Matrix Isopotential Synchronous Fluorescence Research Articles

Derivative Matrix-Isopotential Synchronous Spectrofluorimetry and Hantzsch Reaction: A Direct Route to Simultaneous Determination of Urinary δ-Aminolevulinic Acid and Porphobilinogen.

Early and sensitive detection of δ-aminolevulinic acid (δ-ALA) and porphobilinogen (PBG) is the cornerstone of diagnosis and effective treatment for acute porphyria. However, at present, the quantifying strategies demand multiple solvent extraction steps or chromatographic approaches to separate δ-ALA and PBG prior to quantification. These methods are both time-consuming and laborious. Otherwise, in conventional spectrofluorimetry, the overlapping spectra of the two analytes cause false diagnosis. To overcome this challenge, we present a two-step approach based on derivative matrix-isopotential synchronous fluorescence spectrometry (DMISFS) and the Hantzsch reaction, realizing the simple and simultaneous detection of δ-ALA and PBG in urine samples. The first step is chemical derivatization of the analytes by Hantzsch reaction. The second step is the determination of the target analytes by combining MISFS and the first derivative technique. The proposed approach accomplishes following advantages: 1) The MISFS technique improves the spectral resolution and resolves severe spectral overlap of the analytes, alleviating tedious and complicated pre-separation processes; 2) First derivative technique removes the background interference of δ-ALA on PBG and vice versa, ensuring high sensitivity; 3) Both the analytes can be determined simultaneously via single scanning, enabling rapid detection. The obtained detection limits for δ-ALA and PBG were 0.04 μmol L−1 and 0.3 μmol L−1, respectively. Within-run precisions (intra and inter-day CVs) for both the analytes were <5%. Further, this study would serve to enhance the availability of early and reliable quantitative diagnosis for acute porphyria in both scientific and clinical laboratories.

Derivative matrix-isopotential synchronous spectrofluorimetry: a solution for the direct determination of urinary δ-aminolevulinic acid

The excitation and emission spectra formulated 3D contours, from which isopotential trajectory was selected for the direct detection of urinary δ-aminolevulinic acid, using derivative matrix isopotential synchronous fluorescence spectrometry.

A portable multi-syringe flow system for spectrofluorimetric determination of iodide in seawater

A miniaturized analyzer encompassing a poly(methyl methacrylate) chip with integrated spectrofluorimetric detection and solutions propelling by a multi-syringe module is proposed. Iodide was determined through its catalytic effect on the reaction between Ce(IV) and As(III). Matrix isopotential synchronous fluorescence was explored to set the excitation and emission wavelengths. A two-level full factorial design allowed to evaluate the significance of variables (Ce(IV), As(III) and H2SO4 concentrations) and their interaction effects in the experimental domain. A Doehlert Matrix was applied to identify the critical values. The optimized procedure showed a linear response from 1 to 100μgL−1 (S=53.7+2.61C, in which S is the net fluorescence and C is iodide concentration in μgL−1). Detection limit, coefficient of variation (n=6) and sampling rate were estimated at 0.3 μgL−1, 0.8% and 20h−1, respectively. Recoveries within 90–117% were estimated for iodide spiked to seawater samples. The proposed procedure stands out because of the portability, robustness, and simplicity for in-field analysis of iodide in seawater.

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.

Matrix isopotential synchronous fluorescence spectrometry for the determination of gibberellic acid in watermelon after ultraviolet-irradiation

The determination of gibberellic acid in watermelon was performed by means of first derivative matrix isopotential synchronous fluorescence spectrometry in combination with photochemically induced fluorescence, without the need for tedious pre-separation. Its photochemical reactivity by effect of UV irradiation boosting the fluorescence is in agreement with the occurrence of photoaromatization and photochemical dimerization processes, with loss of carbon dioxide, and the probable formation of several fluorescent photoproducts. Watermelon extract presents a high native fluorescence, which drastically diminishes under UV irradiation treatment. The analyses were performed in 50% (v/v) methanol and pH 5 buffer solution, using 6 min as UV irradiation time in a flow photoreactor. The concentration of gibberellic acid in watermelon samples can be calculated by recording its total luminescence spectrum and applying the isopotential trajectory of the watermelon matrix that cuts the selected band of gibberellic acid. The unknown analytical signal of watermelon is eliminated in the matrix isopotential synchronous fluorescence spectrum obtained, by means of its first derivative. The calibration graph was linear between 40 and 200 ng mL−1 of gibberellic acid with a detection limit of 0.14 ng mL−1 and the obtained value of repeatability of the method was a standard deviation of 1.9%.

Development of a Spectrofluorimetric Method for the Determination of Ofloxacin in Urine

A simple, accurate, rapid, and sensitive spectrofluorimetric method for the determination of ofloxacin in urine was developed by means of first derivative matrix isopotential synchronous fluorescence spectrometry (MISF). The calibration curve was found to be linear in the concentration range 40-320 ng/mL. The method allows the determination of compound 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 urine is suppressed from the MISF spectrum by calculating its first derivative at λex = 319.2 and λem = 465 nm. In order to ensure maximum sensitivity and adequate selectivity, the experimental variables affecting fluorescence intensity were studied in the ofloxacin band centered at λex = 333 and λem = 460 nm. As result, the determination was performed in a water medium at pH 7.2, adjusted by using sodium dihydrogen phosphate as a buffer solution. Calibration graphs were subjected to a comprehensive statistical analysis. The detection limit according to Long and Winefordner was 8.4 ng/mL, and the detection limit proposed by Clayton was 13.9 ng/mL.

Rapid determination of ciprofloxacin in urine by matrix isopotential synchronous spectrometry

A simple, rapid and sensitive synchronous fluorescence method is proposed for the determination of ciprofloxacin in urine. The combination of matrix isopotential synchronous fluorescence (MISF) and first derivative techniques provides good analytical results. MISF spectra are obtained by calculating an isopotential trajectory in the three-dimensional fluorescence spectrum of urine solutions. This trajectory is chosen so that it passes by the fluorescence maxima of ciprofloxacin (λex = 277 nm and λem = 444 nm). Analyses were carried out in an ethanol–water medium (10% v/v), with an apparent pH of 4.8 kept constant by using a sodium acetate/acetic acid buffer solution. The linear range of concentration was between 40 and 400 ng mL−1. Exhaustive statistical analyses were made of all calibration graphs. The detection limit according to the error propagation theory was 5.6 ng mL−1 and the detection limit proposed by Clayton was 13.5 ng mL−1. The repeatability of the method was determined for two series of ten solutions containing 40 and 180 ng mL−1 of ciprofloxacin in urine, respectively. By applying the IUPAC definition, the relative errors were 9.5% and 2.0%, while, based on error propagation, the relative errors were 6.3% and 1.2%, respectively.

A novel rapid method for simultaneous determination of three diagnostically important porphyrins in erythrocytes using hyphenated synchronous fluorescence techniques

The species and concentrations of porphyrins in erythrocytes are of great importance in the clinical screening and diagnosis of porphyrias. However, it is difficult to analyze them simultaneously by conventional spectrofluorimetry. In this paper, we proposed a novel, simple and rapid method for the simultaneous determination of three diagnostically important porphyrins in human erythrocytes, protoporphyrin IX (PP), coproporphyrin III (CP) and zinc protoporphyrin IX (ZnPP), using hyphenated techniques based on derivative matrix isopotential synchronous fluorescence spectrometry and nonlinear variable-angle synchronous fluorescence spectrometry (DMI-NLVASFS). The spectral overlapping problems were well resolved and these three components were determined in one scanning without spectral compensation factors and chromatographic separation. The detection limits were 0.58, 0.21, 0.05nmolL−1 for PP, CP and ZnPP, respectively. Only 30s was needed for a single scanning and the recoveries were from 73% to 105% in erythrocytes. The Bland–Altman analysis indicates no significant difference between the proposed DMI-NLVASFS method and conventional fluorimetry method. The PP level of the erythropoietic protoporphyria (EPP) patients was significantly higher than that of healthy volunteers. This method can determine PP, CP and ZnPP simultaneously in a single scanning, thus providing a potential tool for the clinical analysis of porphyrins in human erythrocytes and the differential diagnosis of porphyrias.

A novel, selective, and rapid fluorimetric method for the simultaneous analysis of coproporphyrin and uroporphyrin in urine

A simple and rapid spectrofluorimetric method is described for the determination of the closely overlapping mixture of coproporphyrin (CP) and uroporphyrin (UP) in urine samples. Matrix Isopotential Synchronous Fluorescence Spectrometry (MISFS) was applied to improve the spectral resolution for the severely overlapped spectra of the urinary porphyrins. First-order derivative technique eliminates the background interference of each component on the other. Using these two techniques together, selectivity was improved, while maintaining a high sensitivity, and time-consuming separation processes and multiple scanning processes were avoided. The limits of detection were 0.15 nmol L −1 and 0.1 nmol L −1 for CP and UP, respectively. The concentrations of CP and UP were determined from the peak amplitudes of the Derivative Matrix Isopotential Synchronous Fluorescence (DMISF) spectra, at their detection points where the interference was suppressed. Porphyrins excretion in urine samples, collected from normal subjects, was studied. A comparison between the new method and the anion-exchange chromatographic method of Martinez and Mills was established using Bland–Altman method and the results indicate that these two methods are in a good agreement with each other.

Determination of Ciprofloxacin, the Major Metabolite of Enrofloxacin, in Milk by Isopotential Fluorimetry

A new method for the determination of ciprofloxacin, the major metabolite of enrofloxacin, for concentrations between 20 and 200 ng/mL by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This new method is useful for the determination of compounds in samples with unknown background fluorescence, such as ciprofloxacin in whey, without the need of tedious preseparation. The determination was performed in an ethanol/water medium (20% v/v) at pH 4.8, provided by adding a sodium acetate/acetic acid buffer solution. Since enrofloxacin is widely used as an antibacterial agent in veterinary medicine, the method was successfully applied to the determination of its main metabolite in milk. 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.

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.

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 and simultaneous determination of coproporphyrin and protoporphyrin in feces by derivative matrix isopotential synchronous fluorescence spectrometry.

Measurement of fecal porphyrins is important in the diagnosis of porphyria, but conventional methods to measure them have drawbacks. We explored the use of derivative matrix isopotential synchronous fluorescence (MISF) spectrometry for the measurement of coproporphyrin and protoporphyrin. The MISF scanning route was selected based on information from the three-dimensional fluorescence spectrum, which was a combination of the contour line of protoporphyrin via a detection point of coproporphyrin and that of coproporphyrin via a detection point of protoporphyrin. Derivative technique eliminated the constant interfering signals. MISF was used to measure porphyrins in stools from 2 pregnant women and 20 healthy volunteers. The coproporphyrin and protoporphyrin spectra were resolved with almost no mutual interference. The amplitudes of the derivative peaks were linearly related to the concentrations of coproporphyrin up to 310 nmol/L and protoporphyrin up to 590 nmol/L. The detection limits for coproporphyrin and protoporphyrin were 1.2 and 1.7 nmol/L, respectively. The within-run imprecision (CV; n = 6) was 2.2% at 175 nmol/L for coproporphyrin and 2.3% at 500 nmol/L for protoporphyrin. Bland-Altman analysis indicated no significant differences between the proposed MISF method and conventional spectrophotometry or fluorimetry. Mean (SD) recoveries of porphyrins added to fecal samples were of 98 (7)% for coproporphyrin and 102 (4)% for protoporphyrin. This technique provides spectral resolution of coproporphyrin and protoporphyrin, obviating the need for chromatographic separation, and measurements can be made in a single scanning. The method also appears suitable for routine testing of large numbers of samples.

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 λ exc=324 nm and λ em=392 nm. In order to ensure maximum sensitivity and adequate selectivity, the experimental variables affecting the fluorescence intensity of the salicylamide band at λ exc=328 nm and λ 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.

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

Urinary 1-hydroxypyrene is a biomarker in the measurement of human exposure to polycyclic aromatic hydrocarbons. A rapid and simple derivative isopotential synchronous fluorescence method was developed for the direct determination of 1-hydroxypyrene in urine. A length of iso-intensity route was scanned on the three-dimensional fluorescence spectrum of urine and this result was combined with that from derivative technique. Thus the strong background signals of urine were removed and the 1-hydroxypyrene can be determined directly in urine without tedious pre-separation. The derivative isopotential synchronous fluorescence spectrum was directly obtained from a single scan on a spectrofluorometer, which further simplified isopotential synchronous fluorescence technique. The recoveries of 93% to 115% were obtained for 1-hydroxypyrene added to urine.

Direct Determination of Dipyridamole in Serum

A new method for the determination of dipyridamole in serum for concentrations between 10 and 100 ng ml−1by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This new method is useful for the determination of compounds in samples with unknown background fluorescence, such as dipyridamole in serum, without the need for tedious preseparation. The determination was performed in an ethanol/water medium (15% v/v) at a pH value of 10.0, provided by adding an ammonium chloride/ammonia buffer solution. Since dipyridamole is a stimulant which is very much used like a doping substance in sport, the method was successfully applied to the determination of dipyridamole in serum. Better sensitivity and repeatability are achieved for these matrices than with the fluorometric ones described in the literature. An exhaustive statistical analysis has been developed for 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 which fits the experimental data. The error propagation has been considered to calculate the detection limit, determination limit, and the repeatability of the method.

Direct determination of amiloride in urine using isopotential fluorimetry.

A method for the determination of amiloride at concentrations between 15 and 152 ng ml-1 by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This method is useful for the determination of compounds in samples with unknown background fluorescence without the need for tedious pre-separation. As amiloride is widely used as a doping substance in sport, the method was successfully applied to the determination of amiloride in urine. To obtain maximum sensitivity and adequate selectivity, factors affecting fluorescence intensity were studied in the amiloride band centered and lambda ex = 362 nm and lambda em = 415 nm. As a result, the determination was performed in an ethanol-water (1 + 1, v/v) medium at pH 6.3, adjusted by using sodium citrate-citric acid (0.1 M) as buffer solution. The concentration of amiloride in urine samples can be calculated by recording its total luminescence spectrum and applying the isopotential trajectory of the urine that cuts the selected band of amiloride. The unknown analytical signal of urine is eliminated in the MISF spectrum obtained, be means of its first derivative. A calibration graph was constructed by measuring first derivative values at lambda ex = 357nm and lambda em = 392 nm. Analytical parameters of the proposed method were calculated according to the error propagation theory. The sensitivity, repeatability, reproducibility and limit of determination achieved with the proposed method are adequate for the determination of amiloride in urine.

Determination of pyridoxal in human serum by matrix isopotential synchronous fluorescence spectrometry

The determination of pyridoxal in human serum was performed using a fluorimetric technique that is useful for the determination of compounds in samples with unknown background fluorescence, based on synchronous scans through a trajectory joining points of equal intensity of a fluorescence matrix three-dimensional spectrum. This technique, called matrix isopotential synchronous fluorescence can be improved by means of the application of derivatives. The determination of pyridoxal in human serum was performed with this technique without any prior separation steps. The validity, applicability and simplicity of the method were demonstrated. The measurements were performed in aqueous medium at pH 7.0 adjusted by adding 0.05 M phosphate buffer solution. A complete statisitical analysis of the experimental data was performed and the results showed that correlation coefficients are between 0.9901 and 0.9958 for all the calibration graphs, and in all cases the intercepts on the ordinate were negligible. The exprimental {ce:inline-formula}F{/ce:inline-formula} (1.72) is smaller than theoretical value (3.89) as expected from an analysis of variance. The reproducibility of the method was tested and the results were a standard deviation of 0.0117 μg ml −1 and a relative error of 4.91%.

Direct determination of triamterene in urine by matrix isopotential synchronous fluorescence spectrometry

A new method for the determination of triamterene for concentrations between 10 and 1000ng ml −1 by means of matrix isopotential synchronous fluorescence spectrometry and derivative techniques is proposed. This new method is useful for the determination of compounds in samples with unknown background fluorescence, such as triamterene in urine, without the need of tedious pre-separation. The determination was performed in an aqueous medium adjusted at a pH value of 6.2, provided by adding sodium citrate-citric acid buffer solution. Since triamterene is widely used as a doping substance in sport, the method was successfully applied to the determination of triamterene in urine. Better sensitivity and repeatability are achieved for these matrices with the proposed method than with the fluorimetric ones described in the literature. An extensive 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 weighted least squares regression has been applied to find the more exact straight line which fits the experimental data. The error propagation has been used for the calculation of the detection limit, determination limit and the repeatability of the method.

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.