The usefulness of derivatization in high-performance liquid chromatography/ion trap tandem mass spectrometry (electro-spray ionization) of conjugated vitamin D metabolites was examined. 24,25-Dihydroxyvitamin D3 [24,25(OH)2D3]-3-and -24-glucuronides (G) were converted to the adducts with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), the methyl esters and the acetates. The combination of the derivatization with PTAD and methylation improved the detection limit in both the positive- and negative-ion modes. The PTAD adducts of 24,25(OH)2D3-3G and -24G also gave a characteris-tic product ion indicating the conjugation position by the positive-MS/MS/MS, which was not observed in the intact forms. 25-Hydroxyvitamin D3 3-sulfate gave the deprotonated ion in the negative-ion mode, but no characteristic ion was observed in the positive-ion mode. On the contrary, its PTAD adduct gave a molecular-related ion in the positive-ion mode, the MS/MS/MS of which provided structural information, that is, the conjugation position and the existence of a sulfuric acid group.
A method for the separation and determination of bile acid 24-glucuronides using liquid chromatography (LC)/electro-spray ionization (ESI)-mass spectrometry (MS) has been developed. In the ESI mode, 24-glucuronides were characterized by an intense deprotonated molecule [M-H]- with a fragment ion [M-H-176]- formed by elimination of the glucuronic acid moiety. The ratio of these negative ions was significantly influenced by the orifice-1 and the ring lens voltages. Bile acid 24-glucuronides in human urine were extracted with an OASIS HLB cartridge and then subjected to the LC/ESI-MS analysis employing an 18O-labeled internal standard. The simultaneous separation was attained on a Develosil ODS-HG-5 column using 20 mM ammonium acetate (pH 6.0)-acetonitrile (3:1→3:2, v/v, 35 min) in a linear gradient elution mode, where the 24-glucuronides were monitored with a characteristic negative ion [M-H]-; the limit of detection was 10 fmol. This newly developed method was applied to the quantitation of bile acid 24-glucuronides in human urine with satisfactory selectivity and reliability.
Reported herein are the adsorption properties of bovine serum albumin to glassy carbon (GC) electrodes coated with poly(phenylene oxide) (PPO) films formed by anodic polymerization of HOC6H4R in phosphate buffer (pH 7.4), where R=o-, m-, and p-MeO- or m-Me(OCH2CH2)nO- (n=1 - 4) group. Evaluation of the voltammetric response of caffeic acid demonstrated that a GC electrode coated with a PPO film derived from m-HOC6H4O(CH2CH2O)4Me not only shows satisfactory electrochemical performance but also resists surface fouling due to protein adsorption. Coating with the film also proved to be useful in preventing the surface of a gold electrode from being fouled by protein adsorption.
Microchannels having a 150×100 µm cross section were fabricated in a quartz glass chip as a component in an integrated flow injection analysis (FIA) system. They were put to use for flow, mixing, reaction, and detection. The reaction system was a chelating reaction of divalent iron ion with o-phenanthroline (o-phen), and a photothermal microscope was applied for the ultra-sensitive detection of the non-fluorescent reaction product. Nano liter levels of solutions were introduced and transported by capillary action and mixed by molecular diffusion. Zeptomole levels of the reaction product were detected quantitatively. This was the first demonstration of an on-chip chemical determination device which integrates the primitive FIA system without using electroosmotic liquid control or fluorometric determination.
We have developed a novel thermal lens microscopy coupled with an optical microscope, and presently applied it to the ultratrace molecule detection in a single biological cell (mouse hybridoma) in-vitro. The determination results obtained for individual cells were calibrated by the average values determined by absorption spectrophotometry. The determination limit of the thermal lens microscope, defined as twice the standard deviation of the calibration curve, was 37.8 amol/cell, which was more than one order smaller than that of a fluorescence microscope under our experimental conditions. This superiority should come from escaping light scattering by a cell membrane and by cytoplasm, which is inevitable in fluorescence spectrometry. The absolute determination limit of the thermal lens microscopy was calculated down to sub-attomole level. In addition, the method is more widely applicable, even to non-fluorescent samples without chemical preparation, and therefore, the thermal lens microscope has proved to be very useful in directly quantifying ultratrace chemical species in a single biological cell in-vitro.
Novel bi-functional extractants, which possess two phosphonic groups on both sides of the extractants, have been synthesized for the separation of Zn(II) and Cu(II). The separation properties and extractability of the novel extractants were investigated using the liquid-liquid extraction technique. These bi-functional extractants provide a high selectivity towards Zn(II) over Cu(II) compared to that of monoacidic phosphorus extractant as an analog. We discussed the extraction behavior of the bi-functional extractants with a computational modeling by means of semi-empirical molecular orbital methods. The calculation suggests that the length of the spacer is one of the decisive factors to enhance the selectivity in the bi-functional extractants. The high separation ability for the bi-functional extractants was related to the high energy difference in the heat of formation calculated by computational modeling. The computational simulation was found to be very useful for the design of novel extractants and prediction of their separation performance.
A novel potentiometric titration of iron(II) and iron(III) is proposed. The method is based on the effect of 1,10-phenanthroline (phen) on the redox reactions of chromium(VI) and iron(III) with cobalt(II). In the presence of phen the conditional redox potential of the Co(III)/Co(II) system falls below those of the Cr(VI)/Cr(III) and Fe(III)/Fe(II) systems at pH around 1. Therefore, chromium(VI) and iron(III) can be titrated with cobalt(II) in the presence of phen, alternatively. Firstly, to a sample solution containing iron(II) and iron(III), a known amount of chromium(VI) is added in excess at pH around 1, so that iron(II) can be oxidized to iron(III) in the absence of phen. After the pretreatment, the sample solution containing the excess of chromium(VI) and iron(III) (total iron) is titrated with a standard cobalt(II) solution in the presence of phen. The concentrations of iron(II) and iron(III) can be calculated from the first and second potential breaks at the equivalence points for the titration. The relative standard deviations of this method were 0.66 and 0.81% for four determinations of 5×10-4 mol dm-3 iron(II) and 1×10-3 mol dm-3 iron(III), respectively. The proposed method was suc-cessfully applied to the determination of total iron in standard iron-ore samples (JSS No. 801-4, 814-1 and 850-4) and to the successive determination of iron(II) and iron(III) in a Mosan iron-ore sample (JSS No. 812-3).
A rapid and sensitive spectrophotometric method is described for the determination of ritodrine hydrochloride (RTH) and amoxicillin (AMX) in both pure and dosage forms. The proposed method uses 3-methyl benzothiazolin-2-one hydrazone as a chromogenic reagent. A mixture of aqueous solutions of the drug and reagent is treated with ammonium cerium(IV) sulfate in an acidic medium to yield pink-colored species. These species exhibit maximum absorption at 520 nm for RTH and at 490 nm for AMX with a molar absorptivity of 2.35×104 and 1.27×104 l mol-1 cm-1 for RTH and AMX, respective-ly. The optimum reaction conditions and other analytical parameters are evaluated. The influence of the substrates commonly employed as excipients with these drugs have been studied. The proposed method was applied to the determination of these drugs in pharmaceutical formulations. The results have demonstrated that the method is equally accurate and reproducible as the official methods.
A monosegmented flow system involving sample and titrant sequential injections and the establishment of a tandem plug between two air bubbles is proposed for automated titrations. The end point is found after successive variations of the sample and titrant volumes, determined by an algorithm based on the one-dimensional optimization method of Fibonacci. The set up includes a multi-port solenoid valve with four inlets and one outlet. The feasibility of the system was demonstrated in the spectrophotometric determination of the total acidity in vinegar with NaOH and phenolphthalein used as the titrant and indicator, respectively. The sample throughput was ca. 30 h-1 and consumption of the sample and titrant was about 0.7 ml. The results were in agreement with the expected values, with relative errors lower than ±0.4%. After 12 successive analyses of typical samples with a 47.1 - 53.4 total acidity, expressed as g l-1 acetic acid, the relative standard deviations of the results were estimated within the 1.2 - 2.1% range.
As(III) (12.5 - 200 ng) in drinking water (25 ml) reacted with 2,3-dimercaptopropane-1-sulfonate (DMPS, 0.60 mg) in ammonium acetate buffer (2.0 mmol, pH 5.5) and formed a complex of As(III)-DMPS. The complex was selectively retained on two Sep-Pak C18 cartridges in series, while As(V) could not be retained. Each cartridge was eluted with methanol (2.00 ml). After addition of Ni2+ (2.0 mg), a portion (20 µl) was introduced into a graphite cuvette and was atomized according to a temperature program. As(V) (up to 100 ng) in drinking water (25 ml) was pre-reduced to As(III) with L-cysteine. The concentration of As(V) was determined by subtracting As(III) from the final total concentration of As(III)+As(V). The method detection limits (3σ) for As(III) and As(V) were 0.11 µg/l and 0.15 µg/l, respectively; the calibration graphs were linear up to 8.00 µg/l for As(III). Accuracies of 97.6 - 99.3% were obtained for As(III) and As(V) in drinking water with a precision within 4.0%. The proposed method could be applied to the determination of As(III) vapor in air with a detection limit of 2.8 ng.
This study presents a semi-empirical equation of the electron number density at the analytical zone of the Ar ICP. This equation was developed from data of a three-stage experiment which demonstrated that the electron number density linearly increased with an increase in the r.f. power, exponentially decreased with an increase in the height above the load coil, and decreased when the quantity of the carrier gas and water molecules increased. This behavior of the electron number density at the analytical zone can be explained by the following equation: ne=kpF[1-k5A-k6W]exp(-kdx), where A=A0(1-k7F)exp(-k8x) and W=W0(1-k9F)exp(-k10x).