Simultaneous determination of urinary tryptophan, tryptophan-related metabolites and creatinine by high performance liquid chromatography with ultraviolet and fluorimetric detection

A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze-thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r)>0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0-106.4%; Kyn, 97.9-106.9%; Kyna, 98.5-105.6%; Trp, 96.7-105.2% and 5-HIAA, 96.1-99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.     

荧光分光光度法测定谷物蛋白质中的酪氨酸

本文探讨了谷物中酪氨酸的荧光测定法。将30毫克风干样置玻璃水解管中,以6Mol盐酸作水解剂,110℃真空水解22小时,水解产物减压脱酸,以去离子双蒸水溶解,在激发波长277nm,发射波长303nm处测其荧光强度。方法精确度良好,重复测定12次变异系数为0.98％。在5ng～2μg/ml标准酪氨酸范围内,回收率为96—105％。用此法测得谷物中酪氨酸与氨基酸分析仪测得值相符。     

Dose response, wavelength dependence and rate of excision of ultraviolet radiation-induced pyrimidine dimers in mouse skin DNA

The yield of thymine-containing dimers produced in mouse skin DNA in vivo by 290 nm ultraviolet radiation was shown to increase with dose up to around 2000 J/m2 and subsequently at a much slower rate up to 8000 J/m2. The study of wavelength dependence of dimer formation in skin indicated that 290 nm was the most effective wavelength of those investigated, followed by 300, 280 and 260 nm, with 310 nm being by far the least effective. A reduction in the number of dimers present in skin DNA was shown to occur by 24 h post-irradiation in a dose-dependent manner. A significant percentage of the dimers was, however, found to persist in the skin until at least 72 h post-irradiation.     

Determination of hexafluoroisopropanol,a sevoflurane urinary metabolite,by 9-fluorenylmethyl chloroformate derivatization

A reversed-phase HPLC method with fluorescence detection for the quantification of hexafluoroisopropanol (HFIP) in urine is presented. HFIP, a metabolite of the inhalation anesthetic sevoflurane, is excreted mainly in urine as glucuronic acid conjugate. After enzymatic hydrolysis of the glucuronate, primary amino groups of interferent urinary compounds are blocked by reaction with o-phthalic dicarboxaldehyde and 3-mercaptopropionic acid, followed by labeling of HFIP with 9-fluorenylmethyl chloroformate. The derivatization reaction proceeds in a water-acetonitrile (1:1) solution at room temperature with a borate buffer of pH 12.5 as a catalyst. A stable fluorescent derivative of HFIP is formed within 5 min. The HFIP-FMOC derivative is separated by reversed-phase chromatography with isocratic elution on an octadecyl silyl column (33x4.6 mm, 3 microm) and guard column (20x4.0 mm, 40 microm), at 35 degrees C, and detected by fluorescence detection at an excitation wavelength of 265 nm and an emission wavelength of 311 nm. The method detection limit is 40 pg, per 10-microl injection volume, corresponding to 16 microg/l of HFIP in urine. The among-series relative standard deviation is <6% at 200 microg/l (n=6). As a preliminary application, the method was used to detect HFIP concentration in the urine of two volunteers exposed for 3 h to an airborne concentration of sevoflurane in the order of 2 ppm.     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.     

Synthesis and properties of a fluorescent derivative of phosphatidylcholine

1-acyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl phosphatidyl choline, (NBD-PC) was prepared by alkylation of ?-amino caproic acid with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1), followed by esterification of lysophosphatidylcholine. The compound was purified by silicic acid chromatography, and exhibited a single spot on thin layer chromatography using acidic, basic and neutral solvent systems, when visualized by UV, molybdate spray, primuline, or charring. The UV-visible absorption spectrum, and the uncorrected fluorescence excitation spectrum of NBD-PC in absolute ethanol showed maxima at approximately 340 and 460 nm, while the fluorescence emission spectrum showed a single peak at 525 nm. Fluorescence intensity and emission maximum wavelength of NBD-PC are strongly dependent on solvent dielectric constant, and the relative fluorescent intensity of NBD-PC in absolute ethanol is directly proportional to its concentration from 1 ng/ml to approximately 3 μg/ml. Incorporation of NBD-PC into membranes of human lymphocytes cultures in the presence or absence of phytohemagglutinin (PHA) resulted in a marked increase in the relative fluorescent intensity of the bound fluorochrome, and a 15 nm blue shift in its emission maximum wavelength. Fluorescence titration data indicate that the unstimulated lymphocytes bound 912 pmoles NBD-PC/mg protein with an association constant of 3.45 × 10⁷ M, while the PHA stimulated cells bound 1200 pmoles NBD-PC/mg protein with an association constant of 2.82 × 10⁷ M. The temperature dependence of the fluorescent intensity of NBD-PC incorporated in control, and PHA stimulated lymphocytes showed discontinuities at 15 and 24 °C respectively. Fluorescence polarization of NBD-PC incorporated in the membranes of stimulated lymphocytes was greater than the polarization of the fluorochrome in non-stimulated cells, suggesting that the plasma membranes of PHA stimulated lymphocytes contain regions of higher microviscosity.     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

基于微孔板的高通量筛选6-羟基烟酸转化菌方法的建立

建立了一种基于96孔板-酶标仪的双波长紫外分光光度法高通量筛选6-羟基烟酸转化菌的方法.实验以251nm为测定波长、231nm为参比波长测定转化样品的6-羟基烟酸含量,6-羟基烟酸与△A251-231在0.5～11 μg/mL浓度范围内有良好的线性关系,服从朗伯-比尔定律,平均回收率为99.11%～100.81%.利用96孔板-酶标仪,每天筛选量可达到2000～5000个反应,达到高通量筛选要求.     

Gold Nanoparticle Interference Study during the Isolation,Quantification, Purity and Integrity Analysis of RNA

Investigations have been conducted regarding the interference of nanoparticles (NPs) with different toxicological assay systems, but there is a lack of validation when conducting routine tests for nucleic acid isolation, quantification, integrity, and purity analyses. The interference of citrate-capped gold nanoparticles (AuNPs) was investigated herein. The AuNPs were added to either BEAS-2B bronchial human cells for 24 h, the isolated pure RNA, or added during the isolation procedure, and the resultant interaction was assessed. Total RNA that was isolated from untreated BEAS-2B cells was spiked with various concentrations (v/v%) of AuNPs and quantified. A decrease in the absorbance spectrum (220–340 nm) was observed in a concentration-dependent manner. The 260 and 280 nm absorbance ratios that traditionally infer RNA purity were also altered. Electrophoresis was performed to determine RNA integrity, but could not differentiate between AuNP-exposed samples. However, the spiked post-isolation samples did produce differences in spectra (190–220 nm), where shifts were observed at a shorter wavelength. These shifts could be due to alterations to chromophores found in nucleic acids. The co-isolation samples, spiked with 100 µL AuNP during the isolation procedure, displayed a peak shift to a longer wavelength and were similar to the results obtained from a 24 h AuNP treatment, under non-cytotoxic test conditions. Moreover, hyperspectral imaging using CytoViva dark field microscopy did not detect AuNP spectral signatures in the RNA isolated from treated cells. However, despite the lack of AuNPs in the final RNA product, structural changes in RNA could still be observed between 190–220 nm. Consequently, full spectral analyses should replace the traditional ratios based on readings at 230, 260, and 280 nm. These are critical points of analyses, validation, and optimization for RNA-based techniques used to assess AuNPs effects.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Quantifying crystal form content in physical mixtures of (±)-tartaric acid and (+)-tartaric acid using near infrared reflectance spectroscopy

The objective of this study was to use diffuse reflectance near infrared spectroscopy (NIRS) to determine racemic compound content in physical mixtures composed primarily of the enantiomorph and to assess the error, instrument reproducibility and limits of detection (LOD) and quantification (LOQ) of the method. Physical mixtures ranging from 0 to 25% (±)-tartaric acid in (+)-tartaric acid were prepared and spectra of the powder samples contained in glass vials were obtained using a Foss NIRSystems Model 5000 monochrometer equipped with a Rapid Content Analyzer scanning from 1100 to 2500 nm. A calibration curve was constructed by plotting (±)-tartaric acid weight percent against the 2^nd derivative values of log (1/R) vs λ at a single wavelength, normalized with a denominator wavelength (1480 nm/1280 nm). Excellent linearity was observed (R²=0.9999). The standard error of calibration (SEC) was 0.07 and the standard error of prediction (SEP) for the validation set was 0.11. Instrument and method errors for samples in the 2% composition range ((±)-tartaric acid in (+)-tartaric acid) were less than 1% RSD and 3% RSD, respectively. The practical LOD and LOQ were 0.1% and 0.5%, respectively, and comparable to the calculated LOD and LOQ. These studies show that NIRS can be used as a rapid and sensitive quantitative method for determining racemate content in the presence of the enatiomerically pure crystal in the solid-state. Published: October 6, 2005     

Effects of UV-irradiation at Various Wavelengths on Sterilizing Drosophila Embryos

Embryos of Drosophila melanogaster at the early intravitelline nuclear multiplication stage were irradiated with UV light at the posterior pole. The sterility and mortality of these embryos were examined in relation to the dose and wavelength of the UV light.
Sterility, expressed either as the frequency of pole-cell-deficient embryos, or as the frequency of agametic adults, was found to be dependent on the wavelength of UV light. UV-irradiation at 280 nm was found moot effective in causing sterility on Drosophila embryos. The minimum dose of radiation to give a 100% sterility was 200 J/m² at 280 nm, and 400 J/m² at 254 nm. In contrast, mortality showed no dependency on the wavelength.
The possibility that nucleic acids in the posterior region is a target of 280 nm radiation is discussed.     

Effect of light source on the microbiological desulfurization in a photobioreactor

Removal rates of a toxic pollutant-hydrogen sulfide were investigated using several light sources in photosynthetic desulfurization. An incandescent bulb has a broad spectrum starting from about 400?nm but emits most of its light energy beyond 800?nm as heat. LED₇₁₀ among those sources saved energy considerably comparing with the incandescent light, but the scattering and absorption was a problem in light transmission within the deep region of a bioreactor due to its own weak light intensity. Fluorescent light was inefficient in desulfurization in comparison with light sources illuminating with wide wavelength range, because of the weak transmittance at the peak wavelength of 460?nm. A combination of LED₇₁₀ and fluorescent lamp was estimated as an optimal light source in this study.     

Simple and sensitive determination of five quinolones in food by liquid chromatography with fluorescence detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method has been developed for the determination of five different quinolones: enrofloxacin, ciprofloxacin, sarafloxacin, oxolinic acid and flumequine in pork and salmon muscle. The method includes one extraction and clean-up step for the five quinolones together which are detected in two separated HPLC runs by means of their fluorescence. The proposed analytical method involves homogenizing of the tissue sample with 0.05 M phosphate buffer, pH 7.4 and clean-up by Discovery DS-18 cartridges. For chromatographic separation a Symmetry C(18) column is used in two different runs: (1) ciprofloxacin, enrofloxacin and sarafloxacin with acetonitrile-0.02 M phosphate buffer pH 3.0 (18:82) as mobile phase and the detector at excitation wavelength: 280 nm and emission wavelength 450 nm; and (2) oxolinic acid and flumequine with acetonitrile-0.02 M phosphate buffer pH 3.0 (34:66) as mobile phase and excitation wavelength: 312 nm and emission wavelength: 366 nm. Detection limit was as low as 5 ng g(-1), except for sarafloxacin which had a limit of 10 ng g(-1). Standard curves using blank muscle tissues spiked at different levels showed a good linear correlation coefficient, r(2) higher than 0.999 for all quinolones.     

The effect of low power laser radiation of blue,green, and red ranges on free radical processes in blood of rats with experimental endotoxic shock

This study was performed to investigate the effects of low power laser radiation in blue (441.2 nm), green (532.5 nm) and red (632.8 nm) wavelength ranges on free radical processes in experimental endotoxic shock in rats. The experimental model was induced by intraperitoneal injection of lipopolysaccharide B (25 mg/kg) (LPS). Functional activity of blood leukocytes was evaluated by the method of luminol-dependent chemiluminescence, plasma superoxide dismutase activity was determined by the nitro blue tetrazolium assay, intensity of lipid peroxidation in erythrocyte membranes was estimated by cis-parinaric acid fluorescence. It was found that the low power laser radiation significantly influenced all investigated processes, in LPS-treated and control animals. The most pronounced effects were observed in all groups of LPS-treated animals, in which the laser radiation increased all investigated parameters. At the radiation dose 0.75 J/cm² green laser was the most effective, while at the dose of 1.5 J/cm² both green and red lasers produced potent effects. Possible mechanisms of the observed phenomena are discussed.     

肾茶熊果酸含量测定的研究

本试验研究用薄层扫描法测定肾茶中熊果酸含量。样品经适当提取后点于硅胶GF254薄层板上,以环已烷-氯仿-乙酸乙酯-甲醇(25:15:5:2)为展开剂,置于紫外光灯(波长254nm)下定位;双波长反射锯齿扫描,λ_S=520nm,λ_R=650nm。结果表明,经方法学考察,点样量在0.5~2.5μg范围内呈现良好的线性关系,其回归方程为y=3658.34X-7857.07,r=0.9998;平均回收率为101.57%,RSD=2.83%。     

Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in saliva

A high performance liquid chromatographic method for determination of moxifloxacin in human saliva was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C18 column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.25 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from saliva was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

The photodynamic activity of 131-[2′-(2-pyridyl)ethylamine] chlorin e6 photosensitizer in human esophageal cancer

A novel 13¹-pyridine substituted chlorin e₆ derivative (Chlorin A) was synthesized. It has characteristic long wavelength absorption at 664?nm and the emission wavelength at 667?nm. The generation rate of singlet oxygen of this compound is higher than Temoporfin. In vitro, Chlorin A showed higher phototoxicity against the human esophageal cancer cells than Temoporfin while with lower dark-toxicity. Its accumulation effect in mitochondria, lysosomes and endoplasmic reticulum was traced in subcellular localization tests. In flow cytometry obvious apoptosis cells were observed after 2?h irradiation. Significant in vivo photodynamic anti-tumor efficacy was also exhibited on mice bearing esophageal cancer. So Chlorin A could be suggested as a promising anti-tumor drug candidate in photodynamic therapy.     

Simple and rapid high-performance liquid chromatography method for the determination of ofloxacin concentrations in plasma and urine

A high-performance liquid chromatographic method for the determination of ofloxacin in human plasma and urine was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was linear from 0.5 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 5%. The average recovery of ofloxacin from plasma was 93%. The method was evaluated in samples from healthy subjects whose drug levels were already measured by microbiological assay.