. Ultraviolet absorption was used for quantification of vitamin E and gamma-oryzanol components. While the level of vitamin E in rice germ was 5 times greater than in rice bran, the level of gamma-oryzanol in rice germ was 5 times lower than in rice bran. Also, the major vitamin E component was alpha-tocopherol in rice germ and gamma-tocotrienol in rice bran. These data suggest that rice bran and germ have significantly different profiles of vitamin E and gamma-oryzanol components. The method enables rapid and direct on-line identification and quantification of the vitamin E and gamma-oryzanol components in rice bran and germ.

In vitro actions on human cancer cells and the liquid chromatography-mass spectrometry/mass spectrometry fingerprint of phytochemicals in rice protein isolate.

Rice protein isolate (RPI) has been reported to reduce the incidence of 7,12-dimethylbenz[a]anthracene-induced mammary tumors in rats. To determine the potential role of phytochemicals associated with the RPI, we studied in vitro antitumor activities of an ether fraction from RPI using human tumor cell lines, including two human breast carcinoma cell lines (MDA-MB-453 and MCF-7) and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent antiproliferative effects of the ether fraction were observed in all cell lines using the standard 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Fraction-induced apoptosis (P < 0.05) was detected in all cell lines, and this was associated with the induction of proapoptotic bax protein and cdk inhibitors (p21) and the suppression of cdk4 and cyclin D1 activity. Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) with both positive and negative modes was used to analyze the phytochemicals in the ether fraction from RPI. Fifty-seven phytochemicals were identified or characterized by their diagnostic fragmentation patterns and direct comparison with the authentic standards on the basis of electrospray ionization-MS/MS data. The major components bound to RPI were lysoglycerophospholipids, fatty acids, and fatty acid 3-[2-(2,3-dihydroxy-propoxycarbonyl)-2-hydroxy-ethoxy]-2-hydroxy-propyl esters.

Inhibition of growth and induction of apoptosis in human cancer cell lines by an ethyl acetate fraction from shiitake mushrooms.

OBJECTIVE: Shiitake (Lentinus edodes) mushrooms have been reported to have cancer-preventing properties. However, little research has been conducted verifying the antitumor activities of "mycochemicals" in shiitake mushrooms. In this study, potential roles of an ethyl acetate fraction from shiitake mushrooms were investigated by in vitro bioassays. DESIGN: The activities of an ethyl acetate fraction were evaluated by [3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide] (MTT), apoptosis bioassay, cell cycle analysis, and Western blot analysis using two human breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human nonmalignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9). RESULTS: Concentration-dependent antiproliferative effects of the fraction were observed in all cell lines using the MTT assay. Approximately 50 mg/L concentration of the fraction induced apoptosis in 50% of the population of four human tumor cell lines and the fraction-induced apoptosis may have been mediated through the pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell cycle arrest by significant decrease of S phase, which was associated with the induction of cdk inhibitors p21 and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumor cells, nonmalignant cells were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and cdk4 expression. A 51% antiproliferative effect occurred at the highest concentration of the fraction (800 mg/L). CONCLUSIONS: These data suggest that inhibition of growth in tumor cells by "mycochemicals" in shiitake mushrooms may result from induction of apoptosis.

Isoflavone conjugates are underestimated in tissues using enzymatic hydrolysis.

Many health effects of soy foods are attributed to isoflavones. Isoflavones upon absorption present as free form, glucuronide, and sulfate conjugates in blood, urine, and bile. Little is known about the molecular forms and the relative concentrations of soy isoflavones in target organs. Acid hydrolysis or enzymatic hydrolysis (glucuronidases and sulfatases) was used to study isoflavone contents in the heart, brain, epididymis, fat, lung, testis, liver, pituitary gland, prostate gland, mammary glands, uterus, and kidney from rats fed diets made with soy protein isolate. The heart had the lowest isoflavone contents (undetectable), and the kidney had the highest (1.8 +/- 0.6 nmol/g total genistein; 3.0 +/- 1.1 nmol/g total daidzein). Acid hydrolysis released 20-60% more aglycon in tissues than enzymatic digestion (p < 0.05), and both hydrolysis methods gave the same level of isoflavones in serum. Approximately 28-44% of the total isoflavone content within the liver was unconjugated aglycon, and the remainder was conjugated mainly as glucuronide. The subcellular distribution of total isoflavones was 55-60% cytosolic and 13-16% in each of the nuclear, mitochondrial, and microsomal fractions. These results demonstrated that (1) soy isoflavones distribute in a wide variety of tissues as aglycon and conjugates and (2) the concentrations of isoflavone aglycons, which are thought to be the bioactive molecules, are in the 0.2-0.25 nmol/g range, far below the concentrations required for most in vitro effects of genistein or daidzein.

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples.

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.

Novel Bioactive Cubé Insecticide Constituents: Isolation and Preparation of 13-homo-13-Oxa-6a,12a-dehydrorotenoids.

Two novel rotenoid-like compounds isolated from cubé resin, used as an insecticide and piscicide, have an unprecedented skeleton consisting of a 1,5-benzodioxepin (A and B rings) and a chromone (C and D rings). Their structures are assigned as (-)-13-homo-13-oxa-6a,12a-dehydrorotenone (5) and 13-homo-13-oxa-6a,12a-dehydrodeguelin (6) based on (1)H and (13)C NMR, MS, UV, and optical rotation. X-ray structure determination confirmed the assignment of 5 and revealed that the 1-methylethenyl group is disordered into two sets of atoms. Compound 5 from cubé resin was identical with a product previously reported as being formed by reaction of rotenone (1) with acetyl chloride and DMF and misassigned as "6a,12a-epoxyrotenone" (7). A modified procedure was developed for preparation of the new oxarotenoid component of the commercial insecticide. The cubé resin from which 5 and 6 were isolated in equal amounts contains 1 and deguelin (2) in a >10:1 ratio; this large difference in ratio indicates that 5 and 6 are not artifacts formed from 1and 2, respectively. Rotenoid 5 acts as a respiratory inhibitor with 50% inhibition of NADH:ubiquinone oxidoreductase activity at 0.11 &mgr;M, of goldfish survival at 1 ppm, and of the viability of three cell lines at 4-8 &mgr;M.

Characterization of isoflavones and their conjugates in female rat urine using LC/MS/MS.

Isoflavone phytoestrogens found in soybeans are the most widely studied phytochemicals in human diets and soy infant formulas. The health benefits of the isoflavones daidzein and genistein have been reported, and concerns about potential adverse effects have also been raised. However, the results of direct analysis of isoflavones and their metabolites in biological fluids after consumption of soy-containing diets are scarce. This study describes an LC/MS/MS method for the analysis of isoflavones and their metabolites in the urine of female rats fed diets made with soy protein isolate. Five isoflavones (daidzein, genistein, glycitein, dihydrodaidzein, and O-desmethylangolensin) were identified by comparison with authentic standards. Seventeen conjugates of isoflavones were characterized in the urine, the most unusual being genistein 5-glucuronide and four glucuronide conjugates of reductive metabolites of daidzein. The application of LC/MS/MS to analyze isoflavone metabolites is simple and sensitive, and appears to be an excellent method for determining the bioavailability and metabolism of food phytochemistry.

LC-MS/MS analysis of lysophospholipids associated with soy protein isolate.

Approximately 20-25% of babies consuming formula are fed formulas containing soy protein as the sole protein source. Isoflavone phytoestrogens are considered as major bioactive components present in soy protein isolate (SPI) and the most widely studied phytochemicals in human diets and soy infant formulas. In the present study, LC-MS/MS analysis of SPI phytochemical extract resulted in identifications of fifty-six lysophospholipids (lyso-PL), including eighteen lysophosphatidylcholines (lyso-PC), twelve lysophosphatidylethanolamines (lyso-PE), eleven lysophosphatidylinositols (lyso-PI), eleven lysophosphatidic acids (lyso-PA) and four lysophosphatidylglycerols (lyso-PG). The LC-MS/MS conditions were first developed using commercially available standard mixtures. Under these conditions, lyso-PL compounds could be separated by LC and yielded unique fragments and neutral losses in the electrospray ionization mass spectrometry (ESI-MS) with both positive- and negative-ion modes, quite indicative of the polar headgroups for different subclasses. This is the first characterization of lyso-PL in SPI. Lyso-PLs are important cell signaling and growth factor molecules experimentally linked to cardiovascular function and disease, cancer, and neurological disorders. These findings suggest that biological effects of PLs associated with SPI in infants clearly needs to be considered as part of an overall evaluation of potential health benefits.

Comprehensive phytochemical profile of soy protein isolate.

Although an FDA health claim for soy protein has been issued, the potential health benefits of soy foods remain controversial among scientists, especially with regard to soy infant formula. The UV detectable isoflavones have been the focus of the majority of studies concerning health-related effects of soy protein isolate (SPI). However, the chemical identities and health effects of other SPI phytochemicals without UV absorption properties are less well-studied. In the current study, we employed liquid chromatography-tandem mass spectrometry methods to reveal a complicated phytochemical profile for SPI consisting of 136 phytochemicals. Also, we have quantitated many of these SPI phytochemicals so that dietary intakes can be estimated for foods containing SPI. On a weight/weight basis, fatty acids are the largest group of phytochemicals in the extract (64.13% total fat), followed by saponins (21.48%), and then isoflavones at 6.82%. Of the 56 lysophospholipids identified in SPI, 0.50% was lysophosphatidylcholines and 0.23% was lysophosphatidylethanolamines.

Characterization of triterpene alcohol and sterol ferulates in rice bran using LC-MS/MS.

Ferulic acid esters of triterpene alcohols and sterols in rice bran oil have been extensively studied and reported to possess important pharmacological actions. Inconsistent results on the numbers and structures of ferulates have been reported, primarily because of the analytical procedures employed. Conventional methods for analysis of phytosterol content in oil are carried out by characterization of trimethylsilylated derivatives (TMS) using GC-EI-MS after saponification of oils or individual compound isolated from oils. This study developed an LC-MS/MS method for the direct analysis of triterpene alcohol and sterol esters in rice bran oil. In addition to verifying the results of previous research, nine new relatively polar triterpene alcohol and sterol esters were characterized by their retention behaviors in LC and ESI-MS data from both negative- and positive-ion mode. This is the first evidence for the presence of hydroxylated ferulate esters and caffeate esters as part of gamma-oryzanol in rice bran. The method enables rapid and direct on-line characterization of triterpene alcohol and sterol esters in oils. LC-MS/MS equipped with reverse-phase LC and ESI-MS should be well-suited for identification and quantification of the polar metabolites of phytosterols in biological fluids after consumption of rice bran oil or other oils.

LC/MS/MS characterization of phenolic constituents in dried plums.

Dried plums are known as a healthy food in the West and used as medicine in India. They have been characterized by high concentrations of phenolic compounds, which are believed to play a crucial role in protection against various age-related diseases. Liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) with four different conditions was used to analyze the phytochemicals in commercial dried plums. The major components were neochlorogenic acid and cryptochlorogenic acid. Forty minor components were characterized by their MS/MS spectra and LC retention time. Six of them are novel ester isomers formed by two caffeic acids and one quinic acid. The diagnostic fragmentation patterns of different phenolics are presented on the basis of electrospray ionization (ESI) MS/MS data of components in dried plums and fourteen authentic standards.