Simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N(4)-acetyl metabolites with gradient liquid chromatography in chicken plasma, tissues, and eggs

A simultaneous determination of sulfamonomethoxine, sulfadimethoxine, and their hydroxy/N⁴-acetyl metabolites in chicken plasma, muscle, liver, and eggs using gradient high-performance liquid chromatography (HPLC) with a photo-diode array detector is developed. All the compounds are extracted by a handheld ultrasonic homogenizer with ethanol followed by centrifugation. The separation is performed by a reversed-phase C4 column with a gradient elution (ethanol:1% (v/v) acetic acid, v/v; 10:90 → 20:80). Average recoveries from samples spiked at 0.1-1.0 μg g⁻¹ or μg ml⁻¹ for each drug were >90% with relative standard deviations within 4%. The limits of quantitation were <30 ng g⁻¹ or ng ml⁻¹.     

High-performance liquid chromatographic method for the simultaneous determination of cyclosporine A and its four major metabolites in whole blood

This study aimed to develop a simple and efficient optimized high-performance liquid chromatograph (HPLC) method for simultaneous determination of cyclosporine A (CyA) and its major, partly active metabolites AM1, AM9, AM4N, and AM19 in whole blood from transplant patients using cyclosporine D (CyD) as internal standard. The method used a CN analytical column maintained at 60 °C with hexan-isopropanol (93:7, v/v) as mobile phase; detection was at 212 nm. Linearity for all five compounds was tested in the range of 31-1500 ng ml⁻¹ for CyA and of 31-1000 ng ml⁻¹ for metabolites. The limit of detection was found to be 15 ng ml⁻¹ for all compounds.This modified, inexpensive method is also suitable for measuring cyclosporine A and metabolite concentrations in routine monitoring of patients undergoing treatment with CyA.     

A rapid determination of propiverine and its N-oxide metabolite in human plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry

A simple, fast and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for determination of propiverine and propiverine N-oxide metabolite in human plasma using oxybutynin as internal standard. Instead of extracting propiverine from plasma using organic solvents, which should be separated from the aqueous phase and evaporated before injecting the sample into the chromatograph, plasma sample containing propiverine and N-oxide was directly injected after precipitating proteins with acetonitrile. Numerous compounds in the plasma did not interfere with the highly specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted propiverine, N-oxide and oxybutynin within 2 min (0.1% formic acid in water/acetonitrile, 25:75, v/v). The LC-MS/MS method and an alternative LC-MS method, using methyl-t-butyl ether extraction and selected ion monitoring, were validated over 1-250 ng ml⁻¹ of propiverine and 2 to 500 ng ml⁻¹ of N-oxide, and successfully applied in a pharmacokinetic study. The lower limit of quantitation was 1 ng ml⁻¹ for propiverine and 2 ng ml⁻¹ for N-oxide in both methods.     

Room-temperature luminescence optosensings based on immobilized active principles actives: Application to nafronyl and naproxen determination in pharmaceutical preparations and biological fluids

This paper presents two easy and selective methods for determining the active principles nafronyl (NFL) and naproxen (NAP), using a flow-through fluorescence optosensor based on the on-line immobilization on a nonionic-exchanger (Silica Gel, Davisil™ and Amberlite XAD 7, respectively) solid support. The determination was performed in 5×10⁻³ M HAc/NaAc buffer solution at pH 5 for NFL and 15×10⁻³ M glycine/HCl buffer solution at pH 2.5 for NAP at a working temperature of 20 °C. The fluorescence intensities were measured at λ_ex/em=294/336 nm and λ_ex/em=332/354 nm for NFL and NAP, respectively. The response time for these optosensors were practically instant, obtaining a linear concentration range between 0 and 700.0 ng ml⁻¹ with a detection limit of 20.8 ng ml⁻¹, an analytical sensitivity of 10.1 ng ml⁻¹ and a standard deviation of 1.27% at a 500 ng ml⁻¹ concentration level for NFL and a linear concentration range between 0 and 200.0 ng ml⁻¹ with the detection limit of 13.3 ng ml⁻¹, an analytical sensitivity of 6.0 ng ml⁻¹ and a standard deviation of 3.52% at a 100 ng ml⁻¹ concentration level for NAP. The proposed methods were satisfactorily applied to real samples (three commercial formulations and urine samples). The effects of the possible interferences were evaluated in all cases.     

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [O,O-dimethyl-O-(4-nitro-m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71-27 ng ml⁻¹ with a limit of detection (LOD) of 0.32 ng ml⁻¹, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC₅₀) was 4.2 ng ml⁻¹. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67-27 ng ml⁻¹ with a LOD of 0.38 ng ml⁻¹ and an IC₅₀ of 3.7 ng ml⁻¹. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml⁻¹ were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.     

A liquid chromatography/positive ion tandem mass spectrometry method for the determination of cilazapril and cilazaprilat in human plasma

A simple, rapid, and sensitive high-performance liquid chromatography (HPLC)-electrospray ionization (ESI) tandem mass spectrometric method (LC-MS/MS) has been developed for simultaneous determination of cilazapril levels and its active metabolite, cilazaprilat, in human plasma using enalapril as internal standard. The acquisition was performed in the multiple reaction monitoring mode; monitoring the transitions: m/z 418.4 > 211.1 for cilazapril and m/z 390.3 > 211.1 for cilazaprilat. The method involves a simple single-step liquid-liquid extraction with ethyl acetate. The analyte was chromatographed on an YMC C₈ reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (10:90, v/v; pH 3.2 with formic acid). Numerous compounds did not interfere with specific multiple reaction monitoring in tandem mass spectrometric detection following C₈ reversed-phase chromatographic separation under conditions that eluted cilazapril, cilazaprilat, and enalapril within 2 min. This method was validated over 0.1-500 ng ml⁻¹ of cilazapril and 0.5-50 ng ml⁻¹ of cilazaprilat. Cilazapril and cilazaprilat were stable in standard solution and in plasma samples under typical storage and processing conditions. The assay was successfully applied to a pharmacokinetic study of cilazapril given as a single oral dose (5 mg) to healthy volunteers.     

Direct spectrophotometric determination of calcium in clinical samples with carboxyazo-p-CH3

A highly sensitive and relatively interference-free spectrophotometric method for determination of calcium is described. The method is based on the reaction between calcium ions and carboxyazo-p-CH₃ in aqueous citrate medium of pH 7, to form a blue complex with maximum absorption at 716 nm. The calibration is linear up to 0.12 μg ml⁻¹ calcium with a repeatability (R.S.D.) of 1.0% at a concentration of 0.04 μg ml⁻¹ (n=5). The molar absorptivity of the complex and Sandell’s sensitivity are 3.5×10⁵ l mol⁻¹ cm⁻¹ and 0.11 ng cm⁻², its 10σ limit of quantification and the 3σ limit of detection were found to be 0.3 ng ml⁻¹ and 0.09 ng ml⁻¹ respectively. The influence of reaction variables and the effect of interfering ions are studied; no interference was observed in clinical samples. The proposed method has been applied directly to the determination of calcium in clinical samples without the need for pre-concentration, masking metal ions and digesting samples.     

Determination of clarithromycin in rat plasma by HPLC-UV method with pre-column derivatization

A novel high-performance liquid chromatographic (HPLC) method using pre-column derivatization and UV detection at 275 nm for the determination of clarithromycin in rat plasma has been validated. Clarithromycin was extracted from plasma sample spiked with internal standard (erythromycin) under alkaline condition with ethyl ether and derivatizated with trimethylbromosilane. The analyses were run on a C₁₈ column, maintained at 40 °C during elution, using a mobile phase comprised of potassium dihydrogen phosphate (50 mM, pH 6.8, contained 0.7% triethylamine), acetonitrile, and methanol (30:45:25, v/v/v). The standard calibration curve for clarithromycin was linear (r² = 0.9998) over the concentration range of 0.1-10 μg ml⁻¹ in rat plasma. The limit of detection (LOD) and limit of quantitation (LOQ) was 30 ng ml⁻¹ and 0.1 μg ml⁻¹ respectively. The intra- and inter-day assay variability range was 2.6-7.4% and 3.3-8.5%, respectively. This method has been successfully applied to a pharmacokinetic study of clarithromycin in rats.     

Use of a solid sensing zone implemented with unsegmented flow analysis for simultaneous determination of thiabendazole and warfarin

For the first time, a solid sensing zone implemented with unsegmented flow analysis is described for the simultaneous determination of two pesticides, thiabendazole and warfarin. The system works as a simple and rapid spectrofluorimetric biparameter sensor. The sensor is based on the retention of the analytes on the sensing solid zone (octadecyl silane C₁₈ gel) placed in the detection zone itself into a quartz flow-cell. A temporary sequentiation in the arrival of the analytes to the sensing zone is achieved by on line separation using a pre-column of the same gel placed just before the flow cell. Thiabendazole is determined the first (using methanol 30% (v/v) as carrier/elution solution) because it passes through the pre-column while warfarin is strongly retained in it. Then, warfarin is conveniently eluted from the pre-column (using methanol 50% (v/v) as carrier/elution solution) the intrinsic fluorescence peak height measured at an excitation wavelength of 309 nm and an emission wavelength of 368 nm is used as analytical signal. Using a low sample volume (40 μl), the analytical signal showed a very good linearity in the range 10-800 ng ml⁻¹ and 2-40 μg ml⁻¹ with detection limits of 2.35 ng ml⁻¹ and 0.54 μg ml⁻¹ for thiabendazole and warfarin, respectively. The sensor was satisfactorily applied to the determination of these two analytes in pesticides and pharmaceutical preparations.     

Resonance Rayleigh scattering, frequency doubling scattering and second-order scattering spectra of the heparin-crystal violet system and their analytical application

In pH 6.0-11.2 Britton-Robinson buffer solution, binding of heparin with crystal violet (CV) can result in a significant enhancement of resonance Rayleigh scattering (RRS) and resonance non-linear scattering, such as frequency doubling scattering (FDS) and second-order scattering (SOS). Their maximum scattering wavelengths, λ_ex/λ_em, appear at 492 nm/492 nm for RRS, 984 nm/492 nm for FDS and 492 nm/984 nm for SOS, respectively. The optimum conditions of the reaction, the influencing factors and the relationship between the three scattering intensities and the concentration of heparin have been investigated. New methods for the determination of trace amounts of heparin based on the RRS, FDS and SOS methods have been developed. The methods exhibit high sensitivities, the detection limit for heparin is 2.9 ng ml⁻¹ for the RRS method, 3.5 ng ml⁻¹ for the FDS method and 3.3 ng ml⁻¹ for the SOS method. The methods have good selectivity and were applied to the determination of heparin in heparin sodium injection samples with satisfactory results.     

A simple method for the study of salbutamol pharmacokinetics by ion chromatography with direct conductivity detection

A simple method for the study of the pharmacokinetics of salbutamol using ion chromatography with direct conductivity detection without chemical suppression is presented in this paper. Baseline separation of salbutamol from plasma components was achieved by using diluted HNO₃ (2 mmol l⁻¹) as mobile phase with acetonitrile (6%, v/v) as modifier. Atenolol was utilized as internal standard. Under the optimal conditions, the linear regression coefficients of the calibration curves are 0.996 within the concentration range of 1000-3 ng ml⁻¹ salbutamol. The detection limit (signal-to-noise ratio = 3) was 1 ng ml⁻¹ salbutamol. The noncompartmental pharmacokinetic parameters for salbutamol following administration of 8 mg salbutamol to 18 subjects were tested. The results coincided most satisfactorily with the results obtained by using reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection.     

A validated liquid chromatographic tandem mass spectrometric method for the determination of mirtazapine and demethylmirtazapine in human plasma: application to a pharmacokinetic study

A new liquid chromatographic tandem mass spectrometric method for the determination of mirtazapine and demethylmirtazapine in human plasma has been developed and fully validated. The article describes in detail the bioanalytical procedure and summarizes the validation results obtained. The samples were extracted using liquid-liquid extraction with a mixture of 1-chlorobutane/isopropanol/ethyl acetate (88:2:10, (v/v/v)). The chromatographic separation was performed on a reversed-phase XTerrra MS C₈ column ( i.d.; 3.5 μm particle size) using a mobile phase consisting of 0.010 M ammonium formate (pH 7.8) and acetonitrile (35:65, (v/v)), pumped at a flow rate of 0.80 ml min⁻¹. The analytes were detected using a Finnigan LCQ advantage ion-trap mass spectrometer with positive electrospray ionization in selected reaction monitoring (SRM) mode. Tandem mass spectrometric detection enabled the quantitation of both compounds down to 0.10 ng ml⁻¹. Calibration graphs were linear (r better than 0.990, n=11), in concentration ranges 0.10 to 200 ng ml⁻¹ for mirtazapine demethylmirtazapine. The intra- and inter-day R.S.D. values were less than 14.8 and 16.6% for mirtazapine and demethylmirtazapine, respectively. The method was successfully applied to a kinetic study in order to assess the main pharmacokinetic parameters of mirtazapine and demethylmirtazapine.     

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.     

Development and validation of a liquid chromatography-electrosprayionization mass spectrometric method for the determination of dexamethasone in sheep plasma

A quantitative liquid chromatographic-electrospray ionization mass spectrometric method for the determination of dexamethasone in sheep plasma has been developed and validated. The samples were extracted using solid-phase extraction cartridges with mixed reversed-phase materials (oasis-HLB). The chromatographic separation was performed on a reversed-phase XTerrra MS C₁₈ column ( mm; 5 μm) using a mobile phase consisting of 65% methanol in water containing 0.1% (v/v) formic acid, pumped at a flow rate of 0.30 ml min⁻¹. The analyte was detected after positive electrospray ionization using selected ion monitoring (SIM) mode. The probe heater temperature was set at 260 °C, the capillary voltage was set at 3.5 kV and the source block voltage (AQA_max) was set at 30 V. The method was fully validated. Calibration graphs were linear (r better than 0.998, n=11), in concentration ranges 6-1000 ng ml⁻¹ for dexamethasone. The intra- and inter-day RSD values were less than 24.1% (n=6). The limits of detection and quantitation for dexamethasone were found to be 1 and 6 ng ml⁻¹, respectively. The efficiency of the solid phase extraction procedure was found to be 92.4% for dexamethasone. The method was further applied to a pilot kinetic study in order to assess the main pharmacokinetic parameters of dexamethasone in sheep.     

Solid phase preconcentration of iron as methylthymol blue complex on naphthalene-tetraoctylammonium bromide adsorbent with subsequent flame atomic absorption determination

A sensitive and selective preconcentration method for the determination of trace amounts of iron by atomic absorption spectrometry has been developed. Iron forms a complex with methylthymol blue at pH=3. This complex is retained by naphthalene tetraoctylammonium bromide adsorbent in a column with a height of about 2 cm. The adsorbed metal complex is then eluted from the column with nitric acid and its iron content is determined by flame atomic absorption spectrometry (FAAS). The effect of different variables such as pH, reagent concentration, flow rate and interfering ions on the recovery of the analyte was investigated. The calibration graph was linear in the range 25-350 ng ml⁻¹ of iron in the initial solution with r=0.9994. The limit of detection based on 3S_b criterion was 12 ng ml⁻¹ and the relative standard deviation for eight replicate measurements of 150 and 300 ng ml⁻¹ of iron was 3.1 and 1.8%, respectively. This procedure was successfully applied to the determination of iron in tap and sewage water samples.     

Flow-through optosensing of 1-naphthaleneacetic acid in water and apples by heavy atom induced-room temperature phosphorescence measurements

A sensitive and selective phosphorimetric method for the determination of 1-naphthaleneacetic acid (1-NAA) based on a flow-injection system connected to a flow cell packed with a solid support and placed in the sample compartment of a conventional luminescence spectrometer is described. A non-ionic solid polymeric resin Amberlite XAD-7 is used for the packing. After injection of the sample, 1-NAA is on-line retained in the packed resin and measurements of the heavy atom induced (HAI)-room temperature phosphorescence (RTP) emission (λ_ex/λ_em = 292/490 nm) from this native luminescent compound are taken.The optimum experimental conditions were investigated by injecting 2 ml samples of an aqueous solution of 1-NAA in the flow system. A concentration 0.15 mol l⁻¹ of thallium(I) ions, as heavy atom, both in the samples and the carrier flow, was finally selected. Also, a concentration of 6 mmol l⁻¹ of sulphite was optimal for ensuring the necessary deoxygenation of the system at the selected flow rate of 0.8 ml min⁻¹. After measurement, the solid support was efficiently regenerated by injecting 1 ml of a mixture water:acetone in a ratio 1:1 (v/v) into the flow.The detection limit (3σ criterion) was 1.2 ng ml⁻¹ of 1-NAA. The repeatability (R.S.D.) for five replicates of a sample containing 50 ng ml⁻¹ of analyte turned out to be ±3% and the calibration graphs proved to be linear up to 500 ng ml⁻¹ of 1-NAA (maximum concentration assayed). The effect of potential interferences from other organic species which can be also used as plant growth regulators, as well as from various inorganic cations and anions, has been investigated as well.The method was successfully applied to the determination of low levels of this plant growth regulator in natural waters (river and fountain waters) and apples.     

Backscattering light detection of nucleic acids with tetraphenylporphyrin-Al(III)-nucleic acids at liquid/liquid interface

A backscattering light (BSL) detection assembly is constructed and applied to the determination of nucleic acids with high sensitivity and selectivity based on the measurements of BSL signals at water/tetrachloromethane (H₂O/CCl₄) interface. In aqueous medium of pH 3, the binary complex of of Al(III)-DNAs could be formed by the interaction of Al(III) with the phosphate group of DNAs, which then could interact with tetraphenylporphyrin (TPP) in tetrachloromethane through liquid/liquid interaction, forming a ternary complex of TPP-Al(III)-DNAs at the interface. It was observed that greatly enhanced BSL signals occurred with maximum peak at 469 nm when the ternary complex of TPP-Al(III)-DNAs were absorbed to the liquid/liquid interface. The enhanced backscattering light intensity (I_BSL) is in proportion to the concentration of calf thymus DNA (ctDNA) and fish sperm DNA (fsDNA) in the range of 0.6-1200 ng ml⁻¹ and 1.1-1200 ng ml⁻¹, respectively. The limits of determination (3σ) are 60 pg ml⁻¹ and 110 pg ml⁻¹, correspondingly. Artificial samples with highly interference backgrounds were determined with the recovery ranging from 94.5 to 106.7%, and relative standard deviation (R.S.D.) less than 2.40%.     

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.     

Potassium permanganate-glyoxal chemiluminescence system for flow injection analysis of cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium in pharmaceutical preparations

A sensitive flow injection chemiluminescence (FL-CL) method for the determination of cephalosporin antibiotics, was developed. The method was based on that cephalosporin antibiotics could enhance the CL reaction of glyoxal and KMnO₄ in sulfuric acid. Method development included the optimization of reagent concentrations and flow-rate. Under the optimized conditions, three cephalosporin antibiotics: cefalexin, cefadroxil, and cefazolin sodium, were determined. The detection limits of the method are 10 ng ml⁻¹ cefalexin, 2 ng ml⁻¹ cefadroxil, and 2 ng ml⁻¹ cefazolin sodium. The method was successfully applied to the determination of three cephalosporin antibiotics in pharmaceutical preparations.     

Microcolumn packed with YPA(4) chelating resin on-line separation/preconcentration combined with graphite furnace atomic absorption spectrometry using Pd as a permanent modifier for the determination of trace mercury in water samples

A new method using a microcolumn packed with YPA₄ chelating resin as solid-phase extractor has been developed for the separation and preconcentration of trace Hg prior to its measurement by GFAAS with Pd as a permanent modifier. Various parameters such as the amount of the modifier, pH, sample flow rate, the concentration and volume of eluent have been studied in order to find the best conditions for the determination of mercury. The detection limit of the method (3σ) for Hg based on an enrichment factor of 100 was 0.2 ng ml⁻¹. A characteristic mass of 114 pg was obtained for mercury using Pd as a permanent modifier. The relative standard deviation was 2.8% at the 10 ng ml⁻¹ level (n = 5). The method has been applied to the determination of trace mercury in environmental water samples and the recoveries for the spiked samples are in the range of 91-105%.