Metabolomic and elemental analysis of camel and bovine urine by GC–MS and ICP–MS

Recent studies from the author’s laboratory indicated that camel urine possesses antiplatelet activity and anti-cancer activity which is not present in bovine urine. The objective of this study is to compare the volatile and elemental components of bovine and camel urine using GC–MS and ICP–MS analysis. We are interested to know the component that performs these biological activities. The freeze dried urine was dissolved in dichloromethane and then derivatization process followed by using BSTFA for GC–MS analysis. Thirty different compounds were analyzed by the derivatization process in full scan mode. For ICP–MS analysis twenty eight important elements were analyzed in both bovine and camel urine. The results of GC–MS and ICP–MS analysis showed marked difference in the urinary metabolites. GC–MS evaluation of camel urine finds a lot of products of metabolism like benzene propanoic acid derivatives, fatty acid derivatives, amino acid derivatives, sugars, prostaglandins and canavanine. Several research reports reveal the metabolomics studies on camel urine but none of them completely reported the pharmacology related metabolomics. The present data of GC–MS suggest and support the previous studies and activities related to camel urine.     

GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-¹⁵N₂]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.     

Metabolomic changes in Caenorhabditis elegans lifespan mutants as evident from GC–EI–MS and GC–APCI–TOF–MS profiling

Lifespan mutants of the nematode Caenorhabditis elegans are a much studied aging model, however, aging-related changes at the metabolome level remain largely unexplored. To identify metabolic features connected to mitochondrial dysfunction, a hallmark of aging and age-related disease, we analyzed a short-lived mitochondrial mutant (mev-1(kn1)), a long-lived mutant with enhanced cellular maintenance (ife-2(ok306)) and the novel double mutant ife-2(ok306);mev-1(kn1) which is normal-lived, possibly through attenuation of the metabolic mev-1 phenotype. Metabolomic analysis involved coupled gas chromatography–mass spectrometry with electron ionization (GC–EI–MS) and, in addition, recently introduced GC with soft atmospheric pressure chemical ionization coupled to time-of-flight mass spectrometry (GC–APCI–TOF–MS) to yield complementary mass spectrometric information for enhanced metabolite annotation. Multivariate analysis allowed distinction of mev-1 and ife-2 mutants from the wild type, while suggesting still another, distinct metabolic phenotype for the ife-2;mev-1 double mutant. In mev-1(kn1), disturbed energy metabolism was indicated by upset TCA cycle homeostasis, elevated glycolytic substrate and lactic acid levels as well as depletion of free amino acids pools. Surprisingly, these mitochondrially related changes were retained in the ife-2;mev-1 mutant, as were highly elevated levels of the dipeptide glycylproline indicative of increased collagen catabolism. However, the double mutant reverted mev-1(kn1) changes in uric acid and long-chain fatty alcohol metabolism, two pathways connected to the peroxisomal compartment. Our results are in line with recent evidence for a critical role of this organelle in aging and demonstrate the usefulness of non-targeted metabolomics approaches for detecting complex metabolic changes in the study of mitochondrial dysfunction.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats

A fast and sensitive HPLC–APCI-MS/MS method was developed for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma. The plasma sample containing ergone and ergosterol (internal standard) were simply treated with acetone to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC–APCI-MS/MS system. Chromatographic separation was performed on a 1.8 μm Zorbax SB-C18 column (100 mm × 3.0 mm) with a 97:3 (v/v) mixed solution of methanol and 0.1% aqueous formic acid being used as mobile phase. Quantification was performed by multiple selected reactions monitoring (MRM) of the transitions with (m/z)⁺ 393–268 for ergone and (m/z)⁺ 379–69 for the IS. The method was validated in the concentration range of 5–1600 ng/mL for ergone. The precision of the assay (RSD%) was less than 10.5% at all concentrations levels within the tested range and adequate accuracy, and the limit of detection was 1.5 ng/mL. The absolute recoveries of both ergone and ergosterol from the plasma were more than 95%. The developed method has been successfully applied to the pharmacokinetic study of the drug in SD rats.     

Sub-proteomic fractionation,iTRAQ, and OFFGEL-LC–MS/MS approaches to cardiac proteomics

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1 h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissues. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of < 5%. Compared to no fractionation prior to LC–MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four-fold. We found that approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.     

BIA–MS–MS: biomolecular interaction analysis for functional proteomics

One of the experimental processes of functional proteomics is the analysis of protein interaction. Here, we review a new analytical platform, BIA–MS, for protein interaction analysis. BIA–MS is an integration of a surface plasmon resonance biosensor for real-time interaction analysis and mass spectrometry for the subsequent identification of interacting molecules.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

BIA–MS–MS: biomolecular interaction analysis for functional proteomics

One of the experimental processes of functional proteomics is the analysis of protein interaction. Here, we review a new analytical platform, BIA–MS, for protein interaction analysis. BIA–MS is an integration of a surface plasmon resonance biosensor for real-time interaction analysis and mass spectrometry for the subsequent identification of interacting molecules.     

Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

Simultaneous HPLC–MS–MS quantification of 8-iso-PGF2α and 8,12-iso-iPF2α in CSF and brain tissue samples with on-line cleanup

Quantitation of isoprostanes such as 8-iso-PGF_2α and 8,12-iso-iPF_2α-VI in biological fluids has been proposed as a reliable test of oxidant stress and inflammation in a variety of disorders. This paper presents a liquid chromatography method with tandem mass spectrometry detection for the simultaneous analysis of these two isoprostanes in human CSF and brain tissue samples. An API 5000 triple quadrupole instrument (AB Sciex, Foster City, CA, USA) with an APCI ion source was used in this study. Aliquots of CSF samples (0.25 mL) were treated with a methanol:zinc sulfate mixture followed by on-line cleanup on an extraction column (Validated-C₁₈) with 0.1% formic acid. The brain tissue samples were homogenized and lipids were extracted using Folch solution. Solid-phase extraction columns (C₁₈) were used for the purification of the brain isoprostane fraction. Chromatographic separation was achieved using an analytical column (Synergi C₁₈ HydroRP) with 0.1% formic acid in water and a mixture of methanol:acetonitrile under isocratic conditions. The mass spectrometer was operated in the MRM scan and negative ion mode. The quadrupoles were set to detect the molecular ions [M?H]^? and high mass fragments of isoprostanes: m/z 353 → 193 amu (8-iso-PGF_2α) and m/z 353 → 115 amu (8,12-iso-iPF_2α-VI) and their deuterated internal standards: m/z 357 → 197 amu (8-iso-PGF_2α-d₄) and m/z 364 → 115 amu (8,12-iso-iPF_2α-VI-d₁₁). The lower limit of quantification was 2.5 pg/mL for 8-iso-PGF_2α and 5.0 pg/mL for 8,12-iso-PF_2α-VI for the CSF method and 10.0 pg/0.1 g of tissue and 30.0 pg/0.1 g of tissue for 8-iso-PGF_2α and 8,12-iso-iPF_2α-VI, respectively, for the brain tissue method. No ion suppression or enhancement of the detection of 8-isoPGF_2α, 8,12-isoPF_2α-VI or both internal standards was found.     

Chemical compositions by using LC–MS/MS and GC–MS and antioxidant activities of methanolic extracts from leaf and flower parts of Scabiosa columbaria subsp. columbaria var. columbaria L.

The members of the Scabiosa genus are one of the traditional medicinal plants used in the treatment of many diseases, in particular the treatment of scabies. In this study, it was aimed to determine antioxidant activities and chemical composition of methanolic extracts of leaves and flowers of Scabiosa columbaria subsp. columbaria var. columbaria. The phenolic contents of both parts of the plant were analyzed by LC–MS/MS. A total of 6 phenolic compounds were determined and chlorogenic acid was the major compound in both flower and leaf parts of the plants, with 5936.052 µg/g and 8021.666 µg/g, respectively. 6 different methods were used to determine the antioxidant activity of the plant parts. Both leaf and flower parts of the plant showed high antioxidant activity in all tested methods and the antioxidant activity values of the leaf part were measured higher than those of the flower part for four tests. The methanol extracts of the plant parts was analyzed with GC–MS and number of the essential oil compounds in the leaf and flower parts were determined as 17 and 13, respectively. Linalool compound was also found to be common in both parts of the plant. The major compounds of the essential oils were identified as 4-Octadecenal (30.01%) in the flower and carvone (35.44%) in the leaf. In addition, terpene derivatives was determined as 90.32% of the highest essential oil group in the leaf, while this value was determined as 1.42% in the flower. For the flower, aromatics were determined as the main component group with 21.31%.     

Quantification of isradipine in human plasma using LC–MS/MS for pharmacokinetic and bioequivalence study

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. The procedure involves a simple liquid–liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r² ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.     

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC–ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD_intra-day 1–8%; RSD_inter-day 5–14%), accurate (intra-day: 95–107%; inter-day: 90–115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24–0.49 μg/ml plasma and 0.78–1.56 μg/ml urine) and lower limits of detection (0.12–0.24 μg/ml plasma and 0.39–0.78 μg/ml urine) were equivalent to quite low absolute on-column amounts (1.1–2.1 pg for plasma and 2.0–3.9 pg for urine). The calibration range (0.24–250 μg/ml plasma and 0.78–200 μg/ml urine) was subdivided into two linear ranges (r² ≥ 0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underling in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.     

Occurrence of Ochratoxin A in Grain and Manufactured Food Products in China Detected by HPLC with Fluorescence Detection and Confirmed by LC–ESI–MS/MS

A total of 110 commercially available samples of manufactured food products including bread, oat, barley, maize, corn, wheat, grape, soluble coffee, soya bean, red wine, and baby food were randomly collected in the northeast of China during the first six months of 2010. Samples were analyzed for the presence of ochratoxin A (OTA) using immunoaffinity column (IAC) clean-up and high-performance liquid chromatography with fluorescence detection (HPLC-FD) and confirmed with LC–ESI–MS/MS. The range of average OTA recoveries was 78.3–103.3% at three spiked levels. The relative standard deviations (RSDs) of recoveries range of 2.1–4.3%. OTA were detected in 13 samples, which were below the maximum allowable limit established by the European Community. The results of this study suggest that those manufactured food products consumed in China present no risk by human exposure to OTA through their consumption.     

Development,validation and comparison of LC–MS/MS and RIA methods for quantification of vertebrates-like sex-steroids in prosobranch molluscs

The role of vertebrate-like sex-steroids (testosterone, T, progesterone, P, and 17β-estradiol, E2) in molluscs is still debated, but they could represent potential biomarkers of endocrine disruption. A radioimmunoassay (RIA) and a liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods have been developed and compared to measure their levels in a gastropod snail Potamopyrgus antipodarum. Both methods showed a good reproducibility despite the complex matrix and the very low levels of vertebrate-like sex-steroids. Only T and P were detected using the LC–MS/MS method, while the RIA method reached lower detection limits and enabled the detection of all three steroids. Results indicated that T and P were mainly present as unconjugated forms. Both methods were compared in the analysis of snails exposed to waste water treatment plant effluents and led to the same conclusions concerning the modulation of steroids levels. Moreover, they both were in agreement concerning T measurements. On the other hand, LC–MS/MS appeared to be more suitable when analyzing P levels due to a low sensitivity of the RIA method. As E2 was not measured using the LC–MS/MS method because of a higher detection limit compared to the other steroids, the results obtained with the RIA method should be interpreted with caution. LC–MS/MS remains the gold standard for sex-steroid determinations, however a relevant and alternative method based on RIA was developed, requiring fewer organisms. RIA seems a promising method as a screening tool for experimental use, allowing comparison of sex-steroid levels in the mudsnail both in laboratory and in field experiments.