Metabolic profiling of urine in young obese men using ultra performance liquid chromatography and Q-TOF mass spectrometry (UPLC/Q-TOF MS)

Obesity is currently epidemic in many countries worldwide. In the young adult, obesity often accompanies hyperlipemia, which is strongly related to the occurrence and development of obesity-related chronic diseases such as diabetes mellitus, hypertension and cardiovascular disease. This study investigated the differences in metabolomic profiling between obese (with hyperlipemia, n=30) and normal-weight (n=30) young men. Anthropometric parameters and conventional metabolites were measured. There were no significant differences between obese and normal-weight young men in age, height and fasting plasma glucose level. Obese young men showed increased weight, body mass index, fat mass, systolic blood pressure, and triglyeride, total cholesterol and insulin levels, and lower levels of testosterone. The endogenous metabolite profile of urine was investigated by UPLC/Q-TOF MS (ultra performance liquid chromatography and Q-TOF mass spectrometry) with electrospray ionization (ESI). Partial least squares (PLS) enabled clusters to be visualized. Eight urine principal metabolites contributing to the clusters were identified; these included increased L-prolyl-L-proline, leucyl-phenylalanine, and decanoylcarnitine in positive ESI mode (m/z 213.1267, 279.1715 and 316.2459, respectively) and N-acetylornithine, 17-hydroxypregnenolone sulfate, 11β-hydroxyprogesterone, 5a-dihydrotestosterone sulfate and glucosylgalactosyl hydroxylysine in negative ESI mode (m/z 173.0931, 411.1883, 331.185, 369.1751 and 485.1875, respectively). These metabolite changes in obese men suggested early changes of metabolism in young-male obesity with hyperlipemia. The study may further aid the clinical prevention and treatment of obesity and related chronic disease.     

Combining ion pairing agents for enhanced analysis of oligonucleotide therapeutics by reversed phase-ion pairing ultra performance liquid chromatography (UPLC)

The burgeoning field of oligonucleotide therapeutics is based upon synthetically derived biopolymers comprised of relatively simple RNA and DNA building blocks. Significant gains in knowledge around mechanisms of action (RNA interference, RNA splicing, etc.) and oligonucleotide design (ASO, siRNA, DsiRNA, miRNA, locked nucleic acid, etc.) have been the main drivers of recent investment for this field [1,2]. As therapeutics, there is currently great interest in oligonucleotides due to the reduced time required to achieve lead molecules and to their potential for treating previously untractable diseases. One of the more challenging areas for the field of oligonucleotide therapeutics is the development of high-quality analysis schemes for the determination of purity in drug substance and product. This, in part, is due to the fact that the synthesis of oligonucleotides results in a significant number of closely related impurities that are not easily removed during purification [1]. As a result, these macromolecules (4000-8000 MW on average, depending on chain length) and their soup of closely related impurities are typically not well resolved from one another via conventional chromatographic approaches. One of the more common chromatographic techniques used for oligonucleotide analysis is reversed phase-ion pairing liquid chromatography (RP-IP). Our research led us to the discovery that the use of multiple ion pairing agents combined in the mobile phase can improve the overall chromatographic resolution and peak shape of the oligonucleotide analytes over the use of a single ion pairing agent alone, resulting in enhanced purity analysis and the opportunity to identify related impurities with greater certainty. In addition, the use of combined ion pairing agents allowed for the development of a "universal" method which has provided superior chromatography for several different oligonucleotide compounds and their related impurities regardless of differences in nucleotide sequence. The RP-IP UPLC method conditions are ESI-MS compatible and have allowed for the mass identification of five positional isomeric impurities chromatographically resolved and present at less than 1% of the nominal parent peak area.     

Quantitative analysis of anandamide and related acylethanolamides in human seminal plasma by ultra performance liquid chromatography tandem mass spectrometry

The endocannabinoids anandamide, palmitoylethanolamide and oleoylethanolamide have been detected in human seminal plasma and are bioactive lipids implicated in regulation of sperm motility, capacitation and acrosome reaction. Several methods exist for endocannabinoid quantification but none have been validated for measurement in human seminal plasma. We describe sensitive, robust, reproducible solid phase and isotope-dilution UHPLC-ESI-MS/MS methods for the extraction and quantification of anandamide, palmitoylethanolamide and oleoylethanolamide in human seminal plasma. Precision and accuracy were evaluated using pooled seminal plasma over a 4 day period. For all analytes, the inter- and intraday precision (CV%) was between 6.6-17.7% and 6.3-12.5%, respectively. Analyses were linear over the range 0.237-19nM for anandamide and oleoylethanolamide and 0.9-76nM for PEA. Limits of detection (signal-to-noise >3) were 50, 100 and 100fmol/mL and limits of quantification (signal-to-noise >10) were 100, 200 and 200fmol/mL, respectively for anandamide, palmitoylethanolamide and oleoylethanolamide. Anandamide and oleoylethanolamide were stable at -80°C for up to 4 weeks, but palmitoylethanolamide declined significantly. We assessed seminal plasma from 40 human donors with normozoospermia and found mean (inter-quartile range) concentrations of 0.21nM (0.09-0.27), 1.785nM (0.48-2.32) and 15.54nM (7.05-16.31) for anandamide, oleoylethanolamide and palmitoylethanolamide, respectively. Consequently, this UHPLC-ESI-MS/MS method represents a rapid, reliable and reproducible technique for the analysis of these endocannabinoids in fresh seminal plasma.     

High resolution "ultra performance" liquid chromatography coupled to oa-TOF mass spectrometry as a tool for differential metabolic pathway profiling in functional genomic studies

The combination of a new 1.7 mum reversed-phase packing material, and a chromatographic system, operating at ca. 12,000 psi, (so-called ultra performance liquid chromatography, UPLC) has enabled dramatic increases in chromatographic performance to be obtained for complex mixture separation. This increase in performance is manifested in improved peak resolution, together with increased speed and sensitivity. Here, we show that UPLC offers significant advantages over conventional reversed-phase HPLC amounting to a more than doubling of peak capacity, an almost 10-fold increase in speed and a 3- to 5-fold increase in sensitivity compared to that generated with a conventional 3.5 microm stationary phase. The first functional genomic application of UPLC-MS technology is illustrated here with respect to multivariate metabolic profiling of urines from males and females of two groups of phenotypically normal mouse strains (C57BL19J and Alpk:ApfCD) and a "nude mouse" strain. We have also compared this technology to conventional HPLC-MS under similar analytical conditions and show improved phenotypic classification capability of UPLC-MS analysis together with increased ability to probe differential pathway activities between strains as a result of improved analytical sensitivity and resolution.     

Quantitation of ursolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry and its pharmacokinetic study

Ursolic acid is a hydroxy pentacyclic triterpene, which proved to have sedation, anti-inflammatory, antibacterial, antiulcer and anti-cancer activities. An ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method with high selectivity, sensitivity and throughput has been established and validated for quantitation of total ursolic acid in human plasma. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate and were chromatographed by an ACQUITY UPLC BEH C(8) column (100 mm×2.1 mm, I.D., 1.7 μm) using mobile phase consisting of acetonitrile and 10 mM ammonium formate (90:10, v/v) at 0.2 mL/min. The duration of chromatography analysis was 3 min. The multiple reaction monitoring (MRM) was performed at m/z 455.1→455.0 for ursolic acid and m/z 469.3→425.2 for glycyrrhetinic acid (internal standard, IS) in the negative ion mode with electrospray ionization (ESI) source. The assay showed good linearity over the range of 10-5000 ng/mL for ursolic acid in human plasma with a lower limit of quantitation of 10 ng/mL. The mean extraction recovery was 73.2±4.5% and the matrix ion suppression ranged from -11.4% to -5.6%. The intra- and inter-day precisions were less than 7.0% and 7.2%, respectively, and the accuracy was within ±2.0%. Ursolic acid was stable during the analysis and the storage period. The validated method has been successfully applied to a pharmacokinetic study after intravenous infusion of Ursolic Acid Nano-liposomes to healthy volunteers.     

Fluorometric analysis of pectenotoxin-2 in microalgal samples by high performance liquid chromatography

A rapid HPLC method with fluorescence detection of pectenotoxin-2 (PTX2), a polyether macrolide toxin, in microalgae is presented. A dienophile reagent, DMEQ-TAD, was used for precolumn fluorescence labeling. PTX2 could be quantitatively detected in the range 1-200 ng. This method confirmed the occurrence of PTX2 in net haul samples mostly composed of dinoflagellates Dinophysisspp. collected in the Adriatic Sea, Italy and Mutsu Bay, Japan.     

Advances in the analysis of amino acid phenylthiohydantoins by high performance liquid chromatography

All the organic soluble amino acid phenylthiohydantoins are eluted (but not totally resolved) in under 40 min on either a Zorbax ODS or Permaphase ETH column. The superior resolution of the Zorbax ODS column and the complimentary characteristic of the Permaphase ETH column allow the isocratic analysis of 15 phenylthiohydantoins in less than 20 min when the columns are operated simultaneously. The water soluble PTH-His and -Arg are analyzed separately in under 10 min with the Zorbax ODS column. Five picomoles of a derivative may be detected with a sample to noise ratio of 6, HPLC is the most sensitive, practical method for PTH analysis.     

Rapid separation of seed gliadins by reversed-phase ultra performance liquid chromatography (RP-UPLC) and its application in wheat cultivar and germplasm identification

To separate gliadin from wheat flour, a novel and stability-indicating reversed-phase ultra performance liquid chromatography (RP-UPLC) method is established and optimized. A comparative analysis of routine capillary electrophoresis (CE), reversed-phase high-performance liquid chromatography (RP-HPLC), and RP-UPLC was performed and the results showed that the resolution and efficiency of RP-UPLC were significantly higher than those of CE and RP-HPLC. Characteristic RP-UPLC patterns of different bread wheat variety and related species were readily identified. These results demonstrated that our RP-UPLC procedure resulted in significant improvements in sensitivity, speed, and resolution, and thus is highly useful in wheat cultivar and germplasm identification.     

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.     

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.     

A comparative multidimensional LC-MS proteomic analysis reveals mechanisms for furan aldehyde detoxification in <Emphasis Type="Italic">Thermoanaerobacter pseudethanolicus</Emphasis> 39E

Background

Chemical and physical pretreatment of lignocellulosic biomass improves substrate reactivity for increased microbial biofuel production, but also restricts growth via the release of furan aldehydes, such as furfural and 5-hydroxymethylfurfural (5-HMF). The physiological effects of these inhibitors on thermophilic, fermentative bacteria are important to understand; especially as cellulolytic strains are being developed for consolidated bioprocessing (CBP) of lignocellulosic feedstocks. Identifying mechanisms for detoxification of aldehydes in naturally resistant strains, such as Thermoanaerobacter spp., may also enable improvements in candidate CBP microorganisms.

Results

Thermoanaerobacter pseudethanolicus 39E, an anaerobic, saccharolytic thermophile, was found to grow readily in the presence of 30 mM furfural and 20 mM 5-HMF and reduce these aldehydes to their respective alcohols in situ. The proteomes of T. pseudethanolicus 39E grown in the presence or absence of 15 mM furfural were compared to identify upregulated enzymes potentially responsible for the observed reduction. A total of 225 proteins were differentially regulated in response to the 15 mM furfural treatment with 152 upregulated versus 73 downregulated. Only 87 proteins exhibited a twofold or greater change in abundance in either direction. Of these, 54 were upregulated in the presence of furfural and 33 were downregulated. Two oxidoreductases were upregulated at least twofold by furfural and were targeted for further investigation. Teth39_1597 encodes a predicted butanol dehydrogenase (BdhA) and Teth39_1598, a predicted aldo/keto reductase (AKR). Both genes were cloned from T. pseudethanolicus 39E, with the respective enzymes overexpressed in E. coli and specific activities determined against a variety of aldehydes. Overexpressed BdhA showed significant activity with all aldehydes tested, including furfural and 5-HMF, using NADPH as the cofactor. Cell extracts with AKR also showed activity with NADPH, but only with four-carbon butyraldehyde and isobutyraldehyde.

Conclusions

T. pseudethanolicus 39E displays intrinsic tolerance to the common pretreatment inhibitors furfural and 5-HMF. Multidimensional proteomic analysis was used as an effective tool to identify putative mechanisms for detoxification of furfural and 5-HMF. T. pseudethanolicus was found to upregulate an NADPH-dependent alcohol dehydrogenase 6.8-fold in response to furfural. In vitro enzyme assays confirmed the reduction of furfural and 5-HMF to their respective alcohols.

     

Application of high performance liquid chromatography and gas chromatography-mass spectrometry to analysis of prostaglandin E1 in biological media.

A method for the quantitation of prostaglandin (PG) E₁ in biological samples of gas chromatography—mass spectrometry has been developed. PGE₁ was separated from PGE₂, 13,14-dihydro-PGE₂, and other potentially interfering prostaglandins by reversed phase high performance liquid chromatography. After conversion of PGE₁ to PGB₁ by treatment with methanolic KOH, PGB₁ was derivatized to the methyl ester trimethylsilyl ether and analyzed by selected ion monitoring using hexadeutero-PGE₁ as an internal standard. Measurable levels of PGE₁ were found in human and rat urine and in incubates of rat and rabbit renal papilla. PGE₁ excretion and production by renal slices was blocked by treatment with indomethacin. A complete mass spectrum of derivatized PGE₁ was obtained from PGE₁ generated by rabbit renal papillary slices.     

Honeydew sugars in wild-caught Phlebotomus ariasi detected by high performance liquid chromatography (HPLC) and gas chromatography (GC)

Phlebotomus ariasi Tonnoir sandflies were caught in light traps hung in oak trees and in a house in the Cévennes focus of leishmaniasis in the South of France. The flies were cryopreserved either immediately on removal from the traps, or after starvation for 6-7 days, or after 6-7 days starvation followed by exposure to oak infested with the aphid genera Lachnus or Thelaxes. After transportation to the laboratory, the sandflies were thawed and aqueous extracts of the crushed flies were analysed for their carbohydrate content using high performance liquid chromatography (HPLC) and gas chromatography (GC). Starved female sandflies lacked significant amounts of any saccharides. Four types of sugar, melezitose and its hydrolysis products turanose, glucose and fructose, were observed in flies which had been starved previously and then placed with Lachnus infested oak. The results also indicate the presence of hydrolysis products of melezitose: (a) in flies previously starved and placed with Thelaxes infested oak, (b) in P.ariasi cryopreserved direct from the light traps hung in oak trees infested with Lachnus and Thelaxes, and (c) flies caught in a house. Unidentifiably small quantities of a trisaccharide were also detected in the latter groups of flies. In previous tests, sugars were detected in P.ariasi after their exposure to aphid-infested oak (Quercus ilex L.), but not when P.ariasi females were exposed to washed oak leaves without aphids. The results indicate that P.ariasi feed on melezitose and/or turanose, the main local source of which is aphid honeydew. A better understanding of sugar meal sources of sandflies using HPLC and GC techniques will assist in our understanding of sandfly/Leishmania relationships, parasite transmission and epidemiology.     

Measurement of cyst(e)amine in physiological samples by high performance liquid chromatography

Two methods for measurement of cyst(e)amine in physiological samples are described. One method involves reduction of disulfides present in the sample with tributylphosphine, reversed phase chromatography of thiols, and electrochemical detection of cysteamine and other thiols. The other method involves reduction of disulfides with dithiothreitol, derivatization of thiols with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, separation of these derivatives by reversed phase chromatography, and fluorometric detection of the thiol adducts. The endogenous concentration of cysteamine in rat liver was estimated to be less than 2.5 nmol/g. Cysteamine is produced in tissues postmortem; rapid sampling/freezing of tissues and rapid inactivation of enzymes during tissue preparation are essential for accurate measurement of endogenous cysteamine concentrations.     

Simultaneous analysis of multiple aminothiols in human plasma by high performance liquid chromatography with fluorescence detection

Aminothiols serve numerous vital functions in biochemistry, including detoxification and regulation of cellular metabolism, enzymatic activity, and protein trafficking and degradation. Plasma aminothiol concentrations are frequently measured for clinical and translational research investigating oxidative stress, and for routine clinical diagnosis and monitoring of vascular injury. Although a variety of techniques are available to measure aminothiol concentrations in plasma, high performance liquid chromatography with fluorescence detection (HPLC–FD) is the most widely used. This review summarizes HPLC–FD methods, including pre-analytical considerations, procedures for sample reduction, derivatization, and chromatographic separation of the primary biological aminothiols cysteine, homocysteine, cysteinylglycine, and glutathione in human plasma.     

Hollow fiber liquid phase microextraction followed by high performance liquid chromatography for determination of ultra-trace levels of Se(IV) after derivatization in urine,plasma and natural water samples

In the present work, a simple and high sensitive method based on hollow fiber liquid phase microextraction (HF-LPME) was developed followed by high performance liquid chromatography (HPLC) for determination of ultra-trace amounts of Se(IV) after derivatization in biological and natural water samples. Se(IV) was complexed with o-phenylenediamine to form piazselenol. The formed piazselenol was extracted into 20 μL of 1-octanol located in the lumen of a hollow fiber and the solution was injected into HPLC-UV for analysis. Using the Taguchi method, an orthogonal array design (OAD), OA₁₆ (4⁵) was employed to optimize the HF-LPME of piazselenol. The effect of five experimental factors (each factor at four levels) including the volume of the organic phase, extraction time, pH of the solution, stirring rate and ionic strength on the extraction efficiency of piazselenol was studied and optimized. The maximum extraction efficiency of piazselenol was obtained at 20 μL of 1-octanol as the extracting solvent, 30 min extraction time, pH 2, stirring rate of 500 rpm and 30% (w/v) NaCl. Under the optimum conditions, preconcentration factors up to 130 were achieved and the relative standard deviation (%RSD) of the method was <3.7% for different concentrations of Se(IV). The calibration curves were obtained in the ranges of 0.2–100 and 0.05–10 μg L^?1 for the 11 and 50 mL of the sample volumes with reasonable linearity, respectively (r² > 0.995). The limits of detection (LOD) were 0.1 and 0.02 μg L^?1 for the 11 and 50 mL sample volumes, respectively (S/N = 3). Finally, the applicability of the proposed method was evaluated by the extraction and determination of Se(IV) in the plasma, urine and water samples.     

Evaluation of a protocol for metabolic profiling studies on human blood plasma by combined ultra-performance liquid chromatography/mass spectrometry: From extraction to data analysis

The investigation presented here describes a protocol designed to perform high-throughput metabolic profiling analysis on human blood plasma by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS). To address whether a previous extraction protocol for gas chromatography (GC)/MS-based metabolic profiling of plasma could be used for UPLC/MS-based analysis, the original protocol was compared with similar methods for extraction of low-molecular-weight compounds from plasma via protein precipitation. Differences between extraction methods could be observed, but the previously published extraction method was considered the best. UPLC columns with three different stationary phases (C8, C18, and phenyl) were used in identical experimental runs consisting of a total of 60 injections of extracted male and female plasma samples. The C8 column was determined to be the best for metabolic profiling analysis on plasma. The acquired UPLC/MS data of extracted male and female plasma samples was subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, a strategy for compound identification was applied here, demonstrating the strength of high-mass-accuracy time-of-flight (TOF)/MS analysis in metabolic profiling.     

Fiber-based liquid-phase micro-extraction of mebeverine enantiomers followed by chiral high-performance liquid chromatography analysis and its application to pharmacokinetics study in rat plasma

The applicability of two-phase liquid-phase micro-extraction (LPME) in porous hollow polypropylene fiber for the sample preparation and the stereoselective pharmacokinetics of mebeverine (MEB) enantiomers (an antispasmodic drug) in rat after intramuscular administration were studied. Plasma was assayed for MEB enantiomer concentrations using stereospecific high-performance liquid chromatography with ultraviolet detection after a simple, inexpensive, and efficient preconcentration and clean-up hollow fiber-based LPME. Under optimized micro-extraction conditions, MEB enantiomers were extracted with 25 μl of 1-octanol within a lumen of a hollow fiber from 0.5 ml of plasma previously diluted with 4.5 ml alkalized water (pH 10). The chromatographic analysis was carried out through chiral liquid chromatography using a DELTA S column and hexane-isopropyl alcohol (85:15 v/v) containing 0.2% triethylamine as mobile phase. The mean recoveries of (+)-MEB and (-)-MEB were 75.5% and 71.0%, respectively. The limit of detection (LOD) was 3.0 ng/ml with linear response over the concentration range of 10-2500 ng/ml with correlation coefficient higher than 0.993 for both enantiomers. The pharmacokinetic studies showed that the mean plasma levels of (+)-MEB were higher than those of (-)-MEB at almost all time points. Also, (+)-MEB exhibited greater t(max) (peak time in concentration-time profile), C(max) (peak concentration in concentration-time profile), t(1/2) (elimination half-life), and AUC(0-240 min) (area under the curve for concentration versus time) and smaller CL (clearance) and V(d) (apparent distribution volume) than its antipode. The obtained results implied that the absorption, distribution, and elimination of (-)-MEB were more rapid than those of (+)-MEB and there were stereoselective differences in pharmacokinetics.     

Quantitative analysis of cytokinins in plants by high performance liquid chromatography: electronspray ionization ion trap mass spectrometry

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