Solid phase extraction using nylon membranes with fluorescence detection as a fast and sensitive method for Amiloride and Furosemide determination in urine samples

A diuretic is a substance widely prohibited in competition sports, by the World Anti-Doping Agency (WADA). In this paper, a sensitive, rapid and convenient analytical solid phase extraction-fluorimetric method for the determination of furosemide (FUR) and amiloride hydrochloride (AMI) diuretics in human urine was developed. FUR and AMI exhibit overlapped fluorescent spectra and urine produces background fluorescence that precludes the direct determination of these diuretics by conventional fluorimetry. AMI was adsorbed using nylon membranes whereas; FUR was detected in aqueous solutions. The optimum analytical conditions for AMI and FUR assay were established. Excitation wavelengths of 365 nm and 237 nm and emission wavelengths of 406 and 415 nm were used for AMI and FUR, respectively. The calibration graphs are linear in the range of 3.7 × 10⁻⁴ to 0.8 μg mL^− 1 and 1.2 × 10^− 3 to 4.0 μg mL^− 1, for AMI and FUR, respectively, with a detection limit of 1.1 × 10^− 4 and 3.5 × 10^− 4 μg mL^− 1 (S/N = 3). These concentrations correspond well with the range of the drugs in urine. AMI and FUR were accurately quantified in doped urine samples and urine of subjects under medical treatment with diuretic. The results were validated by recovery test, being highly satisfactory.     

Analysis of salicylic acid based on the fluorescence enhancement of the As(III)-salicylic acid system

A new, simple and sensitive spectrofluorimetric method for the determination of salicylic acid (λ_ex = 315 nm, λ_em = 408 nm) using As(III) as a sensitizing reagent has been investigated by measuring the increase of fluorescence intensity of salicylic acid due to the complexation of As(III)-salicylic acid in presence of sodium dodecyl sulfate (SDS) 10⁻³ M. Under optimum conditions, a significant relationship was obtained between the fluorescence intensity and salicylic acid concentration. A linear calibration curve was obtained in the range 13.8-13812 μg l⁻¹ with product-moment correlation coefficient (R) 0.99985 and detection limit 4.2 μg l⁻¹. The R.S.D. is 2.35% (n = 5).The method was applied successfully to the determination of salicylic acid in human serum.     

A reversed-phase high performance liquid chromatography coupled with resonance Rayleigh scattering detection for the determination of four tetracycline antibiotics

A new reversed-phase high performance liquid chromatography with resonance Rayleigh scattering detection (HPLC-RRS) was developed for simultaneous separation and determination of four tetracycline antibiotics (TCs). A good chromatographic separation among the compounds was achieved using a Synergi Fusion-RP column (150 mm × 4.6 mm; 4 μm) and a mobile phase consisting of methanol-acetonitrile-oxalic acid (5 mM) at the flow rate of 0.8 mL min⁻¹. Column temperature was 30 °C. The RRS signal was detected at λ_ex = λ_em = 370 nm. The recoveries of sample added standard ranged from 95.3% to 103.5%, and the relative standard deviation was below 2.79%. A detection limit of 2.12-5.12 μg mL⁻¹ was reached and a linear range was found between peak height and concentration in the range of 10.36-518.0 μg mL⁻¹ for oxytetracycline (OTC), 12.11-605.5 μg mL⁻¹ for tetracycline (TC), 11.79-589.5 μg mL⁻¹ for chlortetracycline (CTC) and 10.32-516.0 μg mL⁻¹ for doxycycline (DC). The linear regression coefficients were all above 0.999. The method has been applied successfully to the determination of OTC, TC, CTC, DC in pharmaceutical formulations and in honey. The method was simple, rapid and showed a better linear relation and high repeatability.     

Spectrofluorimetric determination of iridium(IV) traces using 4-pyridone derivatives

A new spectrofluorimetric determination of iridium(IV) with 3-hydroxy-2-methyl-1-phenyl-4-pyridone (HX) or 3-hydroxy-2-methyl-1-(4-tolyl)-4-pyridone (HY) is reported. Iridium(IV) react with HX or HY and chelates were extracted into chloroform or dichloromethane. The organic phase showed fluorescence. The fluorescence measurements to quantify iridium were carried out in its fluorescent band centred at λ_ex=373 nm and λ_em=480 nm. Under optimal conditions, the calibration graphs were linear over the concentration range of 0.1-7.6 μg ml⁻¹ of iridium for Ir(IV)-HX and 0.1-5.8 μg ml⁻¹ for Ir(IV)-HY with a correlation coefficients of 0.999 and 0.992 and relative standard deviation of ±1.1%.The method is free from interference by Rh(III) and Pt(IV), which normally interfere with other methods. Iridium can be determined in the presence of 300-fold excess of rhodium(III) and 10-fold excess of platinum(IV).The method was applied successfully to the determination of iridium in some synthetic mixtures and mineral sample gave satisfactory results.     

Fluorescence and terbium-sensitised luminescence determination of garenoxacin in human urine and serum

Fluorescence and terbium-sensitised luminescence properties of new quinolone garenoxacin have been studied. The fluorimetric method allows the determination of 0.060-0.600 μg ml⁻¹ of garenoxacin in aqueous solution containing HCl/KCl buffer (pH 1.5) with λ_exc=282 nm and λ_em=421 nm. Micellar-enhanced fluorescence was also studied, leading to a higher than 400% increase in analytical signal in presence of 12 mM sodium dodecyl sulphate (SDS), allowing the determination of 0.020-0.750 μg ml⁻¹ of garenoxacin. The terbium-sensitised luminescence method allows the determination of 0.100-1.500 μg ml⁻¹ of garenoxacin in 12 mM SDS solution containing 0.08 M acetic acid/sodium acetate buffer (pH 4.1) and 7.5 mM Na₂SO₃ (chemical deoxygenation agent), with λ_exc=281 nm and λ_em=546 nm. Relative standard deviation (R.S.D.) values for the three methods were in the range 1.0-2.0%. The proposed procedures have been applied to the determination of garenoxacin in spiked human urine and serum.     

A novel spectrofluorimetric method for the ultra trace analysis of nitrite and nitrate in aqueous medium and its application to air, water, soil and forensic samples

A highly sensitive and virtually specific method has been developed for the trace and ultra trace 5 ng ml⁻¹-1 μg ml⁻¹ fluorimetric analysis of nitrite. The method is based on the quenching action of nitrite on the native fluorescence of murexide (ammonium purpurate) [λ_ex=349.0 nm, λ_em=444.5 nm] in the acid range of 0.045-0.315 (M) H₂SO₄. The method is very precise and accurate (S.D.=±0.4877 and R.S.D.=0.4878% for the determination of 0.1 μg ml⁻¹ of nitrite in 11 replicates). Relatively large excesses of over 35 cations and anions do not interfere. The proposed technique has been successfully applied for the determination of nitrite and nitrate in ground water, surface water and sea water, nitrite in soil and nitrate in forensic samples. The method has also been extended for the analysis of NO_x in air.     

Highly sensitive spectrofluorimetric kinetic determination of ultratrace amounts of vanadium(V) based on the oxidation of 1,8-diaminonaphthalene by bromate

A highly sensitive catalytic quenching spectrofluorimetric method was described for the determination of V(V) based on its catalytic effect on the oxidation of 1,8-diaminonaphthalene by potassium bromate with Tiron as an activator in weakly acidic medium and the reaction mechanism was investigated. The reaction was followed spectrofluorimetrically by measuring the fluorescence intensity of 1,8-diaminonathphlene (DAN) (λ_ex=356 nm, λ_em=439 nm) at a fixed time of 5 min from initiation of the reaction. Under the optimum conditions, vanadium(V) can be determined in the range 0.05-50.0 ng ml⁻¹ with a S.D.=0.024 for 15 times measurements. The detection limit of the method was down to 0.0088 ng ml⁻¹ and the catalytic reaction activation energy was found to be 43.92 kJ mol⁻¹. The proposed method was tested for the determination of vanadium(V) in rice and natural water samples.     

Development of a novel flow injection liquid-liquid microextraction method for the on-line separation and preconcentration for determination of zinc(II) using 5-(8-hydroxy-2-quinolinylmethyl)-2,8-dithia-5-aza-2,6-pyridinophane as a sensitive and selective fluorescent chemosensor

A novel flow injection analysis (FIA) system based on liquid-liquid microextraction and fluorimetric determination was developed for the determination of traces of the Zn²⁺ ion using 5-(8-hydroxy-2-quinolinylmethyl)-2,8-dithia-5-aza-2,6-pyridinophane (L) as a sensitive and selective fluorimetric sensor, with λ_ex = 373 nm and λ_em = 530 nm, and hexanol as the extracting organic solvent. In the designed FIA system, the phase separation takes place via gravitation forces in the absence of any segmenter. The influence of pH and ionic strength of the solution, amount of ligand, nature of counter ion, volume of organic solvent, extraction time and coil length was investigated. Under optimized experimental conditions, the calibration curve found to be liner over a concentration range of 0.025-4.53 μg mL⁻¹ (R² = 0.9951) with a limit of detection of 2.3 ng mL⁻¹. The enrichment factor was 45 and relative standard deviation for 7 replicate determinations was 2.43%. The method is very fast and uses low levels of organic solvents. The proposed method was applied successfully to the determination of zinc(II) in human hair, human serum and two inorganic sludge samples.     

Flow-injection determination of hydrogen peroxide based on fluorescence quenching of chromotropic acid catalyzed with Fe(II)

Flow-injection analysis system (FIA system), which was based on Fe(II)-catalyzed oxidation of chromotropic acid with hydrogen peroxide, was developed for the determination of hydrogen peroxide. The chromotropic acid has a fluorescence measured at λ_em = 440 nm (emission wavelength) with λ_ex = 235 nm (excitation wavelength), and the fluorescence intensity at λ_em = 440 nm quietly decreased in the presence of hydrogen peroxide and Fe(II), which was caused by Fe(II)-catalyzed oxidation of chromotropic acid with hydrogen peroxide. By measuring the difference of fluorescence intensity, hydrogen peroxide (1.0 × 10⁻⁸-1.0 × 10⁻³ mol L⁻¹) could be determined by the proposed FIA system, whose analytical throughput was 40 samples h⁻¹. The relative standard deviation (RSD) was 1.03% (n = 10) for 4.0 × 10⁻⁸ mol L⁻¹ hydrogen peroxide. The proposed FIA technique could be applied to the determination of hydrogen peroxide in rain water samples.     

Highly sensitive and selective spectrofluorimetric determination of tolnaftate through the formation of ternary inclusion complex of beta-naphthol/beta-cyclodextrin/anionic surfactant system

An indirect spectrofluorimetric method with high sensitivity and selectivity was developed for the determination of antifungal drug: tolnaftate (TNF), depending on the supramolecualr multirecognition interaction among the anionic surfactant sodium laurylsulfate (SLS), β-cyclodextrin (β-CD) and β-naphthol (ROH). The mechanism of the inclusion was studied and discussed by means of fluorescence spectrum, infra-red spectrograms and ¹HNMR spectroscopy. Results showed that the naphthalene ring of ROH and the hydrophobic hydrocarbon chain of SLS were included into the β-CD's cavity to form a ROH:SLS:β-CD ternary inclusion complex with stoichiometry of 1:1:1 at room temperature, which provided effective protection for the excited state of ROH. At λ_ex/λ_em = 273/360 nm, the fluorescence intensity was linear over a tolnaftate concentration range of 2.46 × 10⁻⁹ to 2.10 × 10⁻⁶ mol L⁻¹. The detection limit and relative standard deviation was 7.50 × 10⁻¹⁰ mol L⁻¹ and 1.4%, respectively. The interference of 31 foreign substances was slight. The proposed method had been successfully applied to the determination of tolnaftate in artificial mixed samples with almost quantitative recovery.     

Normal spectrophotometric and stopped-flow spectrofluorimetric sequential injection methods for the determination of alendronic acid, an anti-osteoporosis amino-bisphosphonate drug, in pharmaceuticals

A normal spectrophotometric and a stopped-flow (SF) spectrofluorimetric method have been developed and optimized for the determination of alendronic acid (ALD) in its pharmaceutical formulations. Both methods are automated using the sequential injection analysis (SIA) principle. The spectrophotometric assay is based on the reaction of the analyte with Cu(II) ions in acidic medium to form an UV-absorbing derivative (λ_max = 240 nm). The SF spectrofluorimetric method is based on the reaction of ALD with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol at basic medium (λ_ex = 340 nm/λ_em = 455 nm). Linear calibration curves were obtained in the range 1.0-60.0 mg l⁻¹ ALD for the UV method, and in the range 0.13-10.0 mg l⁻¹ ALD for the SF spectrofluorimetric one. The sampling rates were 60 and 30 h⁻¹, respectively. The developed assays are critically compared and their advantages are discussed. Both methods were applied to the analysis of an ALD containing pharmaceutical formulation with satisfactory accuracy and precision.     

Rapid and sensitive spectrofluorimetric determination of trace amount of Cr(III) with o-vanillin-8-aminoquinoline

A novel fluorescent reagent o-vanillin-8-aminoquinoline(OVAQ) was synthesized, and its infrared spectrum, elemental analysis and acid-base dissociation constants were obtained. The fluorescent reaction of this reagent with Cr(III) was studied. In acetonitrile-water (1:1, (v/v)) medium of pH 6.00, Cr(III) could react with fluorescent reagent OVAQ (λ_ex/em=280/314 nm) to form a 1:1 non-fluorescent complex. The linear range of the spectrofluorimetric method proposed was from 8.2 to 130 μg l⁻¹, and the detection limit was 2.5 μg l⁻¹. The interferences of 25 foreign ions were also studied. This method could be easily performed and was successfully applied to the determination of Cr(III) and total chromium in domestic and industrial waste water samples.     

Ultrasensitive determination of silver ion based on synchronous fluorescence spectroscopy with nanoparticles

Based on the characteristics of synchronous fluorescence spectroscopy (SFS), a new method with high sensitivity and selectivity was developed for rapid determination of silver ion with functional cadmium sulphide (CdS) nanoparticles as a fluorescence probe. When Δλ (λ_em − λ_ex) = 215 nm, maximum synchronous fluorescence is produced at 304 nm. Under optimal conditions, functional cadmium sulphide displayed a calibration response for silver ion over a wide concentration range from 0.8 × 10⁻¹⁰ to 1.5 × 10⁻⁸ mol L⁻¹. The limit of detection was 0.4 × 10⁻¹⁰ mol L⁻¹ and the relative standard deviation of seven replicate measurements for the lowest concentration (0.8 × 10⁻¹⁰ mol L⁻¹) was 2.8%. Compared with several fluorescence methods, the proposed method had a wider linear range and improved the sensitivity. Furthermore, the concentration dependence of the synchronous fluorescence intensity is effectively described by a Langmuir-type binding isotherm.     

Nickel-aluminum layered double hydroxide as a nanosorbent for selective solid-phase extraction and spectrofluorometric determination of salicylic acid in pharmaceutical and biological samples

The nickel-aluminum layered double hydroxide (Ni-Al LDH) was synthesized by a simple co-precipitation method and used as a solid-phase extraction (SPE) sorbent for separation and pre-concentration of trace levels of salicylic acid (SA) from aqueous solutions. Extraction of analyte is based on the adsorption of salicylate ions on the Ni-Al (NO₃⁻) LDH and/or their exchanging with LDH interlayer NO₃⁻ ions. The retained analyte on the LDH was stripped by 3 mol L⁻¹ NaOH solution and its concentration was subsequently determined spectrofluorometrically at λ_em = 400 nm with excitation at λ_ex = 270 nm. Various parameters affecting the extraction efficiency of SA on the Ni-Al (NO₃⁻) LDH, such as pH, amount of nano-sorbent, sample loading flow rate, elution conditions, sample volume and matrix effects were investigated. In the optimum experimental conditions, the limit of detection (3 s) and enrichment factor were 0.12 μg L⁻¹ and 40, respectively. The relative standard deviation (RSD) for six replicate determinations of 10 μg L⁻¹ SA was 2.3%. The calibration graph using the pre-concentration system was linear in the range of 0.3-45 μg L⁻¹ with a correlation coefficient of 0.9985. The optimized method was successfully applied to the determination of SA in blood serum, willow leaf and aspirin tablet.     

Determination of the antidepressant paroxetine and its three main metabolites in human plasma by liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method has been developed for the determination in human plasma of the specific serotonin reuptake inhibitor (SSRI) antidepressant paroxetine and its three main metabolites (M1, M2, M3). Fluorescence detection was used, exciting at λ = 294 nm and monitoring emission at λ = 330 nm for paroxetine (λ_exc = 280 nm, λ_em = 330 nm for M1 and M2; λ_exc = 268 nm, λ_em = 290 nm for M3). Separation was obtained on a reversed-phase C18 column using a mobile phase composed of 66.7% aqueous phosphate at pH 2.5 and 33.3% acetonitrile. Imipramine (λ_exc = 252 nm, λ_em = 390 nm) was used as the internal standard. A careful pre-treatment of plasma samples was developed, using solid-phase extraction with C8 cartridges (50 mg, 1 mL). The calibration curves were linear over a working range of 2.5-100 ng mL⁻¹ for paroxetine and of 5-100 ng mL⁻¹ for all metabolites. The limit of detection (LOD) was 1.2 ng mL⁻¹ for PRX and 2.0 ng mL⁻¹ for the metabolites. The method was applied with success to plasma samples from depressed patients undergoing treatment with paroxetine. Hence, the method seems to be suitable for the therapeutic drug monitoring of paroxetine and its main metabolites in depressed patients’ plasma.     

Analysis of zearalenone and α-zearalenol in animal feed using high-performance liquid chromatography

Different extraction and clean-up techniques used before HPLC analysis were compared in order to obtain a reliable method for the quantitative determination of zearalenone (ZEA) and α-zearalenol (α-ZOL) in animal feed. Immunoaffinity clean-up was compared to C₁₈ and Florisil column clean-up. Extracted samples were analysed by reversed-phase HPLC with fluorescence detection (λ_ex=274 nm, λ_em=440 nm). A mobile phase of acetonitrile:water (50:50 (v/v)) and a flow-rate of 1.0 ml min⁻¹ resulted in a good separation between ZEA and α-ZOL. Using immunoaffinity clean-up the linear range was between 25 and 600 μg kg⁻¹ for ZEA and α-ZOL in maize. Intra-laboratory coefficients of variation (CV) (under repeatability conditions) were 9.16% for ZEA and 2.18% for α-ZOL. Recoveries for spiked ZEA and α-ZOL samples ranged from 89 to 110% with CVs between 5.2 and 11.2% (under within-laboratory reproducibility conditions). Using C₁₈ and Florisil solid-phase clean-up, matrix interference was too high. Therefore, naturally contaminated animal feed samples were analysed using the developed HPLC method coupled to the immunoaffinity clean-up.     

Two different spectrofluorimetric methods for simultaneous determination of gemfibrozil and rosiglitazone in human plasma

Two accurate, reliable, and highly sensitive spectrofluorimetric methods were developed for simultaneous determination of binary mixture gemfibrozil and rosiglitazone in human plasma without prior separation steps. The first method is based on synchronous fluorescence spectrometry using double scans. At Δλ = 27 nm, gemfibrozil yields detectable signal that is independent of the presence of rosiglitazone. Similarly, at Δλ = 120 nm the signal of rosiglitazone is not influenced by the presence of gemfibrozil. Signals at two wavelengths, 301 (Δλ = 27 nm) and 368 nm (Δλ = 120 nm) vary linearly with gemfibrozil and rosiglitazone concentrations over the range 100-700 ng mL⁻¹ (for gemfibrozil) and 20-140 ng mL⁻¹ (for rosiglitazone), respectively. The limits of detection (LOD) were 2.3 and 2.72 ng mL⁻¹ for gemfibrozil and rosiglitazone, respectively. The second method is based on the technique of simultaneous equations (Vierodt's method), in which 258 nm was selected as the excitation wavelength. Two equations are constructed based on the fact that at (λEm₂=302 nm of gemfibrozil) and (λEm₂=369 nm of rosiglitazone) the fluorescence of the mixture is the sum of the individual fluorescence of gemfibrozil and rosiglitazone. The limits of detection (LOD) were 28.1 and 23.63 ng mL⁻¹ for gemfibrozil and rosiglitazone, respectively. The proposed methods were successfully applied for the determination of the two compounds in synthetic mixtures and in human plasma with a good recovery.     

Spectrofluorimetric determination of aliphatic amines using a new fluorigenic reagent: 2,6-dimethylquinoline-4-(N-succinimidyl)-formate

A new amino fluorescence probe, 2,6-dimethylquinoline-4-(N-succinimidyl) formate (DMQF-OSu) has been synthesized. Based on the selective reaction of DMQF-OSu with primary and secondary aliphatic amines to yield strong fluorescence, a new spectrofluorimetric method for the determination of total aliphatic amines has been developed. At λ_ex/λ_em=324.4/416 nm, the linear calibration range was 6×10⁻⁸-6×10⁻⁶ mol l⁻¹ with the detection limit (3σ) of 1.94×10⁻¹⁰ mol l⁻¹ for the determination of aliphatic amines in weak basic media. The proposed method has been applied to the determination of aliphatic amines in tap water and lake water with the recoveries of 99-104%. Compared with the reported methods, the method presented here is rapid, simple, sensitive and feasible.     

Determination of various alcohols based on a new immobilized enzyme fluorescence capillary analysis

A novel method for the determination of ethanol in tequila based on the immobilized enzyme fluorescence capillary analysis (IE-EFCA) has been proposed. Alcohol dehydrogenase (ADH) was immobilized in inner surface of a capillary and an immobilized enzyme capillary bioreactor (IE-ECBR) was formed. After nicotinamide adenine dinucleotide (NAD⁺) as an oxidizer is mixed with alcohol sample solution, it was sucked into the IE-ECBR. The fluorescence intensity of the mixed solution in the IE-ECBR was detected at λ_ex = 350 nm and λ_em = 459 nm. The experimental conditions are as follows: The reaction time is 20 min; temperature is 40 °C; the concentrations of phosphate buffer solution (pH 7.5) and NAD⁺ are 0.1 mol L⁻¹ and 5 mmol L⁻¹, respectively; immobilization concentration of ADH is 10 U L⁻¹. The determination range of ethanol is 2.0-15.0 g L⁻¹ (F = 10.44C + 6.6002, r > 0.9958); its detection limit is 1.11 g L⁻¹; and relative standard deviation is 1.9%. IE-EFCA method is applicable for the determination of the samples containing alcohol in medicine, industry and environment.     

A sensitive flow analysis system for the fluorimetric determination of low levels of formaldehyde in alcoholic beverages

A sensitive FIA method was developed for the selective determination of formaldehyde in alcoholic beverages. This method is based on the reaction of Fluoral-P (4-amine-3-pentene-2-one) with formaldehyde, leading to the formation of 3,5-diacetyl-1,4-dihydrolutidine (DDL), which fluoresces at λ_ex = 410 nm and λ_em = 510 nm. The analytical parameters were optimized by the response surface method using the Box-Behnken design. The proposed flow injection system allowed for the determination of up to 3.33 × 10⁻⁵ mol L⁻¹ of formaldehyde with R.S.D. < 2.5% and a detection limit of 3.1 ng mL⁻¹. The method was successfully applied to determine formaldehyde in alcoholic beverages, without requiring any sample pretreatment, and the results agreed with the reference at a 95% confidence level by paired t-test. In the optimized condition, the FIA system proved able to analyze up to 60 samples/h.