Reversible pH- and photocontrollable carbohydrate-based surfactants.

The parallel synthesis and properties of a library of photoswitchable surfactants comprising a hydrophobic butylazobenzene tail-group and a hydrophilic carbohydrate head-group, including the first surfactants to exhibit dual photo- and pH-responsive behavior, is reported. This new generation of surfactants shows varying micelle morphologies, photocontrollable surface tension, and pH-induced aggregation and adsorption.

The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota.

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.

Quantification of Gly m 4 protein, a major soybean allergen, by two-dimensional liquid chromatography with ultraviolet and mass spectrometry detection.

Soybean (Glycine max) is considered a major allergenic food. Gly m 4 is one of several soybean allergens that has been identified to cause an allergic reaction, typically the symptoms are localized effects including the skin, gastrointestinal tract, or respiratory tract. Soybean allergens are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting a range of severity from mild rashes up to anaphylaxis. In this study, we have isolated, purified, and characterized an endogenous Gly m 4 protein. The endogenous protein has 88.0% sequence homology with the theoretically predicted Gly m 4 sequence. Following detailed characterization, an assay was developed for quantification of endogenous Gly m 4 using two-dimensional liquid chromatography with ultraviolet and mass spectrometric detection (2DLC-UV/MS). A linear relationship (R(2) > 0.99) was observed over the concentration range of 12.5-531.7 µg/mL. Over the linear range, the assay recoveries (percent relative error, % RE) ranged from -1.5 to 10.8%. The assay precision (percent coefficient of variation, % CV) was measured at three different Gly m 4 levels on each of the 4 days and did not exceed 11.2%. The developed method was successfully applied to quantify Gly m 4 level in 10 commercial soybean lines. To the best of our knowledge, this represents the first quantitative assay for an intact endogenous Gly m 4 protein.

Quantification and characterization of maize lipid transfer protein, a food allergen, by liquid chromatography with ultraviolet and mass spectrometric detection.

Maize (Zea mays) is not considered a major allergenic food; however, when food induced allergenic and immunologic reactions have been implicated to maize, lipid transfer proteins (LTPs) have been identified as major allergens. LTP is an extremely stable protein that is resistant to both proteolytic attack and food processing, which permits the allergen to reach the gastrointestinal immune system in an immunogenic and allergenic conformation, allowing sensitization and induction of systemic symptoms. They are considered a complete food allergen in that they are capable of inducing specific IgE as well as eliciting severe symptoms. We have purified and characterized an endogenous ~9 kDa LTP from maize kernels. The maize LTP consists of 93 amino acid residues and has a M(r) of 9046.1 Da, determined by electrospray ionization mass spectrometry. Following accurate identification and characterization of maize LTP, a highly specific and quantitative assay using liquid chromatography with ultraviolet and mass spectrometric detection was developed. The present assay enables determination of LTP over a concentration range from 29 to 1030 µg/g in maize kernel samples. Assay recovery (percent relative error, % RE) was measured at 11 different concentrations ranging from 4 to 147 µg/mL and did not exceed 5.1%. The precision (percent coefficient of variation, % CV) was measured at 3 concentrations on each of 4 days and did not exceed 14.4%. The method was applied to evaluate the levels of LTP in 14 different maize lines. To our knowledge, this represents the first quantitative liquid chromatography-ultraviolet/mass spectrometry (LC-UV/MS) assay for the determination of LTP for the assessment of a food allergen.

Hypocholesterolemic effects of hydroxypropyl methylcellulose are mediated by altered gene expression in hepatic bile and cholesterol pathways of male hamsters.

Hydroxypropyl methylcellulose (HPMC), a semisynthetic, nonfermentable soluble dietary fiber, is not absorbed by the body, but its presence in the intestinal lumen increases fecal fat, sterol, and bile acid excretions and decreases intestinal cholesterol absorption, all of which may indirectly affect hepatic lipid metabolism. We measured the expression of hepatic genes involved in cholesterol, bile acid, and fatty acid metabolism in hamsters fed diets containing 39% of energy as fat and 5% of weight as HPMC or microcrystalline cellulose (control) for 4 wk. HPMC-fed hamsters gained significantly less body weight than the control group but did not differ in food intake. They had significantly lower plasma triglyceride and total-, VLDL-, HDL-, and LDL-cholesterol concentrations and hepatic total lipid, total and free cholesterol and triglyceride concentrations than controls. Compared with controls, HPMC-fed hamsters had greater levels of mRNA for CYP7A1 (cytochrome P450 7A1; 8-fold of control; P < 0.05), CYP51 (lanosterol 14alpha-demethylase; 5.3-fold of control; P < 0.05), and HMG-CoAR (3-hydroxy-3-methylglutaryl CoA reductase; 1.8-fold of control; P < 0.05). The plasma total cholesterol concentrations from both the control and HPMC groups were inversely correlated with expression of hepatic CYP7A1 (r = -0.54; P < 0.05), CYP51 (r = -0.79; P < 0.005), and HMG-CoAR (r = -0.75; P < 0.005) genes. This suggests that HPMC supplementation affected both cholesterol and bile acid synthesis. Our data confirm that altered hepatic expression of lipid metabolism-related genes, possibly due to modulation of fecal bile acid excretion and intestinal cholesterol absorption, contributes to the lipid-lowering effects of HPMC.

Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using immunoprecipitation liquid chromatography-mass spectrometry/mass spectrometry.

Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety but accurate analysis has been limited by the multiple naturally occurring forms and extensive similarities to gastrin. Changes in levels of one form, CCK-8, a naturally occurring eight amino acid peptide of CCK, have been correlated with satiety responses. Endogenous CCK-8 has not been well characterized in Syrian Golden hamsters, an important model in the study of fat uptake and digestion. We have cloned and sequenced hamster CCK and identified and characterized endogenous CCK-8 from hamster plasma. Hamster CCK-8 is composed of eight amino acid residues which are highly conserved among other species. Following accurate identification and characterization of hamster CCK-8, we have developed a highly specific and sensitive immunoprecipitation based LC-MS/MS assay for its quantification. The present assay enables determination of active CCK-8 over a concentration range from 0.05 to 2.5 ng/mL in hamster plasma samples. This range covers both the basal and postprandial levels of CCK-8. Method performance validation samples were examined at three concentrations replicated over the course of 4 days. The assay accuracy (percent relative error, % RE) average was 11.3% with a precision (percent coefficient of variation, % CV) of 15.5% over all samples in this 4 day period. Additionally, the method was used to determine increases of endogenous plasma CCK-8 in hamsters challenged with a high-fat meal.

Immunodepletion of high abundance proteins coupled on-line with reversed-phase liquid chromatography: a two-dimensional LC sample enrichment and fractionation technique for mammalian proteomics.

Proteomic analysis can be hampered by the large concentration distribution of proteins. Immunoaffinity techniques have been applied to selectively remove high abundant proteins (HAP's) from samples prior to analysis. Although immunodepletion of HAP's has been shown to enable greater detection of low abundance proteins, the resulting fractions are often diluted 5-10-fold during the process. Various concentration techniques can be applied; however, many are incompatible with the high salt content of the fractions. To help overcome this limitation, a two-dimensional liquid chromatography (2D-LC) method was developed which couples an IgY immunodepletion column in the first dimension with a large pore C18 analytical column in the second. A protein trap cartridge serves as an injection loop between the columns to facilitate on-line concentration and desalting. Feasibility of this 2D-LC system was demonstrated for mammalian proteomics. Beyond depletion of interfering proteins, this instrumentation provides four advantages which make immunodepletion technology more convenient, including: (1) on-line desalting (2) automatic buffer exchange (3) facile concentration and (4) fractionation by polarity.

Development of a comprehensive multidimensional liquid chromatography system with tandem mass spectrometry detection for detailed characterization of recombinant proteins.

A multidimensional comprehensive liquid-phase separation system (2DLC) coupled on-line to an electrospray-ionization time-of-flight (ESI-TOF) mass spectrometer (MS) was used to resolve structural alterations and/or post-translational modifications for detailed protein characterization. The system described in this work consists of cation-exchange chromatography in the first dimension and reversed-phase chromatography in the second dimension. A unique spiked gradient was employed in the first dimension to enhance recovery of peptides. This combination of separation followed by MS detection offered the advantages of unique selectivity and high efficiency of the separation methods combined with the mass specificity and sensitivity of MS. During the course of this study it was determined that altered or modified peptides were shown to be better resolved than during a one-dimensional separation. The 2DLC/ESI methodology allowed for a comprehensive evaluation of post-translational modifications and chemical reactions of recombinant proteins, providing a meaningful evaluation of product quality that was not possible with other current analytical approaches. In addition, the system can be used to provide sequence coverage of complex proteins.

Detection of C-terminal peptide of proteins using isotope coding strategies.

The determination of C-terminal peptide sequence is critical since the C-terminal peptide contains biologically relevant information and often undergoes post-translational processing. Another important application is in estimating purity of the biopharmaceuticals, especially for determining the presence of ragged processed ends and for N-terminally blocked polypeptides and proteins. In this paper, different isotope coding strategies in combination with reversed phase chromatography (RPC) coupled with electrospray ionization-mass spectrometry (ESI-MS) were evaluated to detect the C-terminal peptide from proteolytic digests. These were (i) O18 (ii) acylation and (iii) esterification based isotope coding strategies. Using reversed phase chromatography, the C-terminal peptide was resolved from other internal peptides. The isotope coding approaches specifically rendered a characteristic MS signature to the C-terminal peptide, thereby facilitating its detection. The unique MS signature, along with accurate mass data for the C-terminal peptide was found to be sufficient for its detection and identification. The advantages and limitations of the three approaches will be discussed.

Cross species applicability of abundant protein depletion columns for ribulose-1,5-bisphosphate carboxylase/oxygenase.

In plants, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an important enzyme in the Calvin cycle, catalyzing the first step of carbon fixation. Because of its critical role in photosynthesis, RuBisCO comprises 30-60% of the total protein content in green leaf tissue and represents a major protein which can interfere with determination of lower abundance proteins in plant proteomics. A potential solution to aid in the determination of low level proteins in plant proteomics are RuBisCO immunodepletion columns. Two formats, spin and LC, of Seppro IgY RuBisCO depletion columns were evaluated for cross species applicability. The spin and LC columns were found to deplete arabidopsis RuBisCO by greater than 90 and 98%, respectively, and automation could be achieved with the LC format. Canola RuBisCO was depleted to a similar extent, and there was evidence suggesting that corn and tobacco RuBisCO were also highly depleted in flow through fractions. Model proteins were spiked into samples to provide insight into the degree of non-specific binding. Finally, improved detection and identification of lower abundance proteins was demonstrated after depletion.

A novel HPLC-UV-MS method for quantitative analysis of protein glycosylation.

Monoclonal antibody samples derived from transgenic plants (plantibodies) may often contain significant amounts of aglycosylated variants. Because glycosylated and non-/de-glycosylated proteins exhibit different functional and pharmacokinetic properties, accurate measurement of non- and de-glycosylated glycoprotein abundances is important. Glycosylation of plant-derived glycoproteins presents specific challenges. Here we describe a novel method to accurately measure relative and absolute amounts of non-glycosylated, de-glycosylated, and total glycosylated protein using an HPLC-UV-MS methodology. Additionally, these results were compared with glycopeptide profiling by MALDI MS. Our studies demonstrated that the quantitative aspect of HPLC-UV method was superior to MALDI MS profiling, which significantly overestimated the relative amounts of aglycosylated species in the isolated glycopeptide fractions.

Altered hepatic gene expression profiles associated with improved fatty liver, insulin resistance, and intestinal permeability after hydroxypropyl methylcellulose (HPMC) supplementation in diet-induced obese mice.

The effect of hydroxypropyl methylcellulose (HPMC) on hepatic gene expression was analyzed by exon microarray and real-time PCR from livers of diet-induced obese (DIO) mice fed a high-fat (HF) diet supplemented with either 6% HPMC or 6% microcrystalline cellulose (MCC). HPMC-fed mice exhibited significantly reduced body weight gain (55% lower compared to MCC), liver weight (13%), plasma LDL-cholesterol concentration (45%), and HF diet-increased intestinal permeability (48%). HPMC significantly reduced areas under the curve for 2 h insulin and glucose responses, indicating enhanced insulin sensitivity and glucose metabolism. HPMC up-regulated hepatic genes related to fatty acid oxidation, cholesterol and bile acid synthesis, and cellular activation of glucocorticoid (bile acid recycling) and down-regulated genes related to oxidative stress, triglyceride synthesis, and polyunsaturated fatty acid elongation. In conclusion, HPMC consumption ameliorates the effects of a HF diet on intestinal permeability, insulin resistance, hepatic lipid accumulation, glucocorticoid-related bile acid recycling, oxidative stress, and weight gain in DIO mice.

HPMC supplementation reduces abdominal fat content, intestinal permeability, inflammation, and insulin resistance in diet-induced obese mice.

SCOPE: The effects of hydroxypropyl methylcellulose (HPMC), a highly viscous nonfermentable soluble dietary fiber, were evaluated on adipose tissue inflammation and insulin resistance in diet-induced obese (DIO) mice fed a high-fat (HF) diet supplemented with either HPMC or insoluble fiber. METHODS AND RESULTS: DIO C57BL/6J mice were fed a HF diet supplemented with 6% HPMC or 6% microcrystalline cellulose (MCC). Gene expression analyses of epididymal adipose tissue by exon microarray and real-time PCR along with glucose and insulin tolerance and intestinal permeability were assessed. HPMC-fed mice exhibited significantly reduced body weight gain and adipose tissue weight as well as reduced areas under the curve for 2-h insulin and glucose responses. HPMC significantly decreased HF diet-induced intestinal permeability. Overall, HPMC enhanced insulin sensitivity and glucose metabolism and downregulated genes related to inflammation and immune response, adipogenesis, and oxidative stress markers. Pathway analysis of microarray data identified lipid metabolism, inflammatory disease, and acute phase response pathways as being differentially regulated by HPMC. CONCLUSION: These results suggest HPMC consumption ameliorates HF diet effects on obesity-induced insulin resistance, adipose tissue inflammatory and immune responses, weight gain, as well as intestinal permeability.

Effects of cationic hydroxyethyl cellulose on glucose metabolism and obesity in a diet-induced obesity mouse model.

BACKGROUND: To investigate the effect of a new soluble fiber, namely cationic hydroxyethyl cellulose (cHEC), on weight loss and metabolic disorders associated with obesity using a high-fat diet-induced obese mouse model. METHODS: Obese male C57BL/6J (B6) mice were fed high-fat (60% kcal) diets supplemented with cHEC for 5 weeks. Body weight, energy intake, mesenteric adipose and liver weights, plasma cholesterol, plasma insulin, glucose, adiponectin, and leptin were assessed to determine the effects of cHEC. Hepatic and fecal lipids were also analyzed to investigate the effect of cHEC on lipid absorption and metabolism. RESULTS: Supplementation of the high-fat diet with cHEC resulted in significant weight loss in obese mice. In addition, significant decreases were seen in mesenteric adipose and liver weights, as well as concentrations of plasma cholesterol and hepatic lipids. A significant improvement in glucose homeostasis, insulin sensitivity, and leptin concentrations were observed at 4% cHEC. Moreover, increases in fecal excretion of total bile acids, sterols, and fats indicated altered fat absorption when cHEC was supplemented in the diet. CONCLUSIONS: We have shown in the present study that cHEC reduces body weight, improves insulin sensitivity, and prevents the development of metabolic syndrome. Furthermore, the effects of cHEC on glucose and lipid homeostasis in B6 mice are mediated by improvements in leptin sensitivity resulting from reduced fat absorption.

Effects of cationic hydroxyethyl cellulose on dyslipidemia in hamsters.

Cationic hydroxyethyl cellulose (cHEC) was supplemented in a high-fat diet to determine if this new soluble fiber had an effect on hypercholesterolemia and dyslipidemia associated with cardiovascular disease using Golden Syrian hamster as an animal model. Supplementation of 3-5% cHEC in a high-fat diet for 4 weeks led to significant weight gain reduction in hamsters. In addition, significant decreases in adipose and liver weights, concentrations of plasma total, VLDL, and LDL cholesterol, and hepatic lipids were shown. No significant improvements in glucose and insulin levels were observed with cHEC; however, a significant increase in plasma adiponectin and a decrease in leptin were observed. As compared with controls, 8% cHEC-fed hamsters had greater levels of mRNA for CYP7A1 (cytochrome P450 7A1; 2-fold of control; P < 0.05), CYP51 (lanosterol 14α-demethylase; 6-fold of control; P < 0.05), and LDLR (LDL receptor; 1.5-fold of control) in the liver. These findings suggest the possibility of the use of cHEC for cholesterol reduction and beneficial effects on the cardiovascular risk factors.

Two dimensional liquid chromatography-ultraviolet/mass spectrometric (2DLC-UV/MS) analyses for quantitation of intact proteins in complex biological matrices.

A conventional scale online two dimensional liquid chromatography-ultraviolet/mass spectrometric (2DLC-UV/MS) method was developed for simultaneous quantitation of intact proteins. A series of valve switches were utilized between the two LC dimensions and the mass spectrometer to resolve and confirm the proteins of interest from a complex biological matrix. Two model proteins, myoglobin and serum albumin were simultaneously resolved and quantitated from Escherichia coli lysate using a strong anion-exchange chromatography and reversed-phase chromatography as the first and second dimension respectively. The method validation consisted of evaluating linearity, precision, and accuracy. A linear relationship (R(2)>0.99) between the concentrations of the two proteins and peak areas was observed over the concentration range; 12.0-120.4 µg/mL and 8.5-85.4 µg/mL for serum albumin and myoglobin, respectively. The average RSD of peak areas for intra-day and inter-day analyses were 5.9% and 9.4% for myoglobin and 6.2% and 10.1% for serum albumin respectively. Over the linear range, the recoveries ranged from -15.4 to 9.0% for serum albumin and -2.5 to 9.4% for myoglobin. The system presented in this work is amenable to a quality control environment for evaluation and quantitation of expression levels of multiple target proteins. To our knowledge, this represents the first 2DLC-UV/MS method depicting the viability of simultaneous quantitation of more than one intact protein from complex biological mixtures in a single run.

Supplementation of hydroxypropyl methylcellulose into yeast leavened all-whole grain barley bread potentiates cholesterol-lowering effect.

We investigated in Syrian Golden hamsters the biological impact and its underlying mechanism of single whole grain breads supplemented with 2-3% hydroxypropyl methylcellulose (HPMC), a semisynthetic viscous soluble dietary fiber (SDF) as a substitute for gluten. Hamsters were fed high-fat diets supplemented with 48-65% (w/w) differently ground, freeze-dried single grain breads including whole grain wheat, barley, barley supplemented with HPMC, debranned oat, and oat supplemented with HPMC which were compared to a diet containing microcrystalline cellulose (control). All single grain breads significantly lowered plasma LDL-cholesterol concentrations compared to the control. Enrichment with HPMC further lowered plasma and hepatic cholesterol concentrations. Despite the reduced molecular weight of naturally occurring soluble (1--->3),(1--->4)-ß-d-glucan (ß-glucan) caused by the bread-making process, whole grain barley breads downregulated hepatic expression of CYP7A1 and HMG-CoAR genes that are responsible for bile acid and cholesterol synthesis, suggesting a possible role of bioactive compounds such as short-chain fatty acids and phenolic compounds from barley bread. Barley bread enriched with HPMC downregulated expression of ABCG5 gene. Taken together, it appears that distinctive modulation of synthesis and excretion of hepatic cholesterol and bile acid contributes to the cholesterol-lowering properties of whole grain barley breads and breads enriched with HPMC. These data suggests that alternative whole grain breads supplemented with HPMC may provide consumers with a staple food that can assist in cholesterol management.

Effect of hydroxypropyl methylcellulose on obesity and glucose metabolism in a diet-induced obesity mouse model.

BACKGROUND: To investigate the effect of hydroxypropyl methylcellulose (HPMC) on weight loss and metabolic disorders associated with obesity using a high-fat diet-induced obese mouse model under a high-fat diet regimen. METHODS: Obese male C57BL/6J (B6) mice were fed either a high-fat (60% kcal), low-fat (10% kcal), or high-fat diet plus HPMC (4% and 8%) for 5 weeks. Body, mesenteric adipose, and liver weights were determined at the end of the study. In addition, plasma cholesterol, insulin, glucose, adiponectin, and leptin were analyzed to determine the effects of HPMC. Hepatic and fecal lipids were measured to determine the effect of HPMC on lipid absorption and metabolism. RESULTS: Supplementation of the high-fat diet with 4% and 8% HPMC resulted in significant weight loss in obese B6 mice. Furthermore, significant decreases were seen in adipose (30%-40%), liver weights (15%-26%), and concentrations of plasma cholesterol (13%-20%) and hepatic lipids (13%-36%). Supplementation with 8% HPMC led to significant improvements in glucose homeostasis and leptin concentrations. Reductions in plasma cholesterol, glucose, and insulin levels were strongly correlated with reduced leptin concentrations. Moreover, increases in fecal secretion of total bile acids, sterols, and fats indicated altered fat absorption when HPMC was incorporated in the diet. CONCLUSION: The data indicate that HPMC not only reduces body weight, but also normalizes the metabolic abnormalities associated with obesity and suggest that the effects of HPMC on glucose and lipid homeostasis in B6 mice are mediated by improvements in leptin sensitivity resulting from reduced fat absorption.

Dietary hydroxypropyl methylcellulose increases excretion of saturated and trans fats by hamsters fed fast food diets.

In animal studies, hydroxypropyl methylcellulose (HPMC) intake results in increased fecal fat excretion; however, the effects on dietary saturated fatty acids (SATs) and trans-fatty acids (TRANS) remain unknown. This study investigated the effect of HPMC on digestion and absorption of lipids in male Golden Syrian hamsters fed either freeze-dried ground pizza (PZ), pound cake (PC), or hamburger and fries (BF) supplemented with dietary fiber from either HPMC or microcrystalline cellulose (MCC) for 3 weeks. We observed greater excretion of SATs and TRANS by both diets supplemented with HPMC or MCC as compared to the feed. SAT, TRANS, and unsaturated fatty acids (UNSAT) contents of feces of the PZ diet supplemented with HPMC were 5-8 times higher than diets supplemented with MCC and tended to be higher in the PC- and BF-HPMC supplemented diets as well. We also observed significant increases in fecal excretion of bile acids (2.6-3-fold; P < 0.05), sterols (1.1-1.5-fold; P < 0.05), and unsaturated fatty acids (UNSAT, 1.7-4.5-fold; P < 0.05). The animal body weight gain was inversely correlated with the excretion of fecal lipid concentrations of bile acids (r = -0.56; P < 0.005), sterols (r = -0.48; P < 0.005), SAT (r = -0.69; P < 0.005), UNSAT (r = -0.67; P < 0.005), and TRANS (r = -0.62; P < 0.005). Therefore, HPMC may be facilitating fat excretion in a biased manner with preferential fecal excretion of both TRANS and SAT in hamsters fed fast food diets.

Determination of F2-isoprostanes in urine by online solid phase extraction coupled to liquid chromatography with tandem mass spectrometry.

F(2)-isoprostanes are a unique class of prostaglandin-like compounds formed in vivo, which have been established as biomarkers of oxidative stress. Accurate analysis has been challenging due to lack of specificity for the isoforms of isoprostanes and lengthy sample preparation procedures to enable trace quantitative analysis. A quantitative analytical method was developed for the determination of F(2)-isoprostanes in rat and hamster urine by online solid phase extraction (SPE) coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS). The online SPE LC-MS/MS procedure has significant advantages over alternative methods with respect to specificity, sensitivity, simplicity, and speed. The assay enables the detection of iPF(2alpha)-III, iPF(2alpha)-IV, and iPF(2alpha)-VI over a linear dynamic range of 0.1-50 ng/mL in rat urine samples. This range covers the basal levels of these F(2)-isoprostanes. The limit of quantitation (LOQ) for the standard isoprostanes was about 0.3 ng/mL. The average recoveries ranged from 73 to 115% depending upon the individual F(2)-isoprostane isomers in rat urine. Additionally, the method was used to determine increases of endogenous urine iPF(2alpha)-VI and iPF(2alpha)-III in hamsters challenged with either low-fat or high-fat diets.