Determination of Primaquine in Whole Blood and Finger-Pricked Capillary Blood Dried on Filter Paper Using HPLC and LCMS/MS

Primaquine (PQ) is the only 8-aminoquinoline antimalarial drug in clinical use because of its unique action on hypnozoites and gametocytes of Plasmodium species. We report here simple, sensitive and specific assay methods for the determination of PQ in human whole blood and dried blood spot (DBS) samples using high-performance liquid chromatography and liquid chromatography-mass spectrometry, respectively. Sample preparation was performed by a single or two-step liquid-liquid extraction with organic solvents. For whole blood analysis, separation was obtained on a reversed-phase C18 column with the mobile phase consisting of 0.25 % diethylamine and acetonitrile (7:3, v:v) running at a flow rate of 1.0 ml min^?1. UV detection was at the wavelength 263 nm. For DBS analysis, separation was obtained on a reversed-phase column with the mobile phase consisting of methanol and 0.1 formic acid (1:3, v:v) running at a flow rate of 0.5 ml min^?1. The selected ions generated by electrospray ionization were detected using mass spectrometer. Good precision and accuracy (both within-day and day-to-day assays) were obtained at the concentration ranges under investigation. Limits of quantification for PQ were accepted as 25 ng ml^?1 using 500 μl whole blood and 5 ng ml^?1 using 80 μl DBS samples. The mean recoveries for PQ and internal standard pyrimethamine (PYR) for both whole blood and DBS were over 70 %. The methods were successfully applied for a clinical pharmacology study of PQ in patients with Plasmodium vivax. Excellent correlation (r ²  = 0.997) was observed between the analysis of PQ in paired whole blood and DBS samples.     

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.     

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.     

Determination of risedronate in rat plasma samples by ion-pair high-performance liquid chromatography with UV detector

In the present work, an analytical method for determination of risedronate, a member of bisphosphonates, is described for the routine analysis in rat plasma. Sample pre-treatment involves protein precipitation, co-precipitation with calcium at alkaline pH, hydrolysis of possible derivatives of pyrophosphate and reprecipitation. A good separation was obtained by using a reversed-phase column (Hypersil ODS-2 C₁₈, 4.6 mm × 250 mm, 5 μm). The mobile phase was an aqueous solution of buffer (contained 1.5 mM EDTA-2Na, 1 mM sodium etidronate, 11 mM sodium phosphate and 5 mM tetrabutylammonium bromide as ion-pair reagent) - methanol (88:12, v/v) adjusted to pH 6.75 using 1 M NaOH. The flow rate was 1 ml min⁻¹. UV detection (λ = 262 nm) was used to quantitate risedronate in the concentration range of 10-500 ng ml⁻¹. The limit of detection and quantitation for risedronate were 7 and 10 ng ml⁻¹, respectively. The method was applied successfully to plasma samples from Wistar rats undergoing oral administration of risedronate mini-pills. Precision, extraction recoveries, as well as accuracy results, were satisfactory and no interference was found at the retention time of risedronate. Hence, the method is suitable for monitoring risedronate in rat plasma.     

Determination of quinolizidine alkaloids in Sophora medicinal plants by capillary electrophoresis

A new capillary electrophoresis (CE) method for the determination of quinolizidine alkaloids in Sophora medicinal plants was developed. A total of seven alkaloid components (cytisine, sophocarpine, matrine, lehmannine, sophoranol, oxymatrine and oxysophocarpine) were separated within 15 min. The running buffer was a 50 mM phosphate buffer containing 1%HP-β-CD and 3.3% isopropanol. The linear calibration ranges were 5.50-88.0 μg ml⁻¹ for cytisine and lehmannine, 5.00-88.0 μg ml⁻¹ for sophocarpine and sophoranol, 5.60-89.6 μg ml⁻¹ for matrine and oxysophocarpine, and 24.0-384 μg ml⁻¹ for oxymatrine. The recoveries of the seven alkaloids were 96.0-102.9% with relative standard deviations from 1.50 to 3.00% (n = 5). The method was successfully applied to different Sophora medicinal plants including Sophora flavescens, Sophora tonkinensis and Sophora alopecuroides.     

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.     

A rapid spectrophotometric method for the determination of transparent exopolymer particles (TEP) in freshwater

A simple and rapid spectrophotometric method is proposed for the determination of transparent exopolymer particles (TEP) in freshwater samples. In this method, TEP reacts with excess of alcian blue solution yielding a low solubility dye-TEP complex. After centrifugation, the concentration of the remaining dye in the supernatant was determined at 602 nm and its concentration was related to the concentration of TEP in freshwater. The effect of alcian blue concentration from 1.5×10⁻³ to 9.0×10⁻³% (m/v), solution pH from 2.5 to 6.9 and stirring time from 20 to 120 s on the analytical curve was investigated. Under the optimum conditions established, such as alcian blue concentration of 3.0×10⁻³% (m/v); pH of 4.0 (0.2 mol l⁻¹ acetate buffer solution) and stirring time of 1 min, the analytical curve was linear from 0.50 to 10 μg ml⁻¹ (A=0.34−0.037[GX]; r²=0.9999; where A is the absorbance and [GX] the gum xanthan concentration in μg ml⁻¹) with a detection limit of 0.10 μg ml⁻¹. The recovery of TEP (as gum xanthan) for two samples ranged from 95.3 to 108 and the relative standard deviations (R.S.D.s) were lower than 0.8% for gum xanthan solutions at concentrations of 1.0 and 1.5 μg ml⁻¹ (n=8). The results obtained for TEP in freshwater samples using the proposed spectrophotometric method and those obtained using a literature method are in agreement at the 95% confidence level and within an acceptable range of error.     

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.     

Determination of Imidacloprid and metabolites by liquid chromatography with an electrochemical detector and post column photochemical reactor

A procedure for the determination of Imidacloprid and its main metabolites was set up by means of liquid chromatography with an electrochemical detector and post-column photochemical reactor (LC-hν-ED). Sample clean-up was developed for bees, filter paper and maize leaves. Chromatographic conditions were based on a reversed-phase C-18 column operated by phosphate buffer 50 mM/CH₃CN (80/20, v/v) at pH 2.9. Detection of Imidacloprid and its metabolites was performed at a potential of 800 mV after photoactivation at 254 nm. Compared to conventional techniques such as gas chromatography/mass spectrometry (GC/MS) or LC coupled to other detectors, the present method allows simultaneous trace-level determination of both Imidacloprid (0.6 ng ml⁻¹) and its main metabolites (2.4 ng ml⁻¹).     

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.     

Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

A simple high-performance liquid chromatography (HPLC) method is described for the determination of matrine in rat plasma. The plasma was deproteinized with acetonitrile that contained an internal standard (phenacetin) and was separated from the aqueous layer by adding sodium chloride. Matrine was extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: YMC-pack ODS-A, 5 μm, 150×4.6 mm, I.D.; eluent: acetonitrile-0.02 mol ammonium acetate buffer-triethylamine (35:65:0.035, v/v/v) and ultraviolet detection (220 nm). The limit of quantitation for matrine was 200 ng ml⁻¹ in plasma, and the recovery was greater than 89%. The assay was linear from 0.5 to 50.0 μg ml⁻¹. Variation over the range of the standard curve was less than 6%. The method was used to determine the concentration-time profiles of matrine in the plasma following oral administration of matrine aqueous solution or bolus injection from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method for the first time.     

On-line preconcentration and determination of cobalt by chelating microcolumns and flow injection atomic spectrometry

A simple and sensitive flow injection analysis-atomic absorption spectrometric procedure is described for the determination of cobalt. The method is based upon on-line preconcentration of cobalt on a microcolumn of 2-nitroso-1-naphthol immobilized on surfactant coated alumina. The trapped cobalt is then eluted with ethanol (250 μl) and determined by flame atomic absorption spectrometry. The analytical figures of merit for the determination of cobalt are as follows: detection limit (3 S), 0.02 ng ml⁻¹; precision (RSD), 2.8% for 20 ng ml⁻¹ and 1.7% for 70 ng ml⁻¹ of cobalt; enrichment factor, 125 (using 25 ml of sample). The method has been applied to the determination of cobalt in water samples, vitamin B₁₂ and B-complex ampoules and accuracy was assessed through recovery experiment and independent analysis by furnace AAS.     

Chromatographic analysis of serotonin, 5-hydroxyindolacetic acid and homovanillic acid in dried blood spots and platelet poor and rich plasma samples

An isocratic high-performance liquid chromatographic method has been developed for the measurement of serotonin, 5-hydroxyindolacetic and homovanillic acids in dried blood spots and in platelet poor and rich plasma samples. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 13% methanol and 87% aqueous citrate buffer, containing octanesulfonic and ethylendiaminotetracetic acids. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at −0.200 V and the second analytical cell at +0.400 V. For the pre-treatment of biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange Oasis cartridges, was implemented. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0%. The calibration curve was linear over the on-column concentration range of 0.1–22.5 ng mL⁻¹ for serotonin and 5-hydroxyindolacetic acid and of 0.25–22.5 ng mL⁻¹ for homovanillic acid. The sensitivity was good with a limit of detection of 0.05 ng mL⁻¹ for serotonin and 5-hydroxyindolacetic acid and 0.12 ng mL⁻¹ for homovanillic acid. Results were also satisfactory in terms of precision, selectivity and accuracy. The analytical method was successfully applied to human platelet poor and rich plasma samples and to dried blood spots from volunteers and psychiatric patients.     

Flow injection spectrophotometric determination of europium using chlortetracycline

A flow injection (FI) spectrophotometric determination of europium (III) is described, based on the complexation between europium (III), and chlortetracycline (CTC) in a Tris-buffer pH 8.0 medium. The resulting yellow-coloured complex is measured at its absorption maximum of 400 nm after 100 μl of sample or standard solution containing europium (III) are injected into the merged streams of CTC and Tris-buffer solutions. Optimum conditions for determining μg amounts of europium (III) are achieved by univariate method. Various types of reactors are also investigated. It is shown that the use of a single bead string reactor gives rise to the enhancement of peak height. A linear calibration curve over the range of 0.10-0.60 μg ml⁻¹ europium (III) is established with the regression equation (n=6) Y=34.93X+0.01 and the correlation coefficient of 0.9994 is obtained. A detection limit (3σ) of 0.01 μg ml⁻¹ of europium (III) and the relative standard deviation (R.S.D.) of 4.32% for determining 1.0 μg ml⁻¹ of europium (III) (n=7) are obtained. The recommended method has been applied to the quantitation of europium (III) in spiked water and stream sediment samples with average recoveries of 99.9 and 97.5%, respectively. The sampling rate is found to be 85 h⁻¹.     

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.     

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.     

Chemiluminescence determination of barbituric acid using Ru(phen)(3)(2+)-Ce(IV) system

A chemiluminescence (CL) method for the determination of barbituric acid (BA) was proposed, which is based on the enhancement of BA to the CL intensity of Tris-(1,10-phenanthroline)ruthenium(II) (Ru(phen)₃²⁺)-cerium(IV) (Ce(IV)) system. The concentration of BA is proportional to the CL intensity in the range of 5.0×10⁻³-2.0 μg ml⁻¹. The detection limit is 6.9×10⁻⁴ μg ml⁻¹. The relative standard deviation (R.S.D.) of determining 11 samples containing 0.20 μg ml⁻¹ BA is 3.2%. This CL method has been successfully applied to the determination of BA in the synthetic samples. The mechanism of CL reaction was studied.     

Determination of nitrite by simple diazotization method

A simple and rapid spectrophotometric method for the determination of nitrite is described. This is based upon simple diazotization reactions involving p-nitroaniline (PNA) and sulfanilamide (SA) with ethyl acetoacetate (EAA) as the coupling agent. The absorbance was measured at 507 and 356 nm, respectively. The method is optimized for acidity, amount of reagents required, amount of sodium hydroxide and the tolerance amount of other ions. The range of linearity for PNA-EAA couple was 0.05-6.0 μg ml⁻¹ of nitrite with molar absorptivity of 1.59×10⁴ l mol⁻¹ cm⁻¹; while that for SA-EAA couple was found to be 0.2-3.0 μg ml⁻¹ and 1.22×10⁴ l mol⁻¹ cm⁻¹, respectively. The method has been applied to various water and soil samples.     

High-performance liquid chromatographic method for simultaneous determination of honokiol and magnolol in rat plasma

A high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of honokiol and magnolol in rat plasma. The plasma was deproteinized with acetonitrile which contained an internal standard (diphenyl) and was separated from the aqueous layer by adding sodium chloride. Honokiol and magnolol are extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC and ultraviolet detection. The limits of quantitation for honokiol and magnolol were 13 and 25 ng ml⁻¹ in plasma, respectively, and recovery of both analytes was greater than 93%. The assay was linear from 20 to 200 ng ml⁻¹ for honokiol and from 40 to 400 ng ml⁻¹ for magnolol. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of honokiol and magnolol in the plasma following rectal administration of Houpo extract at a dose of 245 mg kg⁻¹, equivalent to 13.5, 24.4 mg kg⁻¹ of honokiol and magnolol, respectively.     

Fluorimetric determination of danthron in pharmaceutical tablets and in urine

A simple, rapid determination is reported for danthron (1,8-dihydroxyanthraquinone) in pharmaceutical tablets. In a flow-injection system, danthron is reduced by sodium dithionite in $\text{1}\text{1}$ methanol/borate buffer to give a fluorescent complex. Linearity ranges from 30 μg ml^?1 to below 0.1 μg ml^?1. In urine samples, danthron is separated from other fluorescing species by reversed-phase high-performance liquid chromatography before its reduction by dithionite in a post-column reactor. Urine preparation requires no extraction. Spiked urine samples were studied in the working range of 0.02–2.0 μg ml^?1 danthron.