Selective detection of azelnidipine in pharmaceuticals via carbon dot mediated spectrofluorimetric method: A green approach.

A spectrofluorimetric method using fluorescent carbon dots (CDs) was developed for the selective detection of azelnidipine (AZEL) pharmaceutical in the presence of other drugs. In this study, N-doped CDs (N-CDs) were synthesized through a single-step hydrothermal process, using citric acid and urea as precursor materials. The prepared N-CDs showed a highly intense blue fluorescence emission at 447 nm, with a photoluminescence quantum yield of ~21.15% and a fluorescence lifetime of 0.47 ns. The N-CDs showed selective fluorescence quenching in the presence of all three antihypertensive drugs, which was used as a successful detection platform for the analysis of AZEL. The photophysical properties, UV-vis light absorbance, fluorescence emission, and lifetime measurements support the interaction between N-CDs and AZEL, leading to fluorescence quenching of N-CDs as a result of ground-state complex formation followed by a static fluorescence quenching phenomenon. The detection platform showed linearity in the range 10-200 µg/ml (R2 = 0.9837). The developed method was effectively utilized for the quantitative analysis of AZEL in commercially available pharmaceutical tablets, yielding results that closely align with those obtained from the standard method (UV spectroscopy). With a score of 0.76 on the 'Analytical GREEnness (AGREE)' scale, the developed analytical method, incorporating 12 distinct green analytical chemistry components, stands out as an important technique for estimating AZEL.

Speciation of essential nutrient trace elements in coconut water.

Coconut water (Cocos Nucifera) is shown to be a source of essential elements present in the form of low-molecular weight stable complexes known for their bio-availability. The total element concentrations were in the range of 0.2-2.7, 0.3-1, 3-14 and 0.5-2 ppm for Fe, Cu, Mn, and Zn, respectively, and varied as a function of the origin of the nut and its maturity. Speciation was investigated by size-exclusion chromatography - inductively coupled plasma mass spectrometry (ICPMS), and hydrophilic interaction liquid chromatography (HILIC) - electrospray-OrbitrapMS. The metal species identified included: iron complexes with citrate and malate: FeIII(Cit)3(Mal), FeIII(Cit)2(Mal)2, FeIII(Mal)2, glutamine: FeIII(Glu)2 and nicotianamine: FeII(NA); copper complexes with phenylanine: CuII(Phe)2 and CuII(Phe)3 and nicotianamine: CuII(NA); zinc complexes with citrate: ZnII(Cit)2 and nicotianamine ZnII(NA) and manganese complex with asparagine MnII(Asp)2. The contributions of the individual species to the total elements concentrations could be estimated by HILIC - ICP MS.

Metabolite Profiling of a Zinc-Accumulating Rice Mutant.

Breeding crops with high zinc (Zn) density is an effective way to alleviate human dietary Zn deficiencies. We characterized a mutant Lilizhi (LLZ) accumulating at least 35% higher Zn concentration in grain than the wild type (WT) in hydroponic experiments. The mutant stored less Zn content in the root and transported more Zn to the grain. Metabolite profiling demonstrated that, with high Zn treatment, the contents of proline, asparagine, citric acid, and malic acid were enhanced in both LLZ and the WT, which were thought to be involved in Zn transport in rice. Furthermore, the contents of cysteine, allothreonine, alanine, tyrosine, homoserine, ß-alanine, and nicotianamine required for the production of many metal-binding proteins were specifically increased in LLZ. LLZ had higher capability of amino acid biosynthesis and metal cation transportation. The current research extends our understanding on the physiological mechanisms of Zn uploading into grain and provides references for further Zn biofortification breeding in rice.

Determination of ferric iron chelators by high-performance liquid chromatography using luminol chemiluminescence detection.

Iron is an essential element for higher plants, and its acquisition and transportation is one of the greatest limiting factors for plant growth because of its low solubility in normal soil pHs. Higher plants biosynthesize ferric iron [Fe(III)] chelator (FIC), which solubilizes the iron and transports it to the rhizosphere. A high-performance liquid chromatography (HPLC) post-column method has been developed for the analysis of FICs using the luminol/H2O2 system for chemiluminescence (CL) detection. A size-exclusion column was the most suited in terms of column efficiency and CL detection efficiency. Mixing of the luminol with H2O2 in a post-column reaction was feasible, and a two-pump system was used to separately deliver the luminol and H2O2 solutions. The luminol and H2O2 concentrations were optimized using Fe(III)-EDTA and Fe(III)-citrate (Cit) solutions as analytes. A strong CL intensity was obtained for Fe(III)-Cit when EDTA was added to the luminol solution, probably because of an exchange of Cit with EDTA after separation on the HPLC column; CL efficiency was much higher for Fe(III)-EDTA than for Fe(III)-Cit with the luminol/H2O2 system. The present method can detect minute levels of Fe(III)-FICs; the detection limits of Fe(III)-EDTA, Fe(III)-Cit and Fe(III)-nicotianamine were 0.77, 2.3 and 1.1pmol, respectively.

Synthesis of [13C4]-labeled 2'-deoxymugineic acid.

The phytosiderophore 2'-deoxymugineic acid (DMA) is exuded via the root system by all grasses (including important crop plants like rice, wheat and barley) to mobilize Fe(III) from soil and improve plant Fe nutrition, crucial for high crop yields. Elucidation of the biogeochemistry of 2'-deoxymugineic acid in the rhizosphere requires its quantification in minute amounts. To this end, (13)C4-DMA was synthesized for the first time, from cheap isotopically labeled starting materials. The synthetic route utilizes L-allyl((13)C2)glycine and L-(2-(13)C)azetidine ((13)C)carboxylic acid as versatile labeled building blocks. The title compound was recently used as an internal standard for analysis of soil and plant samples allowing the first accurate quantification of DMA in these matrices by means of LC-MS/MS. It is furthermore used in tracer experiments investigating biodegradation of DMA in soil.

Accurate LC-ESI-MS/MS quantification of 2'-deoxymugineic acid in soil and root related samples employing porous graphitic carbon as stationary phase and a ¹³C4-labeled internal standard.

For the first time the phytosiderophore 2'-deoxymugineic acid (DMA) could be accurately quantified by LC-MS/MS in plant and soil related samples. For this purpose a novel chromatographic method employing porous graphitic carbon as stationary phase combined with ESI-MS/MS detection in selected reaction monitoring was developed. Isotope dilution was implemented by using in-house synthesized DMA as external calibrant and ¹³C4-labeled DMA as internal standard (concentration levels of standards 0.1-80 µM, determination coefficient of linear regression R² > 0.9995). Sample preparation involved acidification of the samples in order to obtain complete dissociation of metal-DMA complexes. Excellent matrix related LOD and LOQ depending on different experimental setups were obtained in the range of 3-34 nM and 11-113 nM, respectively. Standard addition experiments and the implementation of the internal ¹³C4-DMA standard proved the accuracy of the quantification strategy even in complex matrices such as soil solution. The repeatability of the method, including sample preparation, expressed as short- and long term precision was below 4 and 5% RSD, respectively. Finally, application in the context of plant and soil research to samples from rhizosphere sampling via micro suction cups, from soil solutions and soil adsorption/extraction studies revealed a DMA concentration range from 0.1 to 235 µM.

Mechanisms associated with Fe-deficiency tolerance and signaling in shoots of Pisum sativum.

Mechanisms of Fe-deficiency tolerance and signaling were investigated in shoots of Santi (deficiency tolerant) and Parafield (deficiency intolerant) pea genotypes using metabolomic and physiological approaches. From metabolomic studies, Fe deficiency induced significant increases in N-, S- and tricarboxylic acid cycle metabolites in Santi but not in Parafield. Elevated N metabolites reflect an increase in N-recycling processes. Increased glutathione and S-metabolites suggest better protection of pea plants from Fe-deficiency-induced oxidative stress. Furthermore, Fe-deficiency induced increases in citrate and malate in leaves of Santi suggests long-distance transport of Fe is promoted by better xylem unloading. Supporting a role of citrate in the deficiency tolerance mechanism, physiological experiments showed higher Fe and citrate in the xylem of Santi. Reciprocal-grafting experiments confirm that the Fe-deficiency signal driving root Fe reductase and proton extrusion activity is generated in the shoot. Finally, our studies show that auxin can induce increased Fe-reductase activity and proton extrusion in roots. This article identifies several mechanisms in shoots associated with the differential Fe-deficiency tolerance of genotypes within a species, and provides essential background for future efforts to improve the Fe content and deficiency tolerance in peas.

Determination of nicotianamine in soy sauce and other plant-based foods by LC-MS/MS.

Nicotianamine is a nonproteinogenic amino acid, known to be an important metal chelator in plants. Recently, the antihypertensive effect of nicotianamine was discovered. In this study, a simple method to determine nicotianamine was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a multimode ODS column. This method does not need derivatizing or ion-pairing reagents to retain nicotianamine, which is known for its poor retention on reversed-phase columns because of its high polarity. Moreover, this method showed a sufficient limit of detection (0.5 ng/mL), so it was found to be suitable for the analysis of nicotianamine in soy sauce and other foods, without cleanup. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of nicotianamine in soy sauce ranged from <0.25 to 71 µg/g. Nicotianamine was also determined in other foods, including soy milk, vegetable juice, fruit juice, and bottled tea.

Mechanistic study on degradation of azelnidipine solution under radical initiator-based oxidative conditions.

We identified four degradants (Dg-A, Dg-B, Dg-C, Dg-D) of azelnidipine to be generated under radical initiator-based oxidative conditions and proposed the mechanistic pathway for their formation. 2,2'-Azobisisobutyronitrile was used as a radical initiator. There appeared to be two major pathways in the oxidation of the 1,4-dihydropyridine moiety. One was initiated by hydrogen abstraction from the C-4 position of the dihydropyridine ring, followed by hydrogen abstraction from the N-1 position, leading to aromatization of the dihydropyridine ring and Dg-A generation. The other was initiated by hydrogen abstraction from the N-1 position of the dihydropyridine ring followed by oxidation and hydrolysis to yield Dg-B. Furthermore, Dg-B was subjected to hydrolysis to generate Dg-C and Dg-D. It has been revealed that the rate of the Dg-B degradation was predominantly governed by the water content of the solvent used. Water participation in Dg-B degradation was proved by monitoring the incorporation of heavy oxygen atom ((18)O) into the structure with LC-MS, in which the experiment was carried out in a medium prepared with heavy oxygen water to label (18)O during the hydrolysis.

Use of recombinantly produced 15N3-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry.

Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [(15)N(3)]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots.

A highly sensitive, quick and simple quantification method for nicotianamine and 2'-deoxymugineic acid from minimum samples using LC/ESI-TOF-MS achieves functional analysis of these components in plants.

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) was developed. The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride. The amounts of NA and DMA in 10 mul of xylem sap from rice cultivated under iron (Fe)-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Time-course study and effects of drying method on concentrations of gamma-aminobutyric acid, flavonoids, anthocyanin, and 2''-hydroxynicotianamine in leaves of buckwheats.

Concentrations of gamma-aminobutyric acid (GABA), rutin, minor flavonoids (such as orientin), anthocyanin, and 2''-hydroxynicotianamine (2HN) were quantified in the leaves of common and tartary buckwheat (Fagopyrum esculentum Moench and Fagopyrum tataricum Gaertn., respectively), at 14, 28, and 42 days after sowing (DAS). GABA and rutin concentrations peaked at 42 DAS, whereas anthocyain, 2HN, and minor flavonoid concentrations declined with the age of the plants. However, at 42 DAS, anthocyanin concentrations in the leaves of tartary buckwheat Hokkai T10 leaves were at least 10-fold greater than in the other buckwheats tested. In addition, the effects on target compound concentrations and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity of three different drying methods (20 h at 40 degrees C, 7 h at 70 degrees C, or lyophilization) were investigated. In general, the drying method had no significant effect on the parameters tested. These results indicate that, in terms of GABA, rutin, and anthocyanin concentrations, leaf powder from 42 day old Hokkai T10 has the potential to be a useful food ingredient, such as Ao-jiru juice.

Determination of azelnidipine by LC-ESI-MS and its application to a pharmacokinetic study in healthy Chinese volunteers.

A simple, rapid and sensitive high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) assay for determination of azelnidipine in human plasma using perospirone as the internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide solution, plasma samples were extracted with diethyl ether and separated on a C18 column with a mobile phase of methanol-5 mM ammonium acetate solution (90:10, v/v). The lower limit of quantification (LLOQ) was 0.20 ng/ml. After administration of a single dose of azelnidipine 8mg and 16 mg, respectively; the area under the plasma concentration versus time curve from time 0 h to 96 h (AUC(0-96) were (186 +/- 47) ng ml(-1) h, (429 +/- 145) ng ml(-1) h, respectively; clearance rate (CL/F) were (45.94 +/- 11.61), (42.11 +/- 14.23) L/h, respectively; peak plasma concentration Cmax were (8.66 +/- 1.15), (19.17 +/- 4.13) ng/ml, respectively; apparent volume of distribution (Vd) were (1749 +/- 964), (2480 +/- 2212) L, respectively; time to Cmax (Tmax) were (2.8 +/- 1.2), (3.0 +/- 0.9) h, respectively; elimination half-life (t(1/2beta)) were (22.8 +/- 2.4), (23.5 +/- 4.2) h, respectively; and MRT were (25.7 +/- 1.3), (26.2 +/- 2.2) h, respectively; The essential pharmacokinetic parameters after oral multiple doses (8 mg, q.d.) were as follows: (Cmax) ss, (15.04 +/- 2.27) ng/ml; (Tmax) ss, (2.38 +/- 0.92) h; (Cmin) ss, (3.83 +/- 0.94) ng/ml; C(av), (7.05 +/- 1.54) ng/ml; DF, (1.62 +/- 0.26); AUCss, (169.19 +/- 36.87) ng ml(-1) h.

CE of phytosiderophores and related metal species in plants.

Phytosiderophores (PS) and the closely related substance nicotianamine (NA) are key substances in metal uptake into graminaceous plants. Here, the CE separation of these substances and related metal species is demonstrated. In particular, the three PS 2'-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), and NA, are separated using MES/Tris buffer at pH 7.3. Moreover, three Fe(III) species of the different PS are separated without any stability problems, which are often present in chromatographic analyses. Also divalent metal species of Cu, Ni, and Zn with the ligands DMA and NA are separated with the same method. By using a special, zwitterionic CE capillary, even the separation of two isomeric Fe(III) chelates with the ligand ethylenediamine-N,N'-bis(o-hydroxyphenyl)acetic acid (EDDHA) is possible (i.e., meso-Fe(III)-EDDHA and rac-Fe(III)-EDDHA), and for fast separations of NA and respective divalent and trivalent metal species, a polymer CE microchip with suppressed EOF is described. The proposed CE method is applicable to real plant samples, and enables to detect changes of metal species (Cu-DMA, Ni-NA), which are directly correlated to biological processes.

Highly sensitive quantitative analysis of nicotianamine using LC/ESI-TOF-MS with an internal standard.

A highly sensitive quantitative method for analyzing nicotianamine (NA) by liquid chromatography/electrospray ionization time-of-flight mass spectrometry (LC/ESI-TOF-MS) is reported. Fluorenylmethoxycarbonylation of nicotianamine reduced its polarity and enabled its retention in a reversed-phase column. The adoption of N(epsilon)-nicotyllysine (NL) as an internal standard ensured reliable quantification by giving a linear calibration curve drawn between the NA/NL molar ratios of standard solutions injected and the NA/NL area ratios in mass chromatograms. The high sensitivity of this analytical method allowed us to measure the amount of NA. This analytical method has applications to all research concerning NA.

Anion exchange liquid chromatography--inductively coupled plasma-mass spectrometry detection of the Co2+, Cu2+, Fe3+ and Ni2+ complexes of mugineic and deoxymugineic acid.

Phytosiderophores, such as mugineic and deoxymugineic acid, are produced by graminaceous plant species in response to Fe deficiency conditions normally experienced in calcareous and alkaline non-calcareous soils. As these phytosiderophores have the ability to form thermodynamically stable complexes with other metal cations present in the growing medium, they have also been implicated in the transport and bioavailability of these metals in the environment. However, routine analytical methodology to detect the various metal complexes formed by these phytosiderophores is lacking. Therefore, as these complexes are negatively charged over a wide range of pH values, anion exchange liquid chromatography (AE LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) was investigated as a means to separate and quantify these complexes. The metal-phytosiderophore complexes were prepared at pH 7 and separated by NaOH or NH4NO3 gradient elution on a Dionex AS11 anion exchange column. Of the metals tested only the Co2+ and Ni2+ complexes of mugineic and deoxymugineic acid were detected when using a 0-20mM NaOH gradient elution profile. However, the phytosiderophore complexes of Cu2+ and Fe3+ were also detected when using NH4NO3 as the mobile phase at pH 7. Base-assisted hydrolysis of the latter two complexes is proposed to explain their apparent 'instability' in the high pH NaOH mobile phase. The absolute detection limits of the developed methodologies for these metal complexes ranged from 0.1 to 2.8pmol. As phytosiderophore complexes with Cd2+ and Zn2+ were not detected, it was concluded that the dissociation kinetics of these metal-phytosiderophore complexes were too rapid for these complexes to be observed in the present chromatographic conditions.

[Determination of the azetidine-2-carboxylic acid from lily of the valley].

This paper reports the separation and determination of the azetidine-2-carboxylic acid in lily of the valley by high performance liquid chromatography (HPLC) with post-column derivatization and fluorescence detection. The operating conditions were cation exchange resin column (30 cm x 0.4 cm i.d.) with buffer solution A [1.96% (mass fraction) sodium citrate solution pH 3.10 +/- 0.02] and B [2.1% (mass fraction) sodium nitrate solution pH 9.60 +/- 0.02] as mobile phase at flow-rate of 0.4 mL/min, gradient elution, column temperature 65 degrees C, detection wavelength lambda ex = 338 nm and lambda em = 425 nm. The retention time of azetidine-2-carboxylic acid was 5.86 minutes. The recovery was 96.4% and coefficient of variation was 0.94%. This method is simple, rapid and sensitive.

Azetidine-2-carboxylic acid contaminated dietary proline as a cause of urinary excretion of 4-amino-2-(S-cysteinyl)butyric acid in patients on oral treatment with a synthetic diet.

Three children with branched-chain ketoaciduria (maple syrup urine disease) were found to excrete an abnormal amino acid when they were on an artificial diet. This substance was identified as 4-amino-2-(S-cysteinyl)butyric acid with the use of column liquid chromatography, gas chromatography--mass spectrometry of various derivatives, and 360 MHz 1H-NMR spectroscopy. The same compound was detected in urine samples from subjects undergoing an oral loading test with L-proline. The chromatographic analysis of commercial proline from two sources indicated that one of the batches was contaminated (less than 1%) with L-azetidine-2-carboxylic acid (the homologue of proline with a four-membered ring). The latter compound is probably metabolized by the human via ring-opening and addition of a cysteine moiety. It is highly probable that the artificial diet given to the patients contained the impure proline and that the L-azetidine-2-carboxylic acid in the proline gave rise to the excretion of the 4-amino-2-(S-cysteinyl)butyric acid.