Ultra Performance Liquid Chromatography Coupled To Mass Spectrometry Research Articles

Exploring the wound healing potential of Lobostemon fruticosus using in vitro and in vivo bioassays

Ethnopharmacological relevanceLobostemon fruticosus (L.) H.Buek is a perennial and woody shrub of the Boraginaceae family, found in the Cape region of South Africa. The leaves and twigs are used to treat dermatological conditions such as wounds, burns, ringworm, erysipelas and eczema. Anti-inflammatory, antibacterial, antiviral and anti-proliferative activities of L. fruticosus have been reported. However, there is a void in research which reports on the wound healing properties of this plant. Aim of the studyAligned with the traditional use of L. fruticosus, our study aimed to use in vitro and in vivo bioassays to confirm the wound healing potential of the plant. Materials and methodsAn aqueous methanol extract (80% v/v) of L. fruticosus was prepared using a sample collected from the Western Cape Province of South Africa and chromatographically profiled by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay was performed to determine the non-toxic concentrations of the extract for subsequent use in the in vitro scratch assay. Both the human keratinocyte (HaCaT) and fibroblast (BJ-5ta) cell lines were employed in the in vitro scratch assay. The in vivo caudal fin amputation assay was used to assess the wound healing potential of L. fruticosus, by monitoring fin regeneration in zebrafish larvae treated with the plant extract at various concentrations. ResultsSix major compounds were tentatively identified in the L. fruticosus extract namely; globoidnan A, globoidnan B, rutin, rabdosiin, sagerinic acid and rosmarinic acid. The potentially toxic pyrrolizidine alkaloids were also identified and quantitatively confirmed to be present at a low concentration of 119.58 ppm (m/m). Treatment of HaCaT and BJ-5ta cells with the plant extract in the scratch assay resulted in an increase in cell migration, which translates to accelerated wound closure. After 24 hr treatment with 100 μg/mL of extract, wound closure was recorded to be 91.1 ± 5.7% and 94.1 ± 1.3% for the HaCaT and BJ-5ta cells, respectively, while the untreated (medium) controls showed 72.3 ± 3.3% and 73.0 ± 4.3% for the two cell lines, respectively. Complete wound closure was observed between 24 and 36 hr, while the untreated control group did not achieve 100% wound closure by the end of the observation period (48 hr). In vivo, the crude extract at 100 μg/mL accelerated zebrafish caudal fin regeneration achieving 100.5 ± 3.8% regeneration compared to 68.3 ± 6.6% in the untreated control at two days post amputation. ConclusionsThe study affirms the wound healing properties, as well as low toxicity of L. fruticosus using both in vitro and in vivo assays, which supports the traditional medicinal use. Other in vitro assays that target different mechanisms involved in wound healing should be investigated to support the current findings.

Spray-Dried Microencapsulation of Oregano (Lippia graveolens) Polyphenols with Maltodextrin Enhances Their Stability during In Vitro Digestion

The effect of in vitro gastrointestinal digestion on the release of microencapsulated phenolic compounds was evaluated through an optimized spray drying process. A stock extract of oregano phenolic compounds was developed and microencapsulated in a spray dryer following a central composite rotatable design, controlling the variables, inlet temperature (111.7-168.2°C), and percentage of wall material (5.8-34.1%). Optimum drying conditions for spray drying were decided based on different yield percentages ( Y %) and encapsulated phenolic compounds (EPC). The analyzed physical properties were morphology measured by electron microscopy and humidity; other properties evaluated were the content of total phenolic compounds, antioxidant capacity determined by DPPH and ABTS assays, and phenolic profile by ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). During the gastrointestinal simulation, a stability of 85% was determined in the intestinal stage. Microencapsulation technology by spray drying is an excellent selection to stabilize and protect bioactive compounds of oregano and promote its use as a functional ingredient.

Urinary metabolomics for discovering metabolic biomarkers of bladder cancer by UPLC-MS

Bladder cancer (BC) is one of the most frequent cancer in the world, and its incidence is rising worldwide, especially in developed countries. Urine metabolomics is a powerful approach to discover potential biomarkers for cancer diagnosis. In this study, we applied an ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) method to profile the metabolites in urine from 29 bladder cancer patients and 15 healthy controls. The differential metabolites were extracted and analyzed by univariate and multivariate analysis methods. Together, 19 metabolites were discovered as differently expressed biomarkers in the two groups, which mainly related to the pathways of phenylacetate metabolism, propanoate metabolism, fatty acid metabolism, pyruvate metabolism, arginine and proline metabolism, glycine and serine metabolism, and bile acid biosynthesis. In addition, a subset of 11 metabolites of those 19 ones were further filtered as potential biomarkers for BC diagnosis by using logistic regression model. The results revealed that the area under the curve (AUC) value, sensitivity and specificity of receiving operator characteristic (ROC) curve were 0.983, 95.3% and 100%, respectively, indicating an excellent discrimination power for BC patients from healthy controls. It was the first time to reveal the potential diagnostic markers of BC by metabolomics, and this will provide a new sight for exploring the biomarkers of the other disease in the future work.

Chemical Constituents and Cytotoxic Activity of Rhinella jimi (Anura: Bufonidae)

Rhinella jimi toads (Stevaux, 2002) belong to the Bufonidae family, are endemic in the Brazilian Northeast and are commonly found during rainy periods. In general, amphibians of this family have in their poisons different metabolites that show a diversity of pharmacological activities. The isolation and identification of these compounds are of great importance, and techniques such as high-performance liquid chromatography coupled to mass spectrometry are widely used for the discovery of novel and known compounds in these poisons. For R. jimi poison, the ethyl acetate and methanolic extracts were obtained and thirty compounds were identified by combining ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) with direct infusion atmospheric pressure chemical ionization mass spectrometry (DI-APCI-MS/MS) and direct infusion electrospray mass spectrometry (DI-ESI-MS/MS) for each extract, respectively. Marinobufagin (2) and marinobufotoxin (19) were the majorities of each extract, respectively. In addition, other bufadienolides mainly present in the ethyl acetate extract, such other bufotoxins, alkaloids and arginine diacid derivatives were identified in the methanol extract. In a cytotoxic assay by 3-(4,5-dimethylthiazol-2-yl)-2,5‑diphenyltetrazolium bromide (MTT), the extracts and compound 2 demonstrated half-maximal inhibitory concentration (IC50) values better than the positive control doxorubicin, evidencing excellent cytotoxic. This is the most complete study of the chemical composition of R. jimi toad poison and its respective cytotoxic activity, promoting the enrichment of knowledge about this family and species.

Phenolic compounds and antioxidant properties of wheat fermented with Agaricus brasiliensis and Agaricus bisporus.

Solid-state fermentation with Agaricus brasiliensis and Agaricus bisporus on whole grain wheat was carried out. Phenolic compounds and antioxidant properties of fermented wheat were determined. The results showed that the maximum values of polyphenols contents in wheat fermented with A. brasiliensis and A. bisporus reached, respectively (3.16± 0.21) and (3.93± 0.23) mg GAE/g, which were 2.90 and 3.61 times of unfermented control. By employing ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS), 18 kinds of phenolic compounds were identified from fermented wheat. Compared with control, only 4-hydroxy-benzaldehyde was the same compound. It indicated that fermentation with the two fungi changed polyphenols contents and phenolic compounds composition in wheat to a great extent. Among these phenolic compounds, except for 4-hydroxy-benzaldehyde, 4-hydroxy-benzoic acid and β-N-(γ-glutamyl)-4-formylphenylhydrazine, other 15 kinds of phenolic compounds were first identified from mushroom samples (including fruit bodies, mycelia and fermentation products). DPPH radical scavenging capacity, reducing power, ferrous ion chelating ability and inhibition of lipid peroxidation of fermented wheat were significantly stronger than control (P <0.05).

Pigment production by Fusarium solani BRM054066 and determination of antioxidant and anti-inflammatory properties

The fungal kingdom has been widely studied as a source of bioactive compounds of interest to the pharmaceutical and food industry. This paper studies the production of natural red pigments by Fusarium solani BRM054066 in the submerged fermentation system, using Doehlert experimental design to determine optimal cultivation conditions. The chemical composition of the red pigment was determined by Nuclear Magnetic Resonance spectroscopy (NMR) and Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Antioxidant activity was assessed by the ability to sequester of free radical DPPH. In the analysis of anti-inflammatory activity, murine peritoneal macrophages activated by LPS were used, and the gene expression of TNF-α, IL-1β, IL-6, IL-10 and IL-17 was determined using qPCR. As a result, it was found that agitation at 200 rpm and glucose concentration ≥ 20 g/L promote the best results in the production of red pigment. The chemical compounds identified were two naphthoquinones, fusarubin and dihydrofusarubin, and an anthraquinone, a bostrycoidin, being fusarubin the majority compound. The red pigment showed antioxidant activity by scavenge 50% of the DPPH radical, in a concentration of 24 µg/mL. The pigment also showed an effective anti-inflammatory capacity by reducing the overexpression of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 and promoting the production of anti-inflammatory IL-10 and IL-17, in murine macrophages activated by LPS (p < 0.05). According to the results, the fungus F. solani BRM054066, under optimized conditions of cultivation, proved to be a promising source of biologically active natural pigments with wide industrial applicability.

Phytochemical analysis and hypotensive activity of Ipomoea pes-caprae on blood pressure of normotensive rats

Abstract Ipomoea pes-caprae (Convolvulaceae), popularly known as “salsa-da-praia”, is used in folk medicine for the treatment of several diseases, including hypertension. The aim of this research was to evaluate hypotensive activity attributed to this species and identify the bioactive compounds responsible for this effect. Hydroalcoholic extract of aerial parts of Ipomoea pes-caprae (PIEA) was fractionated by column chromatography with Amberlite XAD-2, thus achieving a methanolic fraction (PIEA-M). The chemical profile investigation of PIEA-M was conducted by Ultra Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS) and compound isolation through High Performance Countercurrent Chromatography (HPCCC). Hemodynamic evaluations of PIEA, PIEA-M and isolated compounds were carried out on normotensive rats. The locomotor activity of PIEA and PIEA-M were performed by Rota Rod test using Swiss mice. UPLC-MS analysis of PIEA-M led to the identification of 11 compounds corresponding to seven phenolic acids and four flavonoids. By means of HPCCC, quercetin 6”-O-acetyl-3-O- glucoside (1), a mixture of quercetin-3-O-galactoside and quercetin-3-O- glucoside (2) and quercetin 3-O-glucoside (3) were isolated. PIEA, PIEA-M and the obtained compounds (1 and 2) significantly reduced blood pressure. Also, PIEA and PIEA-M caused significant impairment on mice locomotor activity. These results indicate the potential of Ipomoea pes-caprae as a source of hypotensive agents suggesting that phenolic compounds present in the species are responsible for its hypotensive activity.

Phenolic Profile, Antioxidant and Anti-Proliferative Activities of Methanolic Extracts from Asclepias linaria Cav. Leaves.

Asclepias linaria Cav. (Apocynaceae) is a shrubby plant endemic of Mexico which has been used in traditional medicine. However, the bioactive potential of this plant remains unexplored. In this study, the phenolic composition, antioxidant, and cytotoxic activities of A. linaria leaves were determined. In order to estimate the phenolic composition of the leaves, the total phenolic, flavonoid, and condensed tannins contents were determined. Furthermore, the antioxidant activity was measured by the scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) and 2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS•+) radicals and the total antioxidant capacity. The phenolic compounds identified in the A. linaria leaves by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) include phenolic acids, such as p-coumaric and ferulic acid, as well as flavonoids, such as rutin and quercetin. The leaves’ extracts of A. linaria showed a high scavenging activity of DPPH• and ABTS•+ radicals (IC50 0.12 ± 0.001 and 0.51 ± 0.003 µg/mL, respectively), high total antioxidant capacity values (99.77 ± 4.32 mg of ascorbic acid equivalents/g of dry tissue), and had a cytotoxic effect against K562 and HL60 hematologic neoplasia cells lines, but no toxicity towards the normal mononuclear cell line was observed. These results highlight the potential of A. linaria and could be considered as a possible alternative source of anticancer compounds.

Los nanomateriales modifican el crecimiento de los cultivos y algunas características de organismos de suelos agrícolas o forestales: Un estudio experimental a nivel laboratorio, invernadero y campo.

Currently, some concerns regarding the potential toxicity of nanoparticles (NP) on the environment have emerged. The effect of ZnO, TiO2, and Fe2O3 NP on corn (Zea mays L.), common beans (Phaseolus vulgaris L.), nanobioremediation of polycyclic aromatic hydrocarbons (PAH), and soil organisms from agricultural or forest soils was studied at laboratory, greenhouse, and land level. The samples were analyzed by X-ray diffraction (XRD), field emission scanning electron microscopy with X-ray energy dispersion spectrometry (FESEM-EDS), scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) gas chromatography (GC), ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) and Fourier transform infrared spectrometry with attenuated total reflectance (FTIR-ATR). ZnO-NP did not harm the mycorrhizal root colonization but, the presence of ZnO-NP decreased the degradation of PAH. The synthesis of metabolites from corn was more affected by the PAH than by ZnONP. FTIR spectra showed that NP affected the synthesis of compounds from specific functional groups in common bean plants. Fe2O3-NP were attached to the body of forestsoil organisms and significantly increased the concentration of Fe in their body, while TiO2-NP changed the morphological tissue of roots and stems of common bean as witnessed by micrographs of longitudinal and cross-sections. The NP used in this research significantly changed some response variables on the experiments carried-out at laboratory, greenhouse, and land level.

Evaluation of cytotoxic, immunomodulatory and antibacterial activities of aqueous extract from leaves of Conocarpus erectus Linnaeus (Combretaceae).

This work evaluated the antibacterial activity, cytotoxicity and immunomodulatory effect on human peripheral blood mononuclear cells (PBMCs) promoted by aqueous extract from Conocarpus erectus leaves (AELCe). The extract was characterized by thin layer chromatography and ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS). Cytotoxicity of AELCe (6.25-50 μg/ml) was investigated using annexin V and propidium iodide. Cytokine and nitric oxide levels in PBMCs culture supernatants exposed or not to AELCe (12.5 μg/ml) were determined, and antibacterial activity was evaluated by disc diffusion and broth microdilution methods. AELCe contained 3',4'-OH flavonoids, phenylpropanoglycosides, saponins, polymeric proanthocyanidins and hydrolysable tannins. Moreover, 10 other compounds were identified through UPLC-MS technique. AELCe did not affect lymphocyte viability at 6.25 and 12.5 μg/ml. IL-2, IL-10, TNF-α, IFN-γ and nitric oxide was produced in higher levels by cells treated with AELCe. Proliferation and activation of CD8+ T lymphocytes were also stimulated. AELCe showed bacteriostatic activity against clinical and antibiotic-resistant strains of Staphylococcus aureus (MIC between 250 and 1000 μg/ml). AELCe showed a moderate bacteriostatic activity and promoted an immunomodulatory status through higher production of Th1 cytokines, nitric oxide release and T CD8+ lymphocytes stimulation.

Prognostic prediction of pemetrexed-platinum chemotherapeutic regimen for NSCLC by serum metabolomics.

e20603 Background: Pemetrexed-platinum chemotherapeutic doublet has become a standard schedule as the first-line treatment for advanced non-squamous NSCLC due to its promising efficacy and a favorable toxicity profile. However, chemotherapy resistance inevitably limits the therapeutic effect of this preferred chemotherapeutic regimen, especially for these intrinsically resistant patients. Development of predictive biomarkers to identify those patients who can benefit from this pemetrexed-based regimen has great significance for personalized chemotherapy. Methods: Total 354 patients who were diagnosed with inoperable stage IIIB or IV lung adenocarcinoma (LADC), assigned to receive pemetrexed-platinum doublet chemotherapy as the first-line treatment were enrolled into this prospective study, including 251 cases diagnosed ahead as training set for predictive model establishment and the subsequently diagnosed 103 cases into validation set. Ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS) analytical platform were applied to perform the metabolic profiling studies of pre-treatment serum samples. Results: In the training set, eight potential biomarkers, comprising of hypotaurine, taurine, choline, betaine, dimethylglycine, uridine, dodecanoylcarnitine and L-palmitoylcarnitine, were discovered to be differentially expressed between the patients in disease progressive group (PD, n = 53) and disease control group (including CR, PR and SD, n = 198). In the validation set (PD vs non-PD: 23 vs 80), targeted quantitative analysis of these metabolic biomarkers revealed similar levels as that observed in the training set. Logistic regression model was accordingly established based on this eight-metabolic biomarker panel, to discriminate patients who might benefit from this chemotherapy regimen with over 90% accuracy. Conclusions: This prospective study identified blood-based biomarker panel by metabonomics analysis could predict the clinical outcome of pemetrexed-platinum chemotherapy prior to treatment for LADC patients, which needed to be validated by clinical trials of large samples.

PUB146 Prognostic Prediction of Pemetrexed-Platinum Chemotherapeutic Regimen by Serum Metabolomics

Lung cancer is a leading cause of cancer deaths worldwide and chemotherapy is the major treatment for advanced stage patients. Currently, pemetrexed combined with platinum (PC regimen), due to its promising efficacy and a favorable toxicity profile, has become a standard schedule as the first-line treatment for advanced non-squamous NSCLC. Nevertheless, like all chemotherapeutic regimens, drug resistance inevitably limits the efficiency of this preferred cytotoxic drug combination. In virtually all initial responders rapidly develop acquired resistance. Besides, some cases are intrinsically resistant to this chemotherapy regimen, and the disease progressed at the first radiological assessment of response after one or two cycles of treatment. Metabolomics, by testing the holistic low-molecular-weight organism metabolites, offers an appropriate strategy to studies of pathophysiological processes, interaction of environment and genotype, drug toxicity and efficacy, biomarker discovery and so on. Metabolites play a substantial role in biological systems. Being the closest biological proximity to the phenotype of the system, metabolomics allows rapid observation of responses to system perturbations in the metabolome. In this work, we hypothesis that the metabolic characteristics of serum may associated with the inherent response (resistance or sensitivity) of patients to cytotoxic drugs. In this study, ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS) analytical platform was applied to perform the metabolic profiling studies of serum samples, aiming to find metabolic biomarkers which could predict the benchmark of pemetrexed and platinum treatment in adeno-NSCLC patients. In the retrospective study, serum samples were subjected to the untargeted metabolomic analysis. The results clearly indicated the different patterns of metabolomics profiles in accordance with the clinical outcomes of progressive disease (PD) or partial response (PR). We then established a prediction model consisting of a robust metabolites-pattern that can predict the response of chemotherapy-naive NSCLC patients before receiving PC chemotherapy regimen. And in prospective study, we employed this new method to select the patients who are more likely to benefit from PC regimen, and gave suggests to take an alternative chemotherapy regimen to the patients who might not sensitive to the PC regimen according to our investigation. The metabolic alterations in serum before pemetrexed and platinum chemotherapy regimen are associated with clinical benefit. This study offers a validated method to identify patients who are unlikely to respond to this treatment and thus can be offered alternative treatments or entry into clinical trials.

Hybrid Quadrupole-Orbitrap mass spectrometry for quantitative measurement of quorum sensing inhibition

Drug resistant bacterial infections cause significant morbidity and mortality worldwide, and new strategies are needed for the treatment of these infections. The anti-virulence approach, which targets non-essential virulence factors in bacteria, has been proposed as one way to combat the problem of antibiotic resistance. Virulence in methicillin-resistant Staphylococcus aureus (MRSA) and many other Gram-positive bacterial pathogens is controlled by the quorum sensing system. Thus, there is excellent therapeutic potential for compounds that target this system. With this project, we have developed and validated a novel approach for measuring quorum sensing inhibition in vitro. Ultraperformance liquid chromatography coupled to mass spectrometry (UPLC-MS) was employed to directly measure one of the important outputs of the quorum sensing system in MRSA, auto-inducing peptide I (AIP I) in bacterial cultures. The method for AIP detection was validated and demonstrated limits of detection and quantification of range of 0.0035μM and 0.10μM, respectively. It was shown that the known quorum sensing inhibitor ambuic acid inhibited AIP I production by a clinically relevant strain of MRSA, with an IC50 value of 2.6±0.2μM. The new method performed similarly to previously published methods using GFP reporter assays, but has the advantage of being applicable without the need for engineering of a reporter strain. Additionally, the mass spectrometry-based method could be applicable in situations where interference by the inhibitor prevents the application of fluorescence-based methods.

Metabolic phenotyping of atherosclerotic plaques reveals latent associations between free cholesterol and ceramide metabolism in atherogenesis.

Current optimum medical treatments have had limited success in the primary prevention of cardiovascular events, underscoring the need for new pharmaceutical targets and enhanced understanding of mechanistic metabolic dysregulation. Here, we use a combination of novel metabolic profiling methodologies, based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) followed by chemometric modeling, data integration, and pathway mapping, to create a systems-level metabolic atlas of atherogenesis. We apply this workflow to compare arterial tissue incorporating plaque lesions to intimal thickening tissue (immediate preplaque stage). We find changes in several metabolite species consistent with well-established pathways in atherosclerosis, such as the cholesterol, purine, pyrimidine, and ceramide pathways. We then illustrate differential levels of previously unassociated lipids to atherogenesis, namely, phosphatidylethanolamine-ceramides (t-test p-values: 3.8 × 10(-6) to 9.8 × 10(-12)). Most importantly, these molecules appear to be interfacing two pathways recognized for their involvement in atherosclerosis: ceramide and cholesterol. Furthermore, we show that β-oxidation intermediates (i.e., acylcarnitines) manifest a pattern indicating truncation of the process and overall dysregulation of fatty acid metabolism and mitochondrial dysfunction. We develop a metabolic framework that offers the ability to map significant statistical associations between detected biomarkers. These dysregulated molecules and consequent pathway modulations may provide novel targets for pharmacotherapeutic intervention.

A non-target chemometric strategy applied to UPLC-MS sphingolipid analysis of a cell line exposed to chlorpyrifos pesticide: A feasibility study

A non-target chemometrics study based on the application of Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method to a data set obtained by ultra-performance liquid chromatography coupled to mass spectrometry (UPLC-MS) has been applied to the study of human prostate cancer (DU145) cell line samples treated with the organophosphate pesticide chlorpyrifos (CPF). Full scan UPLC-MS data sets were segmented in 17 different chromatographic windows and submitted to a non-target detailed study. Every one of these chromatographic windows of the different analyzed samples (treated and non-treated with CPF) was column-wise augmented in a new data matrix with their m/z values in the common column mode to preserve the fulfillment of the assumed spectral bilinear model. MCR-ALS was used to recover the elution and mass spectral profiles of the pure components present in each of the analyzed chromatographic windows. ANOVA (p≤0.05) was then applied to compare the areas under the concentration profiles of the MCR-ALS resolved components in the CPS treated and control samples. This analysis allowed the detection of those sphingolipids having their concentration in cells modified by the presence of CPS compared to control samples where this contaminant was absent. Positively identified sphingolipids included sphingomyelins, dihydrosphingomyelin and C16 ceramide. The strategy described in this work is proposed for a general non-target UPLC-MS MCR-ALS analysis of the effect of environmental contaminants in cells in lipidomic and metabonomic studies.

A metabonomic analysis of urine from rats treated with rhizoma alismatis using ultra‐performance liquid chromatography/mass spectrometry

A metabonomic approach based on ultra-performance liquid chromatography coupled to mass spectrometry (UPLC/MS) was used to study the nephrotoxicity of rhizoma alismatis (RA) in rats. Potential biomarkers of RA toxicity were identified and the toxicological mechanism is discussed. Urine samples were collected from control and treated rats at various stages and analyzed by UPLC/MS in positive ionization mode. Histopathological analysis was used to evaluate renal function. The differences in the metabolic profiles of the control and treated rats were clearly distinguishable with principal components analysis (PCA) of the chromatographic data, and significant changes in 13 metabolite biomarkers were detected in the urine. This metabonomic method combined with PCA could discriminate the treated rats from the control rats on days 60, 120, and 180 after treatment, before serious organic renal damage was apparent on day 180 with histopathology. This research indicates that UPLC/MS-based metabonomic analysis of urine samples can be used to predict the chronic nephrotoxicity induced by rhizoma alismatis.

Optimized Preprocessing of Ultra-Performance Liquid Chromatography/Mass Spectrometry Urinary Metabolic Profiles for Improved Information Recovery

Ultra-performance liquid chromatography coupled to mass spectrometry (UPLC/MS) has been used increasingly for measuring changes of low molecular weight metabolites in biofluids/tissues in response to biological challenges such as drug toxicity and disease processes. Typically samples show high variability in concentration, and the derived metabolic profiles have a heteroscedastic noise structure characterized by increasing variance as a function of increased signal intensity. These sources of experimental and instrumental noise substantially complicate information recovery when statistical tools are used. We apply and compare several preprocessing procedures and introduce a statistical error model to account for these bioanalytical complexities. In particular, the use of total intensity, median fold change, locally weighted scatter plot smoothing, and quantile normalizations to reduce extraneous variance induced by sample dilution were compared. We demonstrate that the UPLC/MS peak intensities of urine samples should respond linearly to variable sample dilution across the intensity range. While all four studied normalization methods performed reasonably well in reducing dilution-induced variation of urine samples in the absence of biological variation, the median fold change normalization is least compromised by the biologically relevant changes in mixture components and is thus preferable. Additionally, the application of a subsequent log-based transformation was successful in stabilizing the variance with respect to peak intensity, confirming the predominant influence of multiplicative noise in peak intensities from UPLC/MS-derived metabolic profile data sets. We demonstrate that variance-stabilizing transformation and normalization are critical preprocessing steps that can benefit greatly metabolic information recovery from such data sets when widely applied chemometric methods are used.