Multicommuted fluorometric multiparameter sensor for simultaneous determination of naproxen and salicylic acid in biological fluids.

A combined approach based on solid-phase optosensing and multicommutation principles has been applied to develop a method for the simultaneous analysis of two pharmaceuticals (naproxen and salicylic acid) in biological fluids. The multicommuted flow-through optosensor was based on direct native fluorescence measurements of both analgesics using a non-polar sorbent (C18 silica gel) as a solid sensing zone. The flow system was controlled by Java-written home-made software and designed using three-way solenoid valves for an independent automated manipulation of sample and carrier solutions. Using an optimized sampling time, the method was calibrated in the range of 1 - 25 and 5 - 200 ng mL(-1). The obtained detection limits were 0.3 and 1.3 ng mL(-1) for naproxen and salicylic acid, respectively, with RSD (%) values of better than 2% for both analytes. The proposed methodology was successfully applied to urine, serum and pharmaceutical preparations. Recovery percentages ranging from 96.1 to 104% were obtained for both analytes.     

Microemulsion electrokinetic chromatography: an application for the simultaneous determination of suspected fragrance allergens in rinse-off products

The feasibility of microwave-accelerated derivatization for capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was evaluated. The derivatization reaction was performed in a domestic microwave oven. Histidine (His), 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) were selected as test analytes and fluorescein isothiocyanate (FITC) was chosen as a fluorescent derivatizing reagent. Parameters that may affect the derivatization reaction and/or subsequent CE separation were systematically investigated. Under optimized conditions, the microwave-accelerated derivatization reaction was successfully completed within 150 s, compared to 4-24 h in a conventional water-bath derivatization process. This will remarkably reduce the overall analysis time and increase sample throughput of CE-LIF. The detection limits of this method were found to be 0.023 ng/mL for His, 0.023 ng/mL for 1-MH, and 0.034 ng/mL for 3-MH, respectively, comparable to those obtained using traditional derivatization protocols. The proposed method was characterized in terms of precision, linearity, accuracy and successfully applied for rapid and sensitive determination of these analytes in human urine.     

Development of HPLC-MS/MS method for the simultaneous determination of metabolites of organophosphate pesticides,synthetic pyrethroids,herbicides and DEET in human urine

We developed an isotopic dilution high-performance liquid chromatography (HPLC)/tandem mass spectrometer (MS/MS) method to rapidly and accurately quantify nine metabolites of several classes of pesticide in 1 mL human urine specimens. The analytes covered in the method are two organophosphate (OP) pesticide metabolites: 3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-6-methyl-4-pyrimidinol (IMPY); three synthetic pyrethroid metabolites: 3-phenoxy benzoic acid (3-PBA), 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA) and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-1(1-cyclopropane) carboxylic acid (t-DCCA); three herbicide metabolites: 2,4-dichlorophenoxyacetic acid (DCPAA), 2,4,5-trichlorophenoxyacetic acid (TCPAA) and atrazine mercapturate; and one insect repellent: N,N-diethyl-meta-toluamide (DEET). The analytes are first deconjugated by incubating with acetate/β-glucuronidase buffer at 37°C for 17 h. The deconjugated analytes are extracted and concentrated from the urine matrix using solid-phase extraction cartridges, separated through C18 reversed phase HPLC, and analysed on MS/MS. The MS/MS was operated in positive and negative electrospray ionisation switch mode. Two ions from each analyte and one from each labelled internal standard are monitored for quantification and confirmation. The limit of detections (LODs) for all the analytes are in the low parts-per-trillion (0.05 ng/mL) except TCPy where it was 0.5 ng/mL) with a wide linear range (0.05 up to 40 ng/mL) and provides high accuracy (recoveries: 90–118%) and high precision (coefficient of variation <15%). The method accuracy was also verified by the analysis of proficiency testing urine samples. We analysed 101 urine samples for a recent California study cohort, and detection frequencies ranged from ~100% to 0%: 3-PBA (98%), IMPY (91%), TCPy, (89%), DCPAA (66%), 4-F-3-PBA (11%), TCPAA (0%).     

Simultaneous determination of salicylic acid and salicylamide in biological fluids

A new methodology for the simultaneous determination of salicylic acid and salicylamide in biological fluids is proposed. The strong overlapping of the fluorescence spectra of both analytes makes impossible the conventional fluorimetric determination. For that reason, the use of fluorescence decay curves to resolve mixtures of analytes is proposed; this is a novel technique that provides the benefits in selectivity and sensitivity of the fluorescence decay curves. In order to assess the goodness of the proposed method, a prediction set of synthetic samples were analyzed obtaining recuperation percentages between 98.2 and 104.6%. Finally, a study of the detection limits was done using a new criterion resulting in values for the detection limits of 8.2 and 11.6 μg L(-1) for salicylic acid and salicylamide respectively. The validity of the method was tested in human serum and human urine spiked with aliquots of the analytes. Recoveries obtained were 96.2 and 94.5% for salicylic acid and salicylamide respectively.     

Stereoselective determination of hydroxychloroquine and its metabolites in human urine by liquid-phase microextraction and CE

Liquid-phase microextraction based on polypropylene hollow fibers and CE were applied for the chiral determination of hydroxychloroquine (HCQ) and its metabolites (desethylchloroquine, DCQ; desethylhydroxychloroquine, DHCQ; bisdesethylchloroquine, BDCQ) in human urine. The analytes were extracted from 3 mL of urine spiked with the internal standard (metoprolol) and alkalinized with 250 muL of 2 M NaOH. The analytes were extracted into 1-octanol impregnated in the pores of the hollow fiber, and into an acid acceptor solution inside the hollow fiber. The electrophoretic separations were carried out in 100 mmol/L Tris buffer (pH adjusted to 9.0 with phosphoric acid) containing 1% w/v S-beta-CD and 30 mg/mL HP-beta-CD with a constant voltage of +18 kV. The method was linear over the concentration range of 10-1000 ng/mL for each HCQ stereoisomer and 21-333 ng/mL for each metabolite stereoisomer. Within-day and between-day assay precision and accuracy for the analytes were studied at three concentration levels for each stereoisomer and were lower than 15%. The developed method was applied for the determination of the cumulative urinary excretion of HCQ, DCQ, and DHCQ after oral administration of rac-HCQ to a health volunteer. The results obtained are in agreement with previous literature data.     

Gas chromatography-mass spectrometry/mass spectrometry analysis to determine natural and post-administration levels of oestrogens in bovine serum and urine

A novel analytical approach has been developed and shown to be capable of detecting the isomers of oestradiol in the low ppt (pg mL(-1)) range in bovine serum and urine. Following extractive derivatisation the analytes were detected as their 3-pentafluorobenzoyl 17-trimethylsilyl ether derivatives by gas chromatography-mass spectrometry/mass spectrometry (GC-MS/MS), using electron capture negative ion chemical ionisation. The isomers of oestradiol were quantified in both blank and post-administration urine and serum samples, with a view to setting action/threshold levels for these compounds, to allow discrimination between normal samples and samples from animals treated with growth promoting ear implants. A non-parametric statistical assessment of the data resulted in proposed action levels (with a false positive probability of 1 in 1000) of 1.6 and 2.7 ng mL(-1) for 17alpha-oestradiol, in male and female urine, respectively, and 40 and 44 pg mL(-1) for 17beta-oestradiol, in male and female urine, respectively. An action level of 20 pg mL(-1) was proposed for 17alpha- and 17beta-oestradiol in male serum. In female serum the proposed action levels were 40 and 20 pg mL(-1) for 17alpha- and 17beta-oestradiol, respectively.     

A solid phase extraction method for the screening and determination of pyrethroid metabolites and organochlorine pesticides in human urine.

A screening method has been developed for the determination of 23 organochlorine pesticides (OCPs) and 3-pyrethroid metabolities [cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid, cis-3-(2,2-dibromovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid and 3-phenoxybenzoic acid] from human urine. OCPs were directly detected in urine samples while pyrethroid metabolites required acid-induced hydrolysis to convert their conjugates into free acids; all compounds were then cleaned-up/preconcentrated using solid phase extraction. Determination and quantitation was achieved by gas chromatography with a mass spectrometer detector operating in selected ion monitoring mode. Limits of detection varied between 0.1 and 0.3 ng/mL with linear ranges from 0.3 to 700 ng/mL; the precision of the method was high (4.3-7.2%). Recoveries of all analytes from urine samples fortified at levels of 30 ng/mL for each OCP and 15 ng/mL for each pyrethroid metabolite ranged from 88 to 101% (captan gave the lowest recovery). The results obtained from the analysis of real urine samples show the suitability of the proposed method for monitoring people exposed to organochlorine and pyrethroid pesticides.     

Study on the ternary mixed ligand complex of palladium(II)-aminophylline-fluorescein sodium by resonance Rayleigh scattering, second-order scattering and frequency doubling scattering spectrum and its analytical application

The interaction between palladium(II)-aminophylline and fluorescein sodium was investigated by resonance Rayleigh scattering, second-order scattering and frequency doubling scattering spectrum. In pH 4.4 Britton-Robinson (BR) buffer medium, aminophylline (Ami) reacted with palladium(II) to form chelate cation([Pd(Ami)]2+), which further reacted with fluorescein sodium (FS) to form ternary mixed ligand complex [Pd(Ami)(FS)2]. As a result, resonance Rayleigh scattering (RRS), second-order scattering (SOS) and frequency doubling scattering spectrum (FDS) were enhanced. The maximum scattering wavelengths of [Pd(Ami)(FS)2] were located at 300 nm (RRS), 650 nm (SOS) and 304 nm (FDS). The scattering intensities were proportional to the Ami concentration in a certain range and the detection limits were 7.3 ng mL(-1) (RRS), 32.9 ng mL(-1) (SOS) and 79.1 ng mL(-1) (FDS), respectively. Based on it, the new simple, rapid, and sensitive scattering methods have been proposed to determine Ami in urine and serum samples. Moreover, the formation mechanism of [Pd(Ami)(FS)2] and the reasons for enhancement of RRS were fully discussed.     

Direct determination of chlorpyrifos and its main metabolite 3,5, 6-trichloro-2-pyridinol in human serum and urine by coupled-column liquid chromatography/electrospray-tandem mass spectrometry

A rapid and sensitive automated coupled-column liquid chromatography/electrospray tandem mass spectrometry (LC/LC/ES-MS/MS) method has been developed for the quantitation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) in both human serum and urine. Human serum was first protein precipitated with acetonitrile, while urine was directly injected into the coupled-column system. A 10 microL aliquot was then analyzed using as first separation column a Discovery C18 5 microm 50 x 2.1 mm; the fraction containing the analyte was transferred on-line to the second column consisting of a ABZ+ 5 microm 100 x 2.1 mm, which was connected to the electrospray source (Z-spray) of a Quattro LC triple-quadrupole instrument. Chlorpyrifos was detected in positive ion mode using four multi reaction monitoring (MRM) transitions while TCP was measured in negative ion mode using three pseudo-MRM transitions. The clean-up performed by the coupled-column approach avoids the use of an internal standard for the correct quantitation of both analytes, and the highly automated procedure renders a sample throughput of more than 100 samples per day. Both compounds can be determined using the same set-up, the only difference in the procedure being the composition of the first mobile phase. The method has proved to be fast, reliable and sensitive, yielding calibration curves for both analytes with correlation coefficients greater than 0.9995. The repeatability and reproducibility at 5 and 50 ng/mL was lower than 8%. The accuracy and precision were evaluated by means of recovery experiments from fortified serum (5-50 ng/mL) and urine (1-10 ng/mL) samples, obtaining satisfactory recoveries for both compounds (87-113% in serum, and 98-109% in urine), with coefficients of variation (CVs) less than 10%. The detection limits were similar for chlorpyrifos and metabolite: 1.5 ng/mL in serum, and 0.5 ng/mL in urine, where no sample handling took place. The validated procedures provide excellent tools for the specific assessment of occupational exposure to the organophosphorus pesticide chlorpyrifos, throughout the analysis of both human serum and urine, and it is more selective and sensitive than the current assay based on the measurement of the decrease in the cholinesterase activity.     

Determination of levodopa and biogenic amines in urine samples using high-performance liquid chromatography

A chromatographic system is developed for the separation and determination of levodopa, biogenic amines, and their metabolites from the catecholamines group: dopamine (DA), epinephrine (E), normetanephrine (NMN), metanephrine (MN), 3,4-dihydroxyphenylacetic acid (DOMA), 3-metoxy-4-hydroxyphenyl-glycol (MHPG), and homovanillic acid (HVA); and indoloamines group: serotonin (5HT) and 5-hydroxyindole-3-acetic acid (5HIAA) in urine. The limit of detection (LOD) and limit of quantitation (LOQ) are determined for all compounds with signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD 10 (ng/mL) and LOQ 30 (ng/mL) are determined for L-DOPA, DOMA, E, NMN, DA, MN, and MHPG, as well as LOD 8 (ng/mL) and LOQ 24 (ng/mL) for HVA, 5HT, and 5HIAA. A fluorescence detector is used. Gradient elution with acetate buffer (pH=4.66) with methanol is applied. In urine samples from patients treated with levodopa, the following concentrations (microg/mL) of analytes are determined: L-DOPA 3.73-46.80, DOMA 1.43-28.43, E 0.83-13.57, NMN 2.58-8.81, DA 24.07-62.11, MN 0.89-66.20, MHPG 6.72-63.64, 5HT 22.96-95.27, 5HIAA 1.45-14.77, and HVA 0.21-15.07.     

Simultaneous determination of tyrosine and dopamine in urine samples using excitation-emission matrix fluorescence coupled with second-order calibration

A "green" and quick analytical method for complex compounds was developed for simultaneous determination of tyrosine (Tyr) and dopamine (DA) in urine samples in this paper. The three-way responsive data recorded by excitation-emission matrix fluorescence (EEM) spectrometer was analyzed using second-order calibration methods based on both parallel factor analysis (PARAFAC) and selfweighted alternating trilinear decomposition (SWATLD) algorithms. The EEM spectra of the analytes were overlapped with the background in urine samples. However the second-order advantage of both PARAFAC and SWATLD methods was exploited, even in the presence of unknown interferences and the satisfactory results can be obtained. Furthermore, the linear ranges of Tyr and DA were determined to be 0.042-6.42 μg/mL and 0.18-4.43 μmg/mL, respectively, and the accuracies of both methods were validated by the analytical figures of merit (FOM).     

Simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene using high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry

A simple and rapid method using reversed-phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the simultaneous determination of the urinary metabolites of benzene, toluene, xylene and styrene in human urine specimens and standard solutions is described. A hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer was compared for the determination of metabolite of aromatic solvents in urine samples. The metabolites selected were: trans,trans-muconic acid, hippuric acid, o-, m- and p-methylhippuric acid and phenylglyoxylic acid. The compounds were well separated from each other on narrow-bore 1-mm i.d. reversed-phase LC C-18 columns. Average recoveries for loading 100 microL of urine samples varied from 88-110% and the quantification limits were less than 30 ng/mL for each analyte (3 ng/mL for trans,trans-muconic acid). The qualitative information obtained (mass accuracy, resolution and full-scan spectra) with the QqTOF mass spectrometer allows a secure identification of analytes in biological matrices.     

Simultaneous Determination of Hydrochlorothiazide and Reserpine in Human Urine by LC with a Simple Pre-Treatment

A simple, selective and sensitive reversed-phase high performance liquid chromatography method for simultaneous analysis of hydrochlorothiazide and reserpine in human urine was developed and subjected to primary pharmacokinetic study. After a simple protein precipitation using methanol and extraction with ethyl acetate, the analytes were separated on an Elite C(18) column at a flow rate of 0.8 mL min(-1). The mobile phase was composed of acetonitrile (A) and 0.2% ammonium chloride solution (B) for a gradient elution starting at A:B at 30:70, v/v for 0~6 min, linearly raising the percent of A from 30% to 50% (6~9 min) and ending at 50:50, v/v (9~25 min). The standard curves were linear over the range of 0.05-20 μg mL(-1) for hydrochlorothiazide and 0.02-5.0 μg mL(-1) for reserpine, respectively (r > 0.999). The limit of detection (LOD) and the limit of quantification (LOQ) were 5.5 ng mL(-1) and 18.2 ng mL(-1) for hydrochlorothiazide, and 7.1 ng mL(-1) and 23.6 ng mL(-1) for reserpine, respectively. The recoveries for both analytes were above 89.0±1.35%. The intra-day and inter-day precision for hydrochlorothiazide were less than 1.91% and 1.38%, and those for reserpine were below 1.61% and 2.64%, respectively. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy, and it was employed successfully for the simultaneous determination of hydrochlorothiazide and reserpine in human urine samples.     

Liquid chromatographic high-throughput analysis of the new ultra-short acting hypnotic 'HIE-124' and its metabolite in mice serum using a monolithic silica column

For the first time, a fast, high-performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the new ultra-short hypnotic HIE-124 and its metabolite in mice serum. Each compound, together with carbamazepine (internal standard) was extracted from the serum matrix using liquid-liquid extraction (LLE). Chromatographic resolution of the analytes was performed on a Chromolith Speed Rod monolithic silica column (100 mm × 4.6 mm i.d.) under isocratic conditions using a mobile phase of 65:35 (v/v), 20 mM phosphate buffer (pH 7.0 adjusted with phosphoric acid)-acetonitrile. The elution of the analytes were monitored at 240 nm and conducted at ambient temperature. Because of high column efficiency the mobile phase was pumped at a flow rate of 2.5 mL min(-1). The total run time of the assay was 2 min. The method was validated over the range of 60-2000 ng mL(-1) for HIE-124 and 200-1600 ng mL(-1) for the metabolite (r(2) = 0.99). The limit of detection (LOD) for HIE-124 and its metabolite were 20 ng mL(-1) and 65 ng mL(-1), respectively. The proposed method was validated in compliance with ICH guidelines, in terms of accuracy, precision, limits of detection and quantitation and other aspects of analytical validation. The developed method could be used for the trace analyses of HIE-124 and its metabolite in serum and was finally used for the pharmacokinetic study investigation of HIE-124 in mice serum.     

Determination of salbutamol using on-line solid-phase extraction and sequential injection analysis. Comparison of chemiluminescence and fluorescence detection

Determination of salbutamol using sequential injection analysis (SIA) with chemiluminescence and fluorescence detection has been devised. The chemiluminescence signal was emitted during the oxidation of salbutamol by potassium permanganate in sulfuric acid medium. Sodium polyphosphate was used as chemiluminescence enhancer. The fluorescence signal (excitation wavelength 230 nm) was also measured in sulfuric acid medium. Both detection techniques were compared with respect to the application of the methods to the determination of salbutamol in biological materials. The sample pre-treatment takes place directly in the SIA system, when salbutamol is adsorbed on the solid-phase (Baker-carboxylic acid) microcolumn integrated into the system. Sulfuric acid serves both as the reagent and the eluent. The lab-made SIA system consisted of a 2.5-mL Cavro syringe pump, ten-port Vici Valco selection valve and Spectra-Physics FS 970 fluorescence detector, which was lab-modified for chemiluminescence detection. The system was controlled by a PC using originally compiled LabVIEW-supported software. Concentrations, volumes of reagents and flow rates were optimised by a simplex method. Salbutamol was determined in the linear range 0.05-10 microg mL(-1) (RSD 1.53%), with the detection limit (3 sigma) 0.03 microg mL(-1) and sample throughput of 42 samples per hour with chemiluminescence detection in standard solutions. The fluorescence detection enabled the determination of salbutamol in standard solutions in the linear range 0.5-100 microg mL(-1) (RSD 2.69%), with the detection limit 0.2 microg mL(-1) and sample throughput of 24 h(-1). The proposed methods were applied to the determination of salbutamol in human serum and urine. However, serum is a very complicated matrix and the SIA-SPE analysis did not provide satisfactory results. It was possible to determine salbutamol in human urine using this technique. Better recovery was achieved with fluorescence detection.     

Cation-selective exhaustive injection and sweeping MEKC for direct analysis of methamphetamine and its metabolites in urine

Direct analysis of methamphetamine, amphetamine, and p-hydroxymethamphetamine in urine was achieved by cation-selective exhaustive injection and sweeping micellar EKC. A bare fused-silica capillary (40 cm, 50 microm id) was filled with phosphate buffer (80 mM, pH 3, containing 20% ACN). Then a high-conductivity buffer (100 mM phosphate, pH 3; 6.9 kPa for 2.5 min) was injected. Samples were loaded using electrokinetic injection (10 kV, 600 s) which created long zones of cationic analytes. To enhance sensitivity by sweeping, the stacking step was performed using a phosphate buffer (50 mM, pH 3, containing 20% ACN and 100 mM SDS) at -20 kV before separation by MEKC. This method was capable of detecting the analytes at ppb levels. The calibration plots were linear (r(2) >or= 0.9948) over a range of 100-5000 ng/mL for methamphetamine, and 100-2000 ng/mL for amphetamine and p-hydroxymethamphetamine. The LODs (S/N = 3) were 20 ng/mL for methamphetamine, and 15 ng/mL for amphetamine and p-hydroxymethamphetamine. The method was applied to analysis of 14 urine samples of addicts and is suitable for screening suspected samples for forensic purposes. The results showed good agreement with fluorescence polarization immunoassay and GC-MS.     

High-performance liquid chromatographic determination of lansoprazole and its metabolites in human serum and urine.

A simple and sensitive high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous determination of lansoprazole and its metabolites in human serum and urine. The analytes in serum or urine were extracted with diethyl ether-dichloromethane (7:3, v/v) followed by evaporation, dissolution and injection into a reversed-phase column. The recoveries of authentic analytes added to serum at 0.05-2 micrograms/ml or to urine at 1-20 micrograms/ml were greater than 88%, with the coefficients of variation less than 7.1%. The minimum determinable concentrations of all analytes were 5 ng/ml in serum and 50 ng/ml in urine. The method was successfully applied to a pharmacokinetic study of lansoprazole in human.     

Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.     

Sensitive quantification of clozapine and its main metabolites norclozapine and clozapine-<Emphasis Type="Italic">N</Emphasis>-oxide in serum and urine using LC-MS/MS after simple liquid–liquid extraction work-up

An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid–liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.     

Mixed hemimicelles SPE based on CTAB-coated Fe3O4/SiO2 NPs for the determination of herbal bioactive constituents from biological samples

In this paper, a solid-phase extraction (SPE) method based on mixed hemimicelles of cetyltrimethyl ammonium bromide (CTAB) on silica-coated magnetic nanoparticles (MNPs) is developed for extraction and preconcentration of compounds from the biological samples. We selected rhein and emodin which are the major active anthraquinones of rhubarb as model analytes. A high performance liquid chromatography-fluorescence detection (HPLC/FLD) method was developed for the determination of rhein and emodin in urine and serum samples. The main factors influencing the extraction efficiency including the amount of surfactant, the concentration of MNPs, the shaking time and the desorption ability of organic solvents were investigated and optimized. No interferences were caused by proteins or endogenous compounds in urine and serum samples. Good linearities (r² > 0.9995) for all calibration curves were obtained, and the limits of detection (LODs) for rhein and emodin were 0.2 and 0.5 ng/mL in urine samples and 7 and 10 ng/mL in serum samples, respectively. Satisfactory recoveries (92.76-109.90% and 97.53-107.72% for rhein and emodin) in the biological matrices were achieved.