Pinelopi C. Ioannou

Application of terbium sensitized fluorescence for the determination of fluoroquinolone antibiotics pefloxacin, ciprofloxacin and norfloxacin in serum

A simple, rapid and sensitive spectrofluorimetric method for the determination of fluoroquinolone antibiotics, norfloxacin (NOR), ciprofloxacin (CIP) and pefloxacin (PEF) is described. The method is based on the radiative energy transfer from fluoroquinolones to terbium ions (Tb3+) in the presence of tri-n-octylphosphine oxide (TOPO) in weakly acidic (pH 5.5) micellar solution of cetylpyridinium chloride (CPCI). Optimum conditions for the formation of the fluoroquinolone-Tb(3+)-TOPO ternary complexes have been investigated. Under optimized conditions the detection limits are 1.7, 1.2 and 4.4 nM for NOR, CIP and PEF, respectively, while the range of application for all three drugs is 0.05-10 microM. The method has been successfully applied to the determination of NOR, CIP and PEF in serum samples after deproteinization with acetonitrile (serum-acetonitrile; 1:2, v/v). The mean recoveries from serum samples spiked with NOR, CIP and PEF (5.0-50.0 microM) were (90.3 +/- 4.9), (105.0 +/- 3.6), and (95.3 +/- 1.5)% respectively. Within-run and day-to-day s, values for 5.0, 25.0 and 50.0 microM of each fluoroquinolone varied from 1.7 to 5.4% and from 3.3 to 12.8%, respectively. The influence of several usually coadministered drugs on the determination of fluoroquinolones in serum has been investigated.

Comparative study of fluorescent ternary terbium complexes. Application in enzyme amplified fluorimetric immunoassay for α-fetoprotein

Abstract A systematic spectrofluorimetric study of the formation of ternary complexes of terbium ions (Tb3+) with ethylenediamine tetraacetic acid (EDTA) and a variety of bidentate organic ligands has been performed. Fourteen ligands (salicylic acid, quinolones and hydroxybenzene sulfonic acid derivatives) were examined as efficient energy donating chelators for Tb3+. The fluorescence properties of the ligands, alone as well as of the ternary complexes with Tb3+ and EDTA have been examined. Optimization studies, including the effect of pH, buffer systems and the use of EDTA analogs, were performed. A possible relationship between the structure and the ability of the ligand to act as an efficient light absorbing and energy donating chelator for Tb3+ is discussed. The phosphate ester of diflunisal, one of the most sensitive ligands for Tb3+, was used as an alkaline-phosphatase substrate for the determination of α-fetoprotein (AFP) in serum by a highly sensitive enzyme amplified lanthanide luminescence immunoassay by means of second derivative synchronous fluorescence spectroscopy as a detection technique. By using this substrate, AFP was determined in serum with a limit of detection (3 × s.d. blank) of 5 pg ml−1, coefficients of variation in the 2.4–5.3% range and mean recovery from four pooled serum samples at four concentration levels (5, 40, 100 and 250 ng ml−1) equal to (99 ± 3)%.

Synchronous scanning second derivative spectrofluorimetry for the simultaneous determination of diflunisal and salicylic acid added to serum and urine as ternary complexes with terbium and EDTA

Abstract By using second derivative synchronous scanning fluorescence spectrometry the simple resolution of two nonsteroidal, antiinflammatory drugs diflunisal and salicylic acid, as ternary complexes with terbium and EDTA, is accomplished. The method developed is simple, sensitive and rapid, and has been successfully applied for the determination of both compounds in 10 μl of untreated human serum and urine samples. The detection limits for diflunisal were 0.9 and 1.8 mg l −1 , and for salicylic acid 1.2 and 1.7 mg l −1 , in serum and urine, respectively. The mean analytical recoveries from serum and urine samples spiked with diflunisal (25–100 mg l −1 ) and salicylic acid (28–138 mg l −1 ) were 99 ± 8% (serum) and 102 ± 8% (urine) for diflunisal and 102 ± 9% (serum) and 95 ± 8% (urine) for salicylic acid. The precision of all determinations varied from 2.5 to 10%.

Simultaneous determination of acetylsalicylic acid and its major metabolites in human serum by second-derivative synchronous fluorescence spectrometry

A method is described for the simultaneous determination of acetylsalicylic, salicylic, gentisic and salicyluric acids (ASA, SA, GA and SU, respectively) in serum, based on their native fluorescence. The ASA-SA-GA-SU-containing serum samples are extracted with chloroform-1% acetic acid solution; ASA and SA are determined in the organic phase, and GA and SU in the aqueous phase, after removal of protein with trichloroacetic acid, at pH 5.0 and 11.6, respectively. The ASA-SA and GA-SU-SA mixtures are resolved using second-derivative fluorescence spectrometry and the appropriate empirical equations involving the effect of each acid on the signal of the other. Recoveries from sera spiked with ASA (1.0-10 micrograms ml-1), SA (25-50 micrograms ml-1), GA (0.05-0.2 micrograms ml-1) and SU (1.0-5.0 micrograms ml-1) ranged from 100 to 104% (mean 101%), from 93 to 99% (mean 97%), from 94 to 104% (mean 99%) and from 94 to 107% (mean 98%), respectively.

Second-derivative synchronous fluorescence spectroscopy for the simultaneous determination of naproxen and salicylic acid in human serum

Second-derivative synchronous fluorescence spectrometry was used to develop a simple, rapid and sensitive spectrofluorimetric method for the simultaneous determination of naproxen and salicylic acid in human serum. The method is based on the intrinsic fluorescence of naproxen and salicylic acid in chloroform-1% acetic acid solution. A delta gamma of 130 nm was used for the direct measurement of salicylic acid in the binary mixture, whereas naproxen was determined from direct measurements at delta gamma = 60 nm and by means of a correction equation which incorporates the concentration of salicylic acid. The range of application is 0-14 mg l-1 for naproxen and 0-13 mg l-1 for salicylic acid. The detection limits for naproxen and salicylic acid are 0.003 and 0.01 mg l-1, respectively. Serum samples are extracted into chloroform-1% acetic acid solution prior to instrumental measurement. Analytical recoveries range from 97 to 105% (mean 102%) for naproxen and from 97 to 112% (mean 103%) for salicylic acid. The within-run precision (RSD) for the method for four naproxen-salicylic acid mixtures varied from 1.2 to 6.7% and the day-to-day precision for mixtures varied from 2.1 to 5.0%.

Second derivative synchronous scanning fluorescence spectrometry as a sensitive detection technique in immunoassays. Application to the determination of α-fetoprotein

Abstract Second derivative synchronous (scanning) fluorescence spectrometry (SDSFS) has been used for the first time as an alternative to time-resolved fluorescence for the detection of Tb 3+ chelates. This approach minimizes the background signal by taking advantage of the large Stokes shift properties of Tb 3+ chelates and offers high sensitivity by narrowing the spectral bands. The analytical performance of this detection technique has been evaluated by choosing the well-defined enzyme-amplified lanthanide luminescence (EALL) immunoassay of α-fetoprotein (AFP) as a model. Monoclonal “capture” antibodies and monoclonal biotin-labelled antibodies in a “sandwich-type” assay configuration in a microwell format have been used. Alkaline phosphatase (ALP) conjugated to an antibiotin antibody was used as an enzyme label. ALP cleaves phosphate from salicylphosphate to produce salicylic acid which forms a highly fluorescent ternary complex with Tb 3+ and EDTA, which is monitored by SDSFS. The method allows the measurement of AFP with a limit of detection of 2 pg ml −1 , coefficients of variation in the range 4.5–9.9% and mean recovery from four pooled serum samples at two concentration levels equal to 94 ± 11%.

Simultaneous determination of naproxen and its desmethyl metabolite in human serum by second-derivative synchronous fluorescence spectrometry

Abstract A simple, rapid, sensitive and selective method for the simultaneous determination of the non-steroidal anti-inflammatory drug naproxen (NAP) and its main metabolite the 6-demethylated derivative (DNAP) in human serum is described. The method is based on the intrinsic fluorescence of both acids in alkaline aqueous solution. The broad-band overlapping conventional spectra of both compounds are resolved by means of second-derivative synchronous fluorescence spectrometry, thus obviating the need for pre-analysis sample separation techniques. NAP and DNAP are determined in serum supernatant solution after removal of proteins with acetonitrile at pH 11.7. Recoveries from sera spiked with NAP (5–70 μg ml −1 ) and DNAP (0.25–5 μg ml −1 ) ranged from 95 to 106% (mean 99.6%) and from 95 to 104% (mean 100.1%), respectively.

Simultaneous determination of acetylsalicylic and salicylic acids in human serum and aspirin formulations by second-derivative synchronous fluorescence spectrometry

A second-derivative synchronous scanning spectrofluorimetric method for the simultaneous determination of acetylsalicylic acid (ASA) and salicylic acid (SA) is described. The method is based on the native fluorescence of both acids in a 1% acetic acid-chloroform solution. Both ASA and SA can be determined within the concentration ranges 0.2-70 and 0.03-10 micrograms ml-1, respectively. The effect of each acid on the signal of the other has been studied in detail. Empirical equations have been used to overcome this effect, thus allowing the accurate determination of both acids in binary mixtures, without a separation step. The method has been applied to the determination of ASA and SA in blood serum and to the determination of SA impurities in aspirin formulations. Recoveries from sera spiked with both ASA (2.5-50 micrograms ml-1) and SA (100-160 micrograms ml-1) varied from 99.5 to 106.7% (mean = 102.6%) and from 93.0 to 98.0% (mean = 95.8%), respectively. Recoveries of SA from spiked aspirin solutions (0.25-1.5 mg g-1 of aspirin) varied from 98.0 to 102.0% (mean = 100.3%).

Simple, rapid and sensitive spectrofluorimetric determination of diflunisal in serum and urine based on its ternary complex with terbium and EDTA

Abstract A very simple, rapid and highly sensitive fluorimetric method for the determination of diflunisal in serum and urine is described. The method is based on the formation of a ternary complex between diflunisal, Tb3+ and EDTA in alkaline aqueous solutions. This complex exhibits very intense terbium ion luminescence with a main emission maximum at 546 nm when excited at 284 nm. Optimum conditions for the complex formation have been investigated. The detection limit for diflunisal is 2.4 μg 1−1, while the range of application is 0.01–6.00 mg 1−1. The method has been successfully applied for the determination of diflunisal in untreated human serum and urine samples. Analytical recoveries from serum and urine samples spiked with diflunisal were in the ranges of 96.8–101.2% and 98.0–102.0%, respectively.

Simultaneous determination of diflunisal and salicylic acid in human serum by second-derivative synchronous fluorescence spectroscopy

Abstract This simple, rapid, sensitive spectrofluorometric method for the simultaneous determination of diflunisal and salicylic acid is based on their intrinsic fluorescence in chloroform-1% acetic acid solution. The broad-band overlapping conventional spectra of both compounds are resolved by means of second-derivative synchronous fluorescence spectroscopy, obviating the need for pre-analysis sample separation techniques. The range of application is 0–25 mg/l for diflunisal and 0–13 mg/l for salicyclic acid. The detection limits for diflunisal and salicylic acid are 0.01 and 0.03 mg/l, respectively. The method has been applied for the determination of diflunisal and salicylic acid in human serum. Serum samples are treated with trichloroacetic acid to remove proteins and the two compounds are extracted into chloroform-1% acetic acid solution prior to instrumental determination. Analytical recoveries range from 97 to 105% (mean 99%) for diflunisal and from 91 to 107% (mean 96%) for salicylic acid, respectively. Within-run CVs for the method for three diflunisal-salicylic acid mixtures varied from 2.6 to 4.8% for mass ratios 1/5 to 5/1. Day-to-day CVs for mixtures varied from 2.7 to 6.8%, respectively.