Structural basis of chorismate isomerization by Arabidopsis ISOCHORISMATE SYNTHASE1.

Salicylic acid (SA) plays a crucial role in plant defense against biotrophic and semi-biotrophic pathogens. In Arabidopsis (Arabidopsis thaliana), isochorismate synthase 1 (AtICS1) is a key enzyme for the pathogen-induced biosynthesis of SA via catalytic conversion of chorismate into isochorismate, an essential precursor for SA synthesis. Despite the extensive knowledge of ICS1-related menaquinone, siderophore, tryptophan (MST) enzymes in bacteria, the structural mechanisms for substrate binding and catalysis in plant isochorismate synthase (ICS) enzymes are unknown. This study reveals that plant ICS enzymes catalyze the isomerization of chorismate through a magnesium-dependent mechanism, with AtICS1 exhibiting the most substantial catalytic activity. Additionally, we present high-resolution crystal structures of apo AtICS1 and its complex with chorismate, offering detailed insights into the mechanisms of substrate recognition and catalysis. Importantly, our investigation indicates the existence of a potential substrate entrance channel and a gating mechanism regulating substrate into the catalytic site. Structural comparisons of AtICS1 with MST enzymes suggest a shared structural framework with conserved gating and catalytic mechanisms. This work provides valuable insights into the structural and regulatory mechanisms governing substrate delivery and catalysis in AtICS1, as well as other plant ICS enzymes.

Proteomic and antimicrobial peptide analyses of Buffalo colostrum and mature Milk whey: A comparative study.

Buffalo colostrum is the initial mammary secretion after parturition, consisting of nutritional and bioactive components. In this study, we conducted a proteomic analysis of buffalo colostrum whey to identify bioactive proteins and peptides. A total of 107 differentially expressed proteins (DEPs) were identified in buffalo colostrum whey compared to those in mature milk. Gene Ontology analysis revealed that DEPs were primarily associated with immune response and tissue development. KEGG pathway enrichment suggested that colostrum actively enhances nascent immunity involved in interleukin and interferon signaling pathways. Furthermore, candidate antimicrobial peptides (AMPs) of whey protein hydrolysates from buffalo colostrum were characterized, which exhibits broad-spectrum activity against gram-positive and gram-negative pathogens. Overall, this study improves our understanding of protein variations in buffalo lactation, and contributes to the development of AMPs from buffalo colostrum.

Deciphering the underlying core microorganisms and the marker compounds of Liupao tea during the pile-fermentation process.

BACKGROUND: Pile fermentation is one of the key steps in developing the Liupao tea (LBT) quality and unique characteristics. The complex biochemical profile of LBT results from microorganisms present during the pile-fermentation process. However, the critical underlying microorganisms and the marker compounds still need to be determined. RESULTS: Staphylococcus, Brevibacterium, Kocuria, Aspergillus, and Blastobotrys were the common dominant microorganisms at the end of the pile fermentation of LBT. Staphylococcus, Aspergillus, Blastobotrys, and nine other genera carried by raw tea are the core microorganisms in the LBT during pile fermentation. A total of 29 critical compounds contributed to the metabolic changes caused by the processing of LBT. Of these, gallic acid, adenine, hypoxanthine, uridine, betaine, 3,4-dihydroxybenzaldehyde, and α-linolenic acid could be characterized as potential marker compounds. Correlation analysis showed that the core microorganisms, including Sphingomonas, Staphylococcus, Kocuria, Aureobasidium, Blastobotrys, Debaryomyce, and Trichomonascus, were closely related to major chemical components and differential compounds. Moreover, the mutually promoting Staphylococcus, Kocuria, Blastobotrys, and Trichomonascus were correlated with the enrichment of marker compounds. Integrated molecular networking and metabolic pathways revealed relevant compounds and enzymes that possibly affect the enrichment of marker compounds. CONCLUSION: This study analyzed the LBT fermentation samples by omics analysis to reveal the stable microbial community structure, critical microorganisms, and markers compounds affecting the quality of LBT, which contributes to a better understanding of pile fermentation of LBT and the fermentation theory of dark tea. © 2023 Society of Chemical Industry.

Fabrication, Evaluation, and Antioxidant Properties of Carrier-Free Curcumin Nanoparticles.

Curcumin (Cur), a natural hydrophobic polyphenolic compound, exhibits multiple beneficial biological activities. However, low water solubility and relative instability hinder its application in food fields. In this study, carrier-free curcumin nanoparticles (CFC NPs) were prepared by adding the DMSO solution of Cur into DI water under continuous rapid stirring. The morphology of CFC NPs was a spherical shape with a diameter of 65.25 ± 2.09 nm (PDI = 0.229 ± 0.107), and the loading capacity (LC) of CFC NPs was as high as 96.68 ± 0.03%. The thermal property and crystallinity of CFC NPs were investigated by XRD. Furthermore, the CFC NPs significantly accelerated the release of Cur in vitro owing to its improved water dispersibility. Importantly, CFC NPs displayed significantly improved DPPH radical scavenging activity. Overall, all these results suggested that CFC NPs would be a promising vehicle to widen the applications of Cur in food fields.

Preparation of carrier-free astaxanthin nanoparticles with improved antioxidant capacity.

Astaxanthin (AST), a red pigment of the carotenoids, has various advantageous biological activities. Nevertheless, the wide application of AST is restricted due to its poor water solubility and highly unsaturated structure. To overcome these limitations, carrier-free astaxanthin nanoparticles (AST-NPs) were fabricated through the anti-solvent precipitation method. The AST-NPs had a small particle size, negative zeta potential and high loading capacity. Analysis of DSC and XRD demonstrated that amorphous AST existed in AST-NPs. In comparison with free AST, AST-NPs displayed enhanced stability during storage. Besides, it also showed outstanding stability when exposed to UV light. Furthermore, the antioxidant capacity of AST-NPs was significantly increased. In vitro release study showed that AST-NPs significantly delayed the release of AST in the releasing medium. These findings indicated that AST-NPs would be an ideal formulation for AST, which could contribute to the development of novel functional foods.

UHPLC-HRMS-based serum lipisdomics reveals novel biomarkers to assist in the discrimination between colorectal adenoma and cancer.

The development of a colorectal adenoma (CA) into carcinoma (CRC) is a long and stealthy process. There remains a lack of reliable biomarkers to distinguish CA from CRC. To effectively explore underlying molecular mechanisms and identify novel lipid biomarkers promising for early diagnosis of CRC, an ultrahigh-performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) method was employed to comprehensively measure lipid species in human serum samples of patients with CA and CRC. Results showed significant differences in serum lipid profiles between CA and CRC groups, and 85 differential lipid species (P < 0.05 and fold change > 1.50 or < 0.67) were discovered. These significantly altered lipid species were mainly involved in fatty acid (FA), phosphatidylcholine (PC), and triacylglycerol (TAG) metabolism with the constituent ratio > 63.50%. After performance evaluation by the receiver operating characteristic (ROC) curve analysis, seven lipid species were ultimately proposed as potential biomarkers with the area under the curve (AUC) > 0.800. Of particular value, a lipid panel containing docosanamide, SM d36:0, PC 36:1e, and triheptanoin was selected as a composite candidate biomarker with excellent performance (AUC = 0.971), and the highest selected frequency to distinguish patients with CA from patients with CRC based on the support vector machine (SVM) classification model. To our knowledge, this study was the first to undertake a lipidomics profile using serum intended to identify screening lipid biomarkers to discriminate between CA and CRC. The lipid panel could potentially serve as a composite biomarker aiding the early diagnosis of CRC. Metabolic dysregulation of FAs, PCs, and TAGs seems likely involved in malignant transformation of CA, which hopefully will provide new clues to understand its underlying mechanism.

Serum untargeted lipidomics by UHPLC-ESI-HRMS aids the biomarker discovery of colorectal adenoma.

BACKGROUND: Colorectal adenoma (CA) is an important precancerous lesion and early screening target of colorectal cancer (CRC). Lipids with numerous physiological functions are proved to be involved in the development of CRC. However, there is no lipidomic study with large-scale serum samples on diagnostic biomarkers for CA. METHODS: The serum lipidomics of CA patients (n = 50) and normal control (NR) (n = 50) was performed by ultra high performance liquid chromatography-high resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). Univariate and multivariate statistical analyses were utilized to screen the differential lipids between groups, and combining the constituent ratio analysis and diagnostic efficiency evaluation by receiver operating characteristic (ROC) curve disclosed the potential mechanism and biomarkers for CA. RESULTS: There were obvious differences in serum lipid profiles between CA and NR groups. Totally, 79 differential lipids were selected by criterion of P < 0.05 and fold change > 1.5 or < 0.67. Triacylglycerols (TAGs) and phosphatidylcholines (PCs) were the major differential lipids with ratio > 60%, indicating these two lipid metabolic pathways showed evident disequilibrium, which could contribute to CA formation. Of them, 12 differential lipids had good diagnostic ability as candidate biomarkers for CA (AUC ≥ 0.900) by ROC analysis. CONCLUSIONS: To our knowledge, this is the first attempt to profile serum lipidomics and explore lipid biomarkers of CA to help early screening of CRC. 12 differential lipids are obtained to act as potential diagnostic markers of CA. PCs and fatty acids were the main dysregulated biomarkers for CA in serum.

Serum Untargeted UHPLC-HRMS-Based Lipidomics to Discover the Potential Biomarker of Colorectal Advanced Adenoma.

BACKGROUND: As a key precancerous lesion, colorectal advanced adenoma (CAA) is closely related to the occurrence and development of colorectal cancer (CRC). Effective identification of CAA-related biomarkers can prevent CRC morbidity and mortality. Lipids, as an important endogenous substance, have been proved to be involved in the occurrence and development of CRC. Lipidomics is an advanced technique that studies lipid metabolism and biomarkers of diseases. However, there are no lipidomics studies based on large serum samples to explore diagnostic biomarkers for CAA. METHODS: An integrated serum lipid profile from 50 normal (NR) and 46 CAA subjects was performed using ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS). Lipidomic data were acquired for negative and positive ionization modes, respectively. Differential lipids were selected by univariate and multivariate statistics analyses. A receiver operator characteristic curve (ROC) analysis was conducted to evaluate the diagnostic performance of differential lipids. RESULTS: A total of 53 differential lipids were obtained by combining univariate and multivariate statistical analyses (P < 0.05 and VIP > 1). In addition, 12 differential lipids showed good diagnostic performance (AUC > 0.90) for the discrimination of NR and CAA by receiver operating characteristic curve (ROC) analysis. Of them, the performance of PC 44:5 and PC 35:6e presented the outstanding performance (AUC = 1.00, (95% CI, 1.00-1.00)). Moreover, triglyceride (TAG) had the highest proportion (37.74%) as the major dysregulated lipids in the CAA. CONCLUSION: This is the first study that profiled serum lipidomics and explored lipid biomarkers with good diagnostic ability of CAA to contribute to the early prevention of CRC. Twelve differential lipids that effectively discriminate between NR and CAA serve as the potential diagnostic markers of CAA. An obvious perturbation of TAG metabolism could be involved in the CAA formation.

Type VI secretion system is not required for virulence on rice but for inter-bacterial competition in Xanthomonas oryzae pv. oryzicola.

The type VI secretion system (T6SS), a multifunctional protein secretion device, plays very important roles in bacterial killing and/or virulence to eukaryotic cells. Although T6SS genes have been found in many Xanthomonas species, the biological function of T6SSs has not been elucidated in most xanthomonads. In this study, we identified two phylogenetically distinct T6SS clusters, T6SS1 and T6SS2, in a newly sequenced Chinese strain GX01 of Xanthomonas oryzea pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) of rice (Oryza sativa L.). Mutational assays demonstrated that T6SS1 and T6SS2 are not required for the virulence of Xoc GX01 on rice. Nevertheless, we found that T6SS2, but not T6SS1, played an important role in bacterial killing. Transcription and secretion analysis revealed that hcp2 gene is actively expressed and that Hcp2 protein is secreted via T6SS. Moreover, several candidate T6SS effectors were predicted by bioinformatics analysis that might play a role in the antibacterial activity of Xoc. This is the first report to investigate the type VI secretion system in Xanthomonas oryzae. We speculate that Xoc T6SS2 might play an important role in inter-bacterial competition, allowing this plant pathogen to gain niche advantage by killing other bacteria.

An ultra high performance liquid chromatography with tandem mass spectrometry method for simultaneous determination of thirteen components extracted from Radix Puerariae in rat plasma and tissues: Application to pharmacokinetic and tissue distribution study.

A rapid and sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was established and validated for simultaneous determination of thirteen bioactive components (gallic acid, protocatechuic acid, puerarin, p-hydroxycinnamic acid, daidzin, ononin, daidzein, naringenin, genistein, apigenin, formononetin, biochanin A, and ß-sitosterol) of Radix Puerariae extract in rat plasma and tissues. The plasma and tissues samples were pretreated by protein precipitation extraction, and umbelliferone and rutin were used as internal standards. Sample separation was performed on a ZORBAX RRHD Eclipse plus C18 column (2.1 mm × 50 mm, 1.8 µm, Agilent) with a mobile phase consisting of methanol-water (containing 0.1% formic acid). The mass spectrometry analysis was conducted in positive and negative ionization modes with multiple reaction monitoring. The lower limit of quantitation range for the 13 analytes was 0.2-35 ng/mL. The intra- and inter-day precision of all the analytes were less than 10.92%, with an accuracy ranging from -13.10 to 11.96%. Both the recovery and matrix effect were within acceptable limits. This method was successfully applied to pharmacokinetic and tissue distribution study of the 13 bioactive components in rats after oral administration of R. Puerariae extract.

Simultaneous determination of fourteen compounds of Hedyotis diffusa Willd extract in rats by UHPLC-MS/MS method: Application to pharmacokinetics and tissue distribution study.

A rapid, sensitive and selective ultra high-performance liquid chromatography-tandem mass spectrometry UHPLC-MS/MS method has been developed and validated for the simultaneous determination of fourteen bioactive ingredients (gallic acid, geniposidic acid, protocatechuic acid, caffeic acid, ferulic acid, scopoletin, apigenin-7-o-glucuronide, daidzein, apigenin, ursolic acid, oleanolic acid, ß-sitosterol, coniferin, and stigmasterol) in the plasma and tissues of rats. Danshensu and icariin were used as internal standards (IS1 and IS2). The chromatographic separation was achieved by using an Agilent ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with gradient elution using mobile phase, which consisted of 0.1% acetic acid water (solvent A) and methanol (solvent B) and pumped at a flow rate of 0.3 mL/min. Mass spectrometric detection was performed in multiple reaction monitoring (MRM) mode utilizing electrospray ionization (ESI) in positive and negative mode. The plasma samples were pretreated via protein precipitation with 300 µL of methanol containing 0.1% (v/v) formic acid and organ homogenates were processed by solid-phase extraction (SPE) with Waters Oasis HLB 3 cc (60 mg), respectively. The intra- and inter- day precisions (RSD%) were less than 10.3%, while the accuracy was ranged from -7.34% to 9.10%. Extraction recovery ranged from 85.02 to 112.0% and the matrix effects ranged from 85.12% to 109.6%. The present method exhibited excellent linearity and the lower limits of quantification (LLOQ) were 30.0 ng/mL, 15.0 ng/mL, 80.0 ng/mL, 30.0 ng/mL, 10.0 ng/mL, 3.0 ng/mL, 2.5 ng/mL, 2.5 ng/mL, 1.5 ng/mL, 15.0 ng/mL, 75.0 ng/mL, 15.0 ng/mL, 30.0 ng/mL, and 20.0 ng/mL for gallic acid, protocatechuic acid, geniposidic acid, caffeic acid, ferulic acid, scopoletin, apigenin-7-o-glucuronide, daidzein, apigenin, ursolic acid, oleanolic acid, ß-sitosterol, coniferin, and stigmasterol, respectively. This analytical method was verified by the FDA guidelines for bioanalytical method validation and applied to investigate the pharmacokinetics and biodistribution of fourteen constituents of Hedyotis diffusa Willd extract in rats. These results provide useful information for improving the pharmacokinetics and biodistribution of fourteen bioactive ingredients of Hedyotis diffusa Willd extract in SD rats, supporting additional clinical application and Chinese herbal medicine safety evaluations.

Simultaneous determination of ten active constituents of Yankening Capsule in rat plasma by ultra high performance liquid chromatography with tandem mass spectrometry.

An ultra high performance liquid chromatography with tandem mass spectrometry (U-HPLC-MS/MS) method was developed for simultaneous determination and pharmacokinetic study of ten active constituents, phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin in rat plasma after oral administration of Yankening Capsule. After mixing with two internal standards tetrahydropalmatine and rutin, plasma samples were pretreated by protein precipitation with anhydrous ethanol-acetonitrile (9:1, v/v). The U-HPLC separation was carried on a ZORBAX RRHD Eclipse Plus C18 column (2.1 mm × 50 mm, 1.8 µm) with gradient elution using a mobile phase composed of methanol and water (containing 0.3% formic acid) at a flow rate of 0.3 mL min(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via electrospray ionization source with positive-negative ionization mode. The calibration curves of ten analytes showed good linearity (r>0.9979). The lower limits of quantification of phellodendrine, coptisine, jatrorrhizine, berberine, palmatine, baicalin, wogonoside, baicalein, wogonin and emodin were 0.50, 0.50, 0.30, 0.30, 0.30, 10, 3.0, 8.0, 1.0, 8.0 µg L(-1), respectively. The relative standard deviation of intra-day precision and inter-day precision were in the range from 1.13% to 5.96% and from 0.65% to 8.85%, respectively. The matrix effects of all analytes were found to be within the acceptable range with a range of 89.99-109.3%. The method is reliable and rapid and has been applied successfully to pharmacokinetic study of the ten active constituents in rat plasma after oral administration of Yankening Capsule.

Understanding the effects of potassium ferricyanide on lead hydride formation in tetrahydroborate system and its application for determination of lead in milk using hydride generation inductively coupled plasma optical emission spectrometry.

To understand the formation of plumbane in the Pb(II)-NaBH4-K3Fe(CN)6 system, the intermediate products produced in the reaction of lead(II) and NaBH4 in the presence of K3Fe(CN)6 were studied. The produced plumbane and elemental lead were measured through continuous flow hydride generation (HG)-inductively coupled plasma optical emission spectrometry (ICP OES) and X-ray diffraction spectrometry techniques, respectively. Based on the experimental results, the explanations can be depicted in the following steps: (1) plumbane and black lead sediment (black Pb) are formed in the reaction of lead(II) and NaBH4; (2) the black Pb is oxidized by K3Fe(CN)6 to form Pb2[Fe(CN)6], which further reacts with NaBH4 to form more plumbane and black Pb; and (3) another round starts in which the produced black Pb from the step 2 is then oxidized continuously by K3Fe(CN)6 to form more Pb2[Fe(CN)6] complex, which would produce more plumbane. In short, the black Pb and Pb2[Fe(CN)6] complex are the key intermediate products for the formation of plumbane in the Pb(II)-NaBH4-K3Fe(CN)6 system. Based on the enhancement effect of potassium ferricyanide and potassium ferrocyanide, a method was developed to analyze lead in milk with HG-ICP OES technique. The detection limit of the method was observed as 0.081 µg L(-1). The linearity range of lead was found between 0.3 and 50,000 µg L(-1) with correlation coefficient of 0.9993. The recovery of lead was determined as 97.6% (n=5) for adding 10 µg L(-1) lead into the milk sample.

Artificial photosynthesis of oxalate and oxalate-based polymer by a photovoltaic reactor.

A photovoltaic reactor was designed for artificial photosynthesis, based on the reactions involved in high energy hydrogen atoms, which were produced from water electrolysis. Water and CO2, under the conditions studied, were converted to oxalate (H2C2O4) and a polymer. This was the first time that the oxalates and oxalate-based polymer were produced from the artificial photosynthesis process.

Functional analysis of an S-adenosylhomocysteine hydrolase homolog of chestnut blight fungus.

S-adenosylhomocysteine (SAH), formed after donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor, is reversibly hydrolyzed to adenosine (ADO) and homocysteine (HCY) by S-adenosylhomocysteine hydrolase (SAHH). In chestnut blight fungus (Cryphonectria parasitica), sahh, a hypovirus-regulated gene that encodes a deduced SAHH protein was shown to have an SAHH enzymatic activity in vitro. Deletion of sahh resulted in the increased accumulation of intracellular SAH and SAM but decreased ADO, and a remarkably increased accumulation of transcripts that encode adenosine kinase, methionine adenosyltransferase, and an O-methyltransferase, key components of the methylation pathway. The Δsahh knockout mutants showed a phenotype of slower growth rate, fewer aerial hyphae, loss of orange pigment, absence of asexual fruiting bodies and conidia, and a significant reduction in virulence. Deletion of sahh significantly reduced the accumulation level of transcripts of the cyp1 that encodes cyclophilin A as well as genes of the heterotrimeric G-protein signaling pathways including cpga1, cpgb1, and cpgc1 and ste12, a target activated by the MAP kinase cascade. Taken together, we demonstrated that SAHH is required for virulence and multiple traits of phenotype in C. parasitica, by regulation of the expression of genes involved in key process of the cell.

Study of the binding equilibrium between Zn(II) and HSA by capillary electrophoresis-inductively coupled plasma optical emission spectrometry.

A new method has been developed for following the interaction between zinc ion and human serum albumin (HSA) by capillary electrophoresis-inductively coupled plasma optical emission spectrometry. Under optimized experimental conditions, the detection limit (3σ) for free Zn(2+) ion was found to be 1.34 µM by running 11 replicates of the reagent blank. The RSD was less than 3% and the recovery was more than 98.13%. The linear range of zinc ion concentration was between 5.1 µM and 0.3M. The measured Zn(II)-HSA combination values of n(1) and K(1) for primary binding of Zn(2+) to HSA were 1.09 and 2.29×10(5) L mol(-1), respectively. The measured values of n(2) and K(2) for the non-specific binding of Zn(2+) to HSA were 8.96 and 6.65×10(3) L mol(-1), respectively. This new method allows rapid analysis of a small amount of sample, simple operation, while avoiding long periods of dialysis and eliminating interference from other metal ions. This method provides a reliable and convenient new way for studying interactions between metal ions and biomolecules.

Cold vapor generation interface for mercury speciation coupling capillary electrophoresis with electrothermal quartz tube furnace atomic absorption spectrometry: determination of mercury and methylmercury.

A novel on-line coupled capillary electrophoresis (CE) cold vapor generation (CVG) with electrothermal quartz tube furnace atomic absorption spectrometry (EQTF-AAS) system for mercury speciation has been developed. The mercury species (inorganic mercury and methylmercury) were completely separated by CE in a 80 cm length x 100 microm i.d. fused-silica capillary at 20 kV and using a buffer of 100 mM boric acid and 10% (v/v) methanol (pH 8.30). The effects of the inner diameter of quartz tube, the acidity of HCl, the NaBH(4) concentration and N(2) flow rate on Hg signal intensity were investigated. Speciation of mercury was highlighted using CE-CVG-EQTF-AAS. The detection limits of methylmercury and mercury were 0.035 and 0.027 microg mL(-1), respectively. The precisions (RSDs) of peak height for six replicate injections of a mixture of 10 microg mL(-1) (as Hg) were better than 4%. The interface was used for speciation analysis of mercury in dry goldfish muscle.

Determination of free calcium and calcium-containing species in human plasma by capillary electrophoresis-inductively coupled plasma optical emission spectrometry.

A new method for the determination of free calcium concentration in human plasma was developed by online coupling capillary electrophoresis (CE) with inductively coupled plasma optical emission spectrometry (ICP-OES). Baseline separation of calcium-containing species was achieved by CE-ICP-OES in a 120-cm-long capillary with 100-microm internal diameter, at 20 kV applied voltage, with a 30 mmol/L Tris-HCl buffer at pH 7.4. A total of eight calcium-containing species were found in human plasma; the concentration of free calcium ion was found to be 41.9 mg/L. The concentrations of calcium for other seven calcium species, estimated from the calibration against Ca(2+) standard, were 3.14-15.6 mg/L. The precision (RSD, n = 10) ranged from 1.2 to 2.7% for the migration time and 2.8 to 3.9% for the peak area. The developed method was also applied to analyze plasma samples with recovery ranged from 94.5 to 102% for samples spiked with 40 mg/L free Ca(2+) ion.

Speciation of magnesium in rat plasma using capillary electrophoresis-inductively coupled plasma-atomic emission spectrometry.

A new method for speciation analysis of magnesium species and quantification of free magnesium concentrations in rat plasma was developed by on-line coupling of CE with inductively coupled plasma-atomic emission spectrometry (ICP-AES). Baseline separation of seven magnesium species was achieved by using a 120 cm (100 microm internal diameter) fused-silica capillary, a 20 kV separation voltage and a solution of 50 mmol/L NaAc-HAc (pH 5.5) as electrolyte buffer. CE-ICP-AES analysis of a rat plasma sample showed the presence of seven magnesium species, one of which was identified as free Mg2+ ion by spiking a Mg2+ standard; the migration time of the Mg2+ peak in the standard and the spiked sample matched with each other. One protein-bound magnesium species in rat plasma is associated with albumin, and the other three species are combined with globulin. The concentration of free magnesium in the plasma was 14.0 mg/L. The other six magnesium species were estimated to be 4-15 mg/L. RSDs of migration time and peak area for the magnesium species from ten replicates were less than 5%. The developed method was also applied to speciation analysis of magnesium species in spiked plasma samples. The recoveries of the free magnesium species in four samples ranged from 95.8 to 103.8%.