Matrix Isopotential Synchronous Fluorescence Spectroscopy for Direct Determination of 1-Hydroxypyrene as a Urinary Biomarker of Exposure to Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are present ubiquitously in the environment and exposure to PAHs is a major environmental and occupational health problem. One important approach in assessing human exposure to PAHs is to measure 1-hydroxypyrene excreted in urine. Its present analytical methods require either chromatographic separation^[1] or solvent extracting^[2]. Matrix isopotential synchronous fluorescence spectroscopy (MISFS) is a new fluorimetric technique which consists of performing synchronous scans through a trajectory joining points of equal intensity on a total fluorescence spectrum^[3]. The technique is very useful for resolving highly-spectrally-overlapped mixtures or detecting analytes in complex fluorescent background without the need for tedious prior separation.     

导数恒基体强度同步荧光分析法同时测定光谱严重重叠的1-氯蒽和9-溴蒽

通过选择适合测定路径,应用恒基体强度同步及荧光法同时测定了两种光谱严重重叠的蒽衍生物1－氯蒽和9－溴蒽。混合物中1－氯蒽和9－溴蒽不必预分离就能直接同时测定。在混合物中1－氯蒽和9－溴蒽的回收率分别为83．5％－94．0％,90．0％－94．0％,检出限分别为0．69μg/L,10μg/L。     

同步荧光分析法的应用及其新进展

同步荧光技术是解决多组分荧光物质同时测定的良好手段之一.本文从恒波长同步荧光法、恒能量同步荧光法、可变角同步荧光法、恒基体同步荧光法以及它们与导数技术、低温技术、化学计量学方法的联用等方面对同步荧光技术领域出现的新技术及应用作一评述.     

Direct determination of nalidixic acid in urine by matrix isopotential synchronous fluorescence spectrometry

A new method for the determination of nalidixic acid in urine is proposed for concentrations between 25 and 1000 ng ml(-1) by means of matrix isopotential synchronous fluorescence spectrometry. This new technique is useful for the determination of compounds in samples with unknown background fluorescence, such as nalidixic acid in urine, without the need for tedious preseparation. The method was performed in ethanol/water medium (80% v/v), at an apparent pH of 2.9 provided by adding sodium monochloracetate/monochloroacetic acid buffer solution. The method was successfully applied to the determination of nalidixic acid in urine. Better sensitivity and reproducibility are achieved in these matrices than with the fluorimetric methods described in the literature.     

Resonance light scattering of 1-hydroxypyrene-ethyl violet-anionic surfactant system and its analytical application.

A novel method for the rapid and sensitive analysis of 1-hydroxypyrene (1-OHP) in human urine has been developed that uses a resonance light scattering (RLS) technique. The assay was based on the interaction of ethyl violet (EV) with 1-hydroxypyrene to form an ion-associate complex, which resulted in the enhancement of RLS intensity and the appearance of new RLS spectra. In the presence of anionic surfactant, the maximum RLS peak of the system was located at 396 nm at pH 8.0. Under the optimum conditions, it was found that the enhanced RLS intensity was directly proportional to the concentration of 1-hydroxypyrene in the range of 4.0 - 982 microg l(-1). The detection limit was 1.2 microg l(-1) and the recoveries of 1-hydroxypyrene were 92.8 - 102.3% (n = 6). The proposed method was successfully applied to the analysis of human urine samples. The results of 1-hydroxypyrene were in agreement with those obtained by the method of high-performance liquid chromatography.     

Direct determination of salicylamide in serum by matrix isopotential synchronous fluorimetry

A method for the determination of salicylamide at concentrations between 25 and 350 ng ml(-1) by use of matrix isopotential synchronous fluorescence spectrometry (MISF) in combination with derivative techniques is proposed. The method allows the determination of compounds in samples with unknown background fluorescence without the need for tedious pre-separation. Synchronous scans are performed along a trajectory that connects points of identical intensity in a three-dimensional fluorescence spectrum. The unknown analytical signal of the serum is suppressed from the MISF spectrum, by calculating its first derivative at lambda(exc)=324 nm and lambda(em)=392 nm. In order to ensure maximum sensitivity and adequate selectivity, the experimental variables affecting the fluorescence intensity of the salicylamide band at lambda(exc)=328 nm and lambda(em)=418 nm were studied. Based on the results, the determination was performed in an aqueous medium at pH 12 that was adjusted with a sodium phosphate/hydrogen phosphate buffer. Calibration graphs were subjected to a comprehensive statistical analysis. The error propagation has been considered in order to calculate the detection limit by the criterium of Clayton.     

Direct determination of closely overlapping drug mixtures of diflunisal and salicylic acid in serum by means of derivative matrix isopotential synchronous fluorescence spectrometry

A direct method for the simultaneous fluorimetric determination of two anti-inflammatory drugs in serum is proposed. The combination of matrix isopotential synchronous fluorescence (MISF) and first derivative technique provides good analytical results and permits the simultaneous determination of diflunisal and salicylic acid in human serum. MISF spectra are obtained by calculating the isopotential trajectory in the three-dimensional fluorescence spectrum for a serum solution. In the spectral contour, the trajectory is taken to be the portion of the line that passes by the fluorescence maxima of both compounds ensuring a sensitivity level similar to that of a direct determination in absence of background fluorescence. Analysis was carried out in water using a pH of 7.2 provides by 0.1 M sodium dihydrogen phosphate buffer. Serum samples are diluted 100 times and provide linear calibration plots at diflunisal and salicylic acid concentrations up to 800 ng mL⁻¹. The goodness of the analytical signal was checked by using variance analysis. Signals recorded throughout the calibration range were subjected to three calibrations per each analyte, both in the absence and in the presence of variable amounts of the other analyte. Differences between individual calibrations and slopes were compared with those within individual calibrations. Based on the results, diflunisal and salicylic acid can be accurately quantified in the presence of each other. The limit of detection calculated according to Clayton who uses error propagation throughout the calibration curve and a non-centralized security factor was 36.8 and 37.3 ng mL⁻¹ for diflunisal and salicylic acid, respectively.     

Rapid resolution of five polynuclear aromatic compounds in a mixture by derivative non-linear variable-angle synchronous fluorescence spectrometry

 A combination of non-linear variable-angle synchronous fluorescence spectrometry with the derivative technique has been developed. A rapid simultaneous identification and quantitative determination of acenaphthene, carbazole, anthracene, 9,10-dimethylanthracene and perylene were achieved from a single spectrum by non-linear variable-angle synchronous fluorescence and combined derivative non-linear variable-angle synchronous fluorescence. The latter approach offered a further improvement in spectral resolution and analytical sensitivity. The usefulness of the proposed method was confirmed by adding known amounts of these five polynuclear aromatic compounds to the extract of waste water samples. Received: 5 August 1996/Revised: 11 September 1996/Accepted: 14 September 1996     

Rapid resolution of five polynuclear aromatic compounds in a mixture by derivative non-linear variable-angle synchronous fluorescence spectrometry

 A combination of non-linear variable-angle synchronous fluorescence spectrometry with the derivative technique has been developed. A rapid simultaneous identification and quantitative determination of acenaphthene, carbazole, anthracene, 9,10-dimethylanthracene and perylene were achieved from a single spectrum by non-linear variable-angle synchronous fluorescence and combined derivative non-linear variable-angle synchronous fluorescence. The latter approach offered a further improvement in spectral resolution and analytical sensitivity. The usefulness of the proposed method was confirmed by adding known amounts of these five polynuclear aromatic compounds to the extract of waste water samples. Received: 5 August 1996/Revised: 11 September 1996/Accepted: 14 September 1996     

导数-可变角同步荧光法用于1-萘酚和2-萘酚的同时测定

同步扫描和导数光谱法是两类很有用的荧光分析新技术。固定波长或恒能量同步荧光法和导数技术的结合可进一步提高光谱分辨率和排除基体干扰。本文将导数技术和可变角同步荧光法结合起来,用于1-萘酚和2-萘酚的同时分析,效果良好。研究表明,导数-可变角同步荧光法可望发展为荧光分析复杂样品的新技术。     

Simultaneous determination of norfloxacin and lomefloxacin in milk by first derivative synchronous fluorescence spectrometry using Al (III) as an enhancer

A novel method for the simultaneous determination of norfloxacin (NFLX) and lomefloxacin (LFLX) in milk samples was developed by using first derivative synchronous fluorimetry. The synchronous fluorescence (Δλ=160 nm) spectra and first derivative synchronous fluorescence spectra of NFLX, LFLX and their mixture were studied. The zero-crossing method was utilized to measure the first derivative value of the derivative spectrum. The zero-crossing points were located at 275.0 nm for NFLX and at 283.8 nm for LFLX, in first derivative synchronous fluorescence spectra. Therefore, 283.8 nm and 275.0 nm were selected for the determination of NFLX and LFLX. The first derivative values varied linearly with the concentrations in the range of 1.68×10(-8)-5.64×10(-6) mol L(-1) for NFLX and 1.89×10(-8)-6.19×10(-6) mol L(-1) for LFLX. The detection limits were 5.03×10(-9) mol L(-1) for NFLX and 7.58×10(-9) mol L(-1) for LFLX. The proposed method is reliable, selective and sensitive, and has been used successfully in the simultaneous determination of NFLX and LFLX in milk samples, whose results were in good agreement with those obtained by HPLC.     

恒波长同步荧光光谱法测定人体尿液中氯波必利的研究

研究发现甲醛溶液可使氯波必利的荧光强度显著增强.在pH为6.6的Britton-Robinson(B-R)缓冲介质中,以恒波长差Δλ=30 nm进行同步荧光扫描,可消除人体尿液中内源性荧光物质的背景干扰,其同步特征峰的强度与氯波必利的质量浓度呈线性关系,据此建立了直接测定尿样中氯波必利的恒波长同步荧光分析方法,并对测定...     

Simultaneous determination of two anti-inflammatory drugs in serum using isopotential fluorimetry

The simultaneous determination of 6-methoxy-2-naphthylacetic acid (6MNA) and diflunisal in serum samples using the combination of matrix isopotential synchronous fluorescence (MISF) and first derivative technique is proposed. 6MNA and diflunisal exhibit overlapped spectra and serum produces background fluorescence that precludes the direct determination of these anti-inflammatory drugs by conventional fluorimetry. This method provides good analytical results for determination of compounds in samples with unknown background fluorescence. The method was applied for the simultaneous determination of 6MNA and diflunisal in serum samples at concentrations between 20-200 and 100-1000 ng mL⁻¹, respectively, by means of absolute values of first derivative of synchronous scan at 247.9/364.0 and 262.6/392.4 nm for 6MNA and diflunisal, respectively. In order to obtain maximum sensitivity and adequate selectivity, factors affecting fluorescence intensity were studied. As a result, the analyses were performed in water at a pH of 7.2, adjusted by using sodium dihydrogen phosphate/hydrogen phosphate (0.1 M) as a buffer solution. Serum samples were diluted 200 times. Analytical parameters of the proposed method were calculated according to the error propagation theory. The limit of detection calculated according to Clayton was 15.8 and 63.0 ng mL⁻¹ for 6MNA and diflunisal, respectively. The sensitivity, repeatability and reproducibility achieved with the proposed method were adequate for the determination of these anti-inflammatory agents in serum samples.     

Rapid simultaneous analysis of 1-naphthol and 2-naphthol in water samples by derivative variable-offset synchronous fluorescence spectroscopy

Derivative variable-offset synchronous fluorescence spectroscopy is developed to improve the spectral resolution and the selectivity of fluorescence measurements. 1-naphthol and 2-naphthol are employed to evaluate the proposed coupled technique and the various spectral comparisons are conducted. Second derivative variable-offset synchronous scanning permits the rapid simultaneous identification and quantitative determination of 1-naphthol and 2-naphthol in a mixture from a single spectrum. 6.7-2000 ng/ml 1-naphthol and 3.6-500 ng/ml 2-naphthol can be quantified with 1-naphthol and 2-naphthol ratios of 40: 1-1: 10. The determination of 1-naphthol and 2-naphthol in various spiked water samples gave a mean recovery of 100.7% with a relative standard deviation of 2.8% for 1-naphthol and mean recovery of 99.7% with a relative standard deviation (RSD of 2.6% for 2-naphthol, respectively.     

Determination of 1-hydroxypyrene in human urine by high-performance liquid chromatography with fluorescence detection using a deuterated internal standard

A high-performance liquid chromatographic method has been developed for the quantification of 1-hydroxypyrene (1-OHP) in human urine using deuterated 1-hydroxypyrene ([2H9]1-OHP) as an internal standard with fluorescence detection. [2H9]1-OHP was prepared enzymatically from deuterated pyrene ([2H10]Pyr) with cytochrome P450 1A1. It eluted immediately prior to non-deuterated 1-OHP on alkylamide-type reversed-phase columns and had nearly the same fluorescence characteristics as non-deuterated 1-OHP. The detection limit was 0.1 microg/L and the calibration range was from 1 to 100 nmol/L. Urine sample treatment involved enzymatic hydrolysis followed by solid-phase extraction using Sep-Pak C18 cartridges. The proposed method was used to determine urinary 1-OHP in smokers and non-smokers.     

Determination of nafcillin and methicillin by different spectrofluorimetric techniques

In order to explore the possibilities of combining synchronous fluorescence and derivative spectrometry and to establish a methodology for this type of technique, nafcillin and methicillin were determined using these techniques. Several methods for resolving binary mixtures of these penicillins using first derivative spectrofluorimetry, first derivative constant wavelength synchronous luminescence spectrometry and constant energy synchronous luminescence spectrometry are described. The analyses were performed in aqueous medium at pH 6.20 provided by the addition of phosphate buffer solution. A complete and exhaustive statistical analysis of the experimental data was performed to demonstrate the validity of these methods, which obtained good results when applied for determining nafcillin and methicillin synthetic and real mixtures.     

Direct determination of antibacterial norfloxacin in urine by isopotential fluorimetry

A novel and simple method is presented for the determination of norfloxacin in human urine by matrix isopotential synchronous fluorescence spectrometry (MISF). This method is useful for the determination of compounds in samples with unknown background fluorescence, such as norfloxacin in urine, without the need for tedious sample pre-treatment. The method was performed in ethanol-water medium (10% v/v), at an apparent pH of 4.8 provided by adding sodium acetate-acetic acid buffer solution. In the determination of norfloxacin in urine the fluorescent intensity varied linearly with its concentration from 20 to 200 ng/mL. An exhaustive statistical analysis has been developed to all calibration graphs, this treatment includes robust regression such as least median of squares, which also detects outliers and leverage points. The overall least-squares regression has been applied to find the more exact straight line that fits the experimental data. The error propagation has been considered to calculate the detection limit and the repeatability of the method. The method shows very low detection limits with acceptable recoveries and precisions. The applicability of the proposed method was illustrated in the determination of norfloxacin in human urine sample without sample pre-treatment.     

Direct Determination of Salicylic Acid in Human Serum by Matrix Isopotential Synchronous Fluorescence

A new spectrofluorimetric method has been applied for determining salicylic acid in human serum for concentrations between 0.020 and 0.252 ppm. The method was performed in an aqueous medium at pH 6.0 attained by addition of phosphate buffer solution. The method is based on the use of a new technique called matrix isopotential synchronous fluorescence, which enables us to determine analytes in complex fluorescent matrices without the need for tedious prior separation. The sensitivity and reproducibility achieved in these matrices are better than those achieved with the fluorimetric methods described in the references.     

二阶导数同步荧光光谱法同时直接测定厚朴酚及和厚朴酚

研究了厚朴酚与和厚朴酚及其混合溶液的二阶导数同步荧光光谱,结果两者的二阶导数同步荧光光谱得到完全分离,消除了彼此间的干扰,据此建立了一种二阶导数同步荧光光谱法同时直接测定混合物中厚朴酚与和厚朴酚的新方法.厚朴酚与和厚朴酚的线性范围分别为2.8～500.0 μg/L和4.3～560.0 μg/L;检出限分别为0.84和1.30 μg/L,回收率分别为94.65%～105.58%和95.09%～104.51%; 相对标准偏差均低于4.08%.本方法用于同时直接测定厚朴药材及其提取物中厚朴酚与和厚朴酚含量,结果令人满意.     

Enhanced synchronous spectrofluorimetric determination of aflatoxin B1 in pistachio samples using multivariate analysis

The determination of aflatoxin B₁ (AFB1) in pistachio has been accomplished by normal and synchronous fluorimetry in combination with some multivariate calibration methods and derivative techniques. Extending the two-dimensional synchronous fluorescence scan to a three-dimensional total synchronous fluorescence scan was used to obtain the optimized Δλ for AFB1 in pistachio sample. The methods are based on the enhanced fluorescence of AFB1 by β-cyclodextrine in 10% (v/v) methanol-water solution. Twenty-six pistachio samples containing AFB1 in the range 0-15 ppb were used as calibration set. Eighteen combinational methods were tested to make best model for prediction of AFB1 and finally best results obtained using a method based on synchronous fluorimetry in combination with multiple linear regressions (MLR). For concentrations ranging from 0 to 15 ppb of AFB1 in 22 pistachio samples as prediction set, the values of root mean square difference (RMSD) and relative error of prediction (REP) using MLR were 0.328 and 4.453%, respectively were observed. Two naturally contaminated pistachio samples were analyzed by synchronous fluorimetry using MLR and compared with HPLC results.