Eunice Yanez Barrientos

Determination of SeMet and Se(IV) in biofortified yeast by ion-pair reversed phase liquid chromatography-hydride generation-microwave induced nitrogen plasma atomic emission spectrometry (HPLC-HG-MP-AES)

Selenium biofortified yeast is the most common dietary Se supplement in human nutrition and in farm animals. Therefore, the production and routine quality control of commercial products are highly demanded. In this work, a simple and cost-effective procedure is proposed for the determination of SeMet and Se(IV) in hydrolyzed yeast, consisting of ion-pair reversed phase separation, post-column hydride generation and Se quantification by atomic emission spectrometry with microwave plasma sustained by nitrogen (HPLC-HG-MP-AES). Freeze-dried biomass was hydrolyzed with methanesulfonic acid; chromatographic separation was performed with a mobile phase containing 0.08% v/v heptafluorobutyric and methanol (92:8) at a flow rate of 1 mL min−1; the column effluent was on-line mixed with an alkaline solution of potassium persulfate (K2S2O8 6% m/v, NaOH 3% m/v), passed through a reaction coil submerged in a water bath at 60 °C, and then 10 M hydrochloric acid was added prior to hydride generation in the MP-AES multimode sample introduction system (NaBH4 2% m/v, NaOH 0.3% m/v). The total chromatographic run was accomplished in 5 min and the evaluated on-column quantification limits were 59 ng Se mL−1 for Se(IV) and 0.52 μg mL−1 for SeMet. The procedure was tested using standardized Seleno Excell® high selenium yeast and then applied for the analysis of Saccharomyces cerevisiae biofortified under different fermentation and exposure conditions. The procedure was capable of detecting differences in selenium concentration among cultures and the results were consistent with those obtained while coupling HPLC separation directly to ICP-MS detection.

Mechanistic insight into chromium(VI) reduction by oxalic acid in the presence of manganese(II)

Over the past few decades, reduction of hexavalent chromium (Cr(VI)) has been studied in many physicochemical contexts. In this research, we reveal the mechanism underlying the favorable effect of Mn(II) observed during Cr(VI) reduction by oxalic acid using liquid chromatography with spectrophotometric diode array detector (HPLC-DAD), nitrogen microwave plasma atomic emission spectrometry (HPLC-MP-AES), and high resolution mass spectrometry (ESI-QTOFMS). Both reaction mixtures contained potassium dichromate (0.67 mM Cr(VI)) and oxalic acid (13.3mM), pH 3, one reaction mixture contained manganese sulfate (0.33 mM Mn(II)). In the absence of Mn(II) only trace amounts of reaction intermediates were generated, most likely in the following pathways: (1) Cr(VI)→ Cr(IV) and (2) Cr(VI)+Cr(IV)→ 2Cr(V). In the presence of Mn(II), the active reducing species appeared to be Mn(II) bis-oxalato complex (J); the proposed reaction mechanism involves a one-electron transfer from J to any chromium compound containing CrO bond, which is reduced to CrOH, and the generation of Mn(III) bis-oxalato complex (K). Conversion of K to J was observed, confirming the catalytic role of Mn(II). Since no additional acidification was required, the results obtained in this study may be helpful in designing a new, environmentally friendly strategy for the remediation of environments contaminated with Cr(VI).

Effect of different glycation agents on Cu(II) binding to human serum albumin, studied by liquid chromatography, nitrogen microwave-plasma atomic-emission spectrometry, inductively-coupled-plasma mass spectrometry, and high-resolution molecular-mass spectrometry.

AbstractThe ability of human serum albumin to capture unbound copper under different clinical conditions is an important variable potentially affecting homeostasis of this element. Here, we propose a simple procedure based on size-exclusion chromatography with on-line UV and nitrogen microwave-plasma atomic-emission spectrometry (MP-AES) for quantitative evaluation of Cu(II) binding to HSA upon its glycation in vitro. The Cu-to-protein molar ratio for non-glycated albumin was 0.98 ± 0.09; for HSA modified with glyoxal (GO), methylglyoxal (MGO), oxoacetic acid (GA), and glucose (Glc), the ratios were 1.30 ± 0.22, 0.72 ± 0.14, 0.50 ± 0.06, and 0.95 ± 0.12, respectively. The results were confirmed by using ICP-MS as an alternative detection system. A reduced ability of glycated protein to coordinate Cu(II) was associated with alteration of the N-terminal metal-binding site during incubation with MGO and GA. In contrast, glycation with GO seemed to generate new binding sites as a result of tertiary structural changes in HSA. Capillary reversed-phase liquid chromatography with electrospray-ionization quadrupole-time-of-flight tandem mass spectrometry enabled detection and identification of Cu(II) coordinated to the N-terminal metal-binding site (Cu(II)–DAHK) in all tryptic digests analyzed. This is the first report confirming Cu(II)–DAHK species in HSA by means of high-resolution tandem mass spectrometry, and the first report on the use of MP-AES in combination with chromatographic separation. Graphical AbstractGeneral scheme designed to study Cu(II) binding to glycated versus non-glycated albumin and MPAESinstrumentation used

Melatonin reduces lead levels in blood, brain and bone and increases lead excretion in rats subjected to subacute lead treatment.

Melatonin, a hormone known for its effects on free radical scavenging and antioxidant activity, can reduce lead toxicity in vivo and in vitro.We examined the effects of melatonin on lead bio-distribution. Rats were intraperitoneally injected with lead acetate (10, 15 or 20mg/kg/day) with or without melatonin (10mg/kg/day) daily for 10 days. In rats intoxicated with the highest lead doses, those treated with melatonin had lower lead levels in blood and higher levels in urine and feces than those treated with lead alone, suggesting that melatonin increases lead excretion. To explore the mechanism underlying this effect, we first assessed whether lead/melatonin complexes were formed directly. Electronic density functional (DFT) calculations showed that a lead/melatonin complex is energetically feasible; however, UV spectroscopy and NMR analysis showed no evidence of such complexes. Next, we examined the liver mRNA levels of metallothioneins (MT) 1 and 2. Melatonin cotreatment increased the MT2 mRNA expression in the liver of rats that received the highest doses of lead. The potential effects of MTs on the tissue distribution and excretion of lead are not well understood. This is the first report to suggest that melatonin directly affects lead levels in organisms exposed to subacute lead intoxication.

Monitoring of phosphorus oxide ion for analytical speciation of phosphite and phosphate in transgenic plants by high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Large amounts of phosphate fertilizers utilized in agriculture and their relatively poor efficiency are of high ecological and economic concern. Therefore, transgenic plants capable of metabolizing phosphite are being engineered. In support of this biotechnological task, analytical speciation of phosphorus in biological tissues is required. In this study, plant extracts were analyzed by liquid chromatography-inductively coupled plasma mass spectrometry at m/z of elemental phosphorus and phosphorus oxide ions. Using polymeric-based anion exchange column and millimolar concentration of nitric acid in potassium phthalate mobile phase (pH 2.5), phosphite and phosphate ions were baseline resolved with retention times 6.95 ± 0.03 and 7.90 ± 0.03 min and with a total chromatographic run time 10 min. The detection limits were 1.58 and 1.74 μg P L(-1) at m/z 47, as compared to 2.18 and 2.04 μg P L(-1) at m/z 31, respectively. The results obtained in real world samples for the two detection modes were in good agreement, yet signal acquisition at m/z 47 enabled better precision without collision/reaction cell (RSD below 2%) as compared to RSD around 4% obtained at m/z 31 using He-pressurized cell (3.5 mL min(-1)).

Determination of total arsenic and speciation analysis in Mexican maize tortillas by hydride generation – microwave plasma atomic emission spectrometry and high performance liquid chromatography – inductively coupled plasma – mass spectrometry

The determination of total arsenic (tAs), inorganic As (iAs = As(III) + As(V)) and As speciation in Mexican maize tortillas has been undertaken for the first time. Hydride generation – microwave plasma atomic emission spectrometry (HG-MP-AES) with tellurium (sodium tellurite Te(IV)) as the internal standard (IS) yielded tAs results in good agreement with those provided by inductively coupled plasma mass spectrometry (ICP-MS). In seven products from local markets, tAs was found in the range of 21.8–192 μg As kg−1. Analysis of As(III), As(V), MMAs(V), and DMAs(V) by an established anion exchange liquid chromatography – ICP-MS procedure showed that iAs corresponded to 72.3–98.0% of tAs. A non-chromatographic procedure was envisioned for iAs: diluted tortilla extract or calibration solution (pH 9.0) was loaded on a strong anion exchange cartridge modified with silver chloride, iAs fraction was collected and analyzed by HG-MP-AES with IS. For tAs by HG-MP-AES (with IS) the method quantification limit was 47.6 μg As kg−1 whereas for iAs it got worse (95 μg As kg−1) due to the low recovery of this As form in SPE ( 93% while analyzing natural water with off-line quantification by any of the two atomic spectrometry techniques, indicating that the chemical matrix of tortillas was responsible for poor SPE performance. It is concluded that HG-MP-AES with IS is a cost-effective alternative for the determination of tAs that offers improved precision under a non-rigorous protocol; however, efficient separation/preconcentration of iAs by SPE is still a challenge.

Determination of copper and lead in tequila by conventional MALDI-TOFMS and partial least square regression

RATIONALE Quantification of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is challenging yet attractive, due to micro-scale procedural simplicity, high throughput and lack of memory effects. Since these features are important while analyzing trace elements in quality control schemes, MALDI-TOFMS was used for the determination of copper (Cu) and lead (Pb) in tequila with quantification carried out by partial least squares regression (PLS2) and by univariate calibration (UC). METHODS In the proposed procedure, Bi(III) was added as internal standard (IS), diethyldithiocarbamate complexes were formed (pH 7.4) and extracted into chloroform; after solvent evaporation and re-constitution in acetonitrile, the sample was co-crystallized with α-cyano-4-hydroxycinnamic acid on a steel target. From the acquired mass spectra, UC was performed using IS-normalized signals of the monoisotopic ions of analytes, and the m/z range 350-513 was used for PLS2. Accuracy was tested by recovery experiments and by inductively coupled plasma (ICP)-MS analysis. RESULTS When compared with direct analyte signal measurements, application of IS yielded enhanced analytical performance using either UC or PLS2; the method quantification limits were: 11.1 μg L-1 , 23.4 μg L-1 for Cu and 89.8 μg L-1 , 97.1 μg L-1 for Pb, respectively. In tequila, MALDI-TOFMS and ICP-MS provided consistent results for Cu (165-2599 μg L-1 ); Pb was not detected in any sample by MALDI-TOFMS, yet recoveries obtained after standard addition were indicative of acceptable accuracy (400 μg L-1 Pb added; recoveries: 91.2-108% for UC and 98.8-120% for PLS2). CONCLUSIONS New experimental evidence has been provided supporting the inclusion of trace metals quantification within a range of MALDI-TOFMS applications. Slightly better results were obtained for UC as compared with PLS2 yet both methods can be recommended for testing the compliance of Cu and Pb levels with Official Mexican Norm. Of note, while using PLS2, there is no need for signal integration nor for IS normalization.

Changes of Metabolomic Profile in Helianthus annuus under Exposure to Chromium(VI) Studied by capHPLC-ESI-QTOF-MS and MS/MS

The application of capHPLC-ESI-QTOF-MS and MS/MS to study the impact of Cr(VI) on metabolites profile in Helianthus annuus is reported. Germinated seeds were grown hydroponically in the presence of Cr(VI) (25 mgCr/L) and root extracts of the exposed and control plants were analyzed by untargeted metabolomic approach. The main goal was to detect which metabolite groups were mostly affected by Cr(VI) stress; two data analysis tools (ProfileAnalysis, Bruker, and online XCMS) were used under criteria of intensity threshold 5 · 104 cps, fold change ≥ 5, and p ≤ 0.01, yielding precursor ions. Molecular formulas were assigned based on data processing with two computational tools (SIRIUS and MS-Finder); annotation of candidate structures was performed by database search using CSI:FingerID and MS-Finder. Even though ultimate identification has not been achieved, it was demonstrated that secondary metabolism became activated under Cr(VI) stress. Among 42 candidate compounds returned from database search for seven molecular formulas, ten structures corresponded to isocoumarin derivatives and eleven were sesquiterpenes or sesquiterpene lactones; three benzofurans and four glycoside or pyrane derivatives of phenolic compounds were also suggested. To gain further insight on the effect of Cr(VI) in sunflower, isocoumarins and sesquiterpenes were selected as the target compounds for future study.