In vivo solid-phase microextraction for single rodent pharmacokinetics studies of carbamazepine and carbamazepine-10,11-epoxide in mice

The use of solid-phase microextraction (SPME) for in vivo sampling of drugs and metabolites in the bloodstream of freely moving animals eliminates the need for blood withdrawal in order to generate pharmacokinetics (PK) profiles in support of pharmaceutical drug discovery studies. In this study, SPME was applied for in vivo sampling in mice for the first time and enables the use of a single animal to construct the entire PK profile. In vivo SPME sampling procedure used commercial prototype single-use in vivo SPME probes with a biocompatible extractive coating and a polyurethane sampling interface designed to facilitate repeated sampling from the same animal. Pre-equilibrium in vivo SPME sampling, kinetic on-fibre standardization calibration and liquid chromatography–tandem mass spectrometry analysis (LC–MS/MS) were used to determine unbound and total circulating concentrations of carbamazepine (CBZ) and its active metabolite carbamazepine-10,11-epoxide (CBZEP) in mice (n = 7) after 2 mg/kg intravenous dosing. The method was linear in the range of 1–2000 ng/mL CBZ in whole blood with acceptable accuracy (93–97%) and precision (<17% RSD). The single dose PK results obtained using in vivo SPME sampling compare well to results obtained by serial automated blood sampling as well as by the more conventional method of terminal blood collection from multiple animals/time point. In vivo SPME offers the advantages of serial and repeated sampling from the same animal, speed, improved sample clean-up, decreased animal use and the ability to obtain both free and total drug concentrations from the same experiment.     

Routine monitoring of carbamazepine and carbamazepine-10,11-epoxide in plasma by high-performance liquid chromatography using 10-methoxycarbamazepine as internal standard

Carbamazepine and carbamazepine-10,11-epoxide were separated by high-performance liquid chromatography (HPLC) with acetonitrile-water as mobile phase, and detection was effected by UV absorption at 215 nm with a total retention time of less than 10 min. Plasma samples were extracted with dichloromethane and 4 M sodium hydroxide, and 10-methoxy-carbamazepine was added as internal standard. Other commonly used anticonvulsant drugs present in plasma showed no significant interference. The within-batch coefficient of variation for carbamazepine was 4.9% and carbamazepine-10,11-epoxide 5.9%. Between-batch coefficients of variation were 3.7% and 5.3%, respectively. Mean recovery for carbamazepine was 100.2% and for carbamazepine-10,11-epoxide 100.6%. This HPLC method was compared with both an enzyme immunoassay procedure (EMIT) and a gas-liquid chromatographic (GLC) method. Correlation coefficient between HPLC/EMIT for carbamazepine was 0.983, HPLC/GLC carbamazepine 0.988 and HPLC/GLC carbamazepine-10,11-epoxide 0.981.     

Simultaneous quantitation of salivary carbamazepine, carbamazepine-10,11-epoxide, phenytoin and phenobarbitone by high-performance liquid chromatography

A rapid, sensitive and accurate high-performance liquid chromatographic method for the simultaneous quantitation of phenobarbitone, phenytoin, carbamazepine and carbamazepine-10,11-epoxide in saliva is described. Only small volumes of saliva (100 microliters) are required. Separation of the drugs is achieved by reversed-phase chromatography on a Nova-Pak C18 column, with a mobile phase of acetonitrile-phosphate buffer at a flow-rate of 2.0 ml/min. Detection is effected by ultra-violet absorption at 215 nm. The total run time is under 12.5 min per assay. A precipitation but no extraction step is involved, simplifying the assay method. Salivary concentrations in the range 0.25-25 micrograms/ml for carbamazepine, 0.5-20 micrograms/ml for phenytoin and phenobarbitone and 0.4-20 micrograms/ml for carbamazepine-10,11-epoxide can be measured. Recovery varies from 94 to 108%. The method has been used for routine measurements of anticonvulsants in saliva collected daily from patients with intractable epilepsy.     

Quantitation of plasma and biliary cefpiramide concentrations in human samples using high-performance liquid chromatography

Cefpiramide is frequently used to treat biliary infections. However, no bioanalytical method has been validated to quantitate cefpiramide in human samples, particularly in bile. Therefore, this study was conducted to develop a simple, selective and validated high-performance liquid chromatographic method to determine cefpiramide in human plasma and bile. A protein precipitation procedure was used to extract cefpiramide and cefoperazone (internal standard, IS) from 200 μl of plasma and bile. Utilizing a Capcell Pak C₁₈ column (4.6 × 250 mm), cefpiramide and IS were separated using the timed-gradient mobile phase consisting of 0.1 m sodium acetate (pH 5.2) and acetonitrile at a flow rate of 1 ml/min with photodiode array detector (wavelength set at 273 nm). The calibration curves showed linearity at concentrations ranging from 1 to 150 μg/ml in both plasma and bile (r² > 0.999). The within- and between-run coefficients of variation (CVs) for plasma samples were 0.570–4.43 and 1.10–2.76%, respectively; for bile samples, the within- and between-day precision (CV) was 0.814–6.34 and 2.05–4.00%, respectively. Our newly developed bioanalytical method was successfully employed to quantify cefpiramide concentrations in both plasma and bile at multiple time points in patients with acute cholangitis.     

Determination of cisapride in human plasma by high-performance liquid chromatography with fluorescence detection

Summary A new sensitive HPLC-FLD method has been developed and validated for the determination of cisapride in human plasma for a bioequivalence study. A gradient method was used to remove late-eluting plasma components of no interest. The separation was performed on a Li-ChroCART 250-4 Purospher RP-18 (5 μm particle) analytical column fitted with a LiChroCART 4-4 Purospher RP-18 endcapped (5 μm particle) guard column. The excitation and emission wavelengths were 295 and 350 nm during fluorescence detection. The calibration plot was linear in the range of 5–200 ng mL⁻¹. A demethoxy analogue of cisapride was used as internal standard.     

Analysis of sulfamerazine in pond water and several fishes by high-performance liquid chromatography using molecularly imprinted solid-phase extraction

The synthesis and evaluation of a molecularly imprinted polymer (MIP) used as a selective solid-phase extraction sorbent and coupled to high-performance liquid chromatography (HPLC) for the efficient determination of sulfamerazine (SMR) in pond water and three fishes are reported. The polymer was prepared using SMR as the template molecule, methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the crosslinking monomer in the presence of tetrahydrofuran as the solvent. The SMR-imprinted polymers and nonimprinted polymers were characterized by FT-IR and static adsorption experiments. The prepared SMR-imprinted material showed a high adsorption capacity, significant selectivity and good site accessibility. The maximum static adsorption capacities of the SMR-imprinted and nonimprinted materials for SMR were 108.8 and 79.6 mg g⁻¹, respectively. The relative selectivity factor of this SMR-imprinted material was 1.6. Several parameters influencing the solid-phase extraction process were optimized. Finally, the SMR-imprinted polymers were used as the sorbent in solid-phase extraction to determine SMR in pond water and three fishes with satisfactory recovery. The average recoveries of the MIP-SPE method were 94.0% in ultrapure water and 95.8% in pond water. Relative standard deviations ranging from 0.3% to 5.2% in MIP were acquired. The results for the SMR concentrations in crucian, carp and wuchang fish were 66.0, 127.1 and 51.5 ng g⁻¹, respectively. The RSDs (n = 5) were 3.51%, 0.53% and 5.08%, respectively. The limit of detection (LOD) for SMR was 1 ng g⁻¹ and the limit of quantitation (LOQ) was 3.5 ng g⁻¹.     

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Summary Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns.Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 m particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (7525, v:v) and switched to a CN column (10 cm long, 5 m particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 m particle diameter) with acetonitrile-0.088 M acetate buffer (7525, v:v) as mobile phase.Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (_ex= 225 nm; _em > 320 nm).Presented at the 14th International Symposium on Chromatography London, September, 1982     

A new high-speed hollow fiber based liquid phase microextraction method using volatile organic solvent for determination of aromatic amines in environmental water samples prior to high-performance liquid chromatography

A new and fast hollow fiber based liquid phase microextraction (HF-LPME) method using volatile organic solvents coupled with high-performance liquid chromatography (HPLC) was developed for determination of aromatic amines in the environmental water samples. Analytes including 3-nitroaniline, 3-chloroaniline and 4-bromoaniline were extracted from 6 mL basic aqueous sample solution (donor phase, NaOH 1 mol L⁻¹) into the thin film of organic solvent that surrounded and impregnated the pores of the polypropylene hollow fiber wall (toluene, 20 μL), then back-extracted into the 6 μL acidified aqueous solution (acceptor phase, HCl 0.5 mol L⁻¹) in the lumen of the two-end sealed hollow fiber. After the extraction, 5 μL of the acceptor phase was withdrawn into the syringe and injected directly into the HPLC system for the analysis. The parameters influencing the extraction efficiency including the kind of organic solvent and its volume, composition of donor and acceptor phases and the volume ratio between them, extraction time, stirring rate, salt addition and the effect of the analyte complexation with 18-crown-6 ether were investigated and optimized. Under the optimal conditions (donor phase: 6 mL of 1 mol L⁻¹ NaOH with 10% NaCl; organic phase: 20 μL of toluene; acceptor phase: 6 μL of 0.5 mol L⁻¹ HCl and 600 m mol L⁻¹ 18-crown-6 ether; pre-extraction and back-extraction times: 75 s and 10 min, respectively; stirring rate: 800 rpm), the obtained EFs were between 259 and 674, dynamic linear ranges were 0.1-1000 μg L⁻¹ (R > 0.9991), and also the limits of detection were in the range of 0.01-0.1 μg L⁻¹. The proposed procedure worked very well for real environmental water samples with microgram per liter level of the analytes, and good relative recoveries (91-102%) were obtained for the spiked sample solutions.     

Simultaneous determination of rifampicin and sulbactam in mouse plasma by high-performance liquid chromatography

A simple and rapid high-performance liquid chromatographic (HPLC) method with ultraviolet detection has been developed and validated for the simultaneous determination of rifampicin and sulbactam in mouse plasma. Plasma samples were deproteinized with acetonitrile and separated by HPLC on a RP-18 (125 x 4 mm, 5 microm) column and gradient elution with potassium dihydrogen phosphate solution (pH 4.5; 50 mm) and acetonitrile at a flow-rate of 1.0 mL/min. Rifampicin and sulbactam were monitored at 230 nm and confirmed by means of their UV spectra using a diode-array detector. The method was linear at plasma levels from 1 to 100 microg/mL for rifampicin and from 5 to 200 microg/mL for sulbactam. The limits of quantification were 0.6 microg/mL for rifampicin and 4.2 microg/mL for sulbactam. The intra- and inter-day precisions of the method (RSD) were lower than 5% for both compounds. Average recoveries of rifampicin and sulbactam from mice plasma were 98.2 and 89.3%, respectively. The developed method was successfully applied to the determination of the pharmacokinetic profile of both compounds in mice.     

Determination of carbamazepine and carbamazepine-10,11-epoxide in human serum and plasma by micellar electrokinetic capillary chromatography in the absence of electroosmosis

Therapeutic drug monitoring of carbamazepine (CBZ), a widely used antiepileptic drug, is required for optimization of pharmacotherapy with this drug and for assessment of the patient's compliance to therapy. The suitability of employing micellar electrokinetic capillary chromatography (MEKC) in the absence of electroosmosis for the determination of CBZ and its main metabolite carbamazepine-10,11-epoxide (CBZE) in extracts of human serum and plasma is reported. Using micelles formed by dodecyl sulfate, analyses performed in untreated fused-silica capillaries at acidic pH and in commercially available coated capillaries under application of reversed polarity are compared. Uncoated and polyvinyl alcohol coated capillaries proved to be unsuitable for this purpose, whereas capillaries coated with linear polyacrylamide and N-acryloylaminoethoxyethanol and operated at pH 7.6 are shown to provide high-quality and reliable data on a short time scale. Assay performance is discussed via statistical analysis of the data produced from a set of quality control sera that contain up to 14 different drugs and via analysis of patient samples. Intraday and interday imprecision data for concentrations between 4.0 and 84 microM are demonstrated to be < 10%. Run times are shown to be < 50% compared to those observed in conventional MEKC at alkaline pH (i.e., in the presence of electroosmosis).     

Simultaneous determination of amiodarone and its metabolite desethylamiodarone by high-performance liquid chromatography with chemiluminescent detection

A novel method was developed for the determination of amiodarone and desethylamiodarone by high-performance liquid chromatography (HPLC) coupled with chemiluminescent (CL) detection. The procedure is based on the post-column photolysis of the analytes into photoproducts which are active in the tris(2,2′-bipyridyl)ruthenium(III) [Ru(bpy)₃³⁺] CL system. Ru(bpy)₃³⁺ was on-line generated by photo-oxidation of the Ru(II) complex in the presence of peroxydisulfate. The separation was carried out on a Mediterranea C₁₈ column with isocratic elution using a mixture of methanol and 0.017 mol L⁻¹ ammonium sulfate buffer of pH 6.8. Under the optimum conditions, analytical curves, based on standard solutions, were linear over the range 0.1-50 μg mL⁻¹ for amiodarone and 0.5-25 μg mL⁻¹ for desethylamiodarone. The detection limits of amiodarone and desethylamiodarone were 0.02 and 0.11 μg mL⁻¹, respectively. Intra- and inter-day precision values of 0.9% relative standard deviation (R.S.D.) (n = 10) and 1.6% R.S.D. (n = 15), respectively, were obtained. The method was applied successfully to the determination of these compounds in serum and pharmaceutical formulations.     

Determination of fluoxetine and its N-desmethyl metabolite in human plasma by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic method has been developed for the simultaneous determination of the antidepressant fluoxetine and its active metabolite norfluoxetine in human plasma using paroxetine as internal standard. After liquid-liquid extraction, the compounds were separated on a C18 column using as mobile phase acetonitrile and 40 mM potassium dihydrogen phosphate buffer (pH 2.3) in the ratio 31:69 (v/v). The quantification of fluoxetine and norfluoxetine was made by fluorescence detection at Ex/Em 230/312 nm. The assay for each analyte was linear over the ranges 1-39 and 0.9-36 ng/ml, respectively. For both compounds intra- and inter-day accuracy and precision ranged between −7.9-12.4 and 0.7-14.7%, respectively. The method was applied to the analysis of plasma samples obtained from healthy subjects treated with one single oral dose of 40 mg fluoxetine.     

Analysis of meloxicam by high-performance liquid chromatography with cloud-point extraction

A simple cloud-point extraction method for the determination of meloxicam in human serum was developed. Meloxicam was extracted from serum sample after adding 1 mL of 3% (v/v) Triton X-114 aqueous solution in the presence of 1M HCl and 60 mg NaCl. The meloxicam, present in the surfactant-rich phase, was enriched again with acetonitrile. Tenoxicam was used as the external standard. The separation was achieved on a C18 analytical column with a mobile phase consisting of aqueous acetic acid (1%, v/v) and acetonitrile (54:46, v/v). UV detection was performed at 360 nm. The response was linear over the range 45–2000 ng mL⁻¹ in human serum, and intra- and interday precisions of less than 15.0% were obtained. The relative error was within ±3.0%. The recoveries of meloxicam were larger than 92.0%. The method was compared with liquid–liquid extraction. The results showed that the new method has a considerable LOQ and higher recoveries but poorer precision than liquid–liquid extraction, which exhibited poor recoveries of less than 86.0%, precisions of less than 5.0% and relative errors of less than 7.0%. The method was used for the determination of meloxicam in healthy human volunteers.     

Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent

A sensitive and selective high-performance liquid chromatographic method has been developed and validated for the determination of nateglinide in human plasma. Nateglinide and the internal standard, undecylenic acid, were extracted from plasma by liquid-liquid extraction using a mixture of ethyl acetate-diethyl ether, 50:50 (v/v). Pre-column derivatization reaction was performed using a coumarin-type fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The derivatization proceeded in acetone in the presence of potassium carbonate and catalyzed by 18-crown-6 ether. The fluorescent derivatives were separated under isocratic conditions on a Hypersil BDS-C8 analytical column (250.0 mm × 2.1 mm i.d., particle size 5 μm) with a mobile phase that consisted of 65% acetonitrile in water and pumped at a flow rate of 0.50 mL min⁻¹. The excitation and emission wavelengths were set at 345 and 435 nm, respectively. The assay was linear over a concentration range of 0.05-16.00 μg mL⁻¹ for nateglinide with a limit of quantitation of 0.05 μg mL⁻¹. Quality control samples (0.05, 4.50 and 16.00 μg mL⁻¹) in five replicates from five different runs of analysis demonstrated intra-assay precision (%coefficient of variation <6.8%), inter-assay precision (%coefficient of variation <1.6%) and an overall accuracy (%relative error) less than −3.4%. The method can be used to quantify nateglinide in human plasma covering a variety of pharmacokinetic or bioequivalence studies.     

The determination of atmospheric concentrations of the active ingredients of pesticide formulations by high-performance liquid chromatography

Summary Methods of sampling atmospheres contaminated by pesticides in factory and agricultural environments, and subsequent analysis by HPLC, are discussed. Air sampling is carried out using porous polymer or filter collection media, usually a 100 dm³ air volume is suitable. Detection limits with ultra-violet detection are in the range 0.1 to 10 g m^–3.Presented at the 14th International Symposium on Chromatography London, September, 1982     

Analysis of triazine in water samples by solid-phase microextraction coupled with high-performance liquid chromatography

Solid-phase microextraction (SPME) coupled with high-performance liquid chromatography (HPLC) for the determination of triazine is described. Carbowax/templated resin (CW/TPR, 50 μm), polydimethylsiloxane/divinylbenzene (PDMS/DVB, 60 μm), polydimethylsiloxane (PDMS, 100 μm), and polyacrylate (PA, 85 μm) fibers were evaluated for extraction of the triazines. CW/TPR and PDMS/DVB fibers were selected for further study. Several parameters of the extraction and desorption procedure were studied and optimized (such as types of fibers, desorption mode, desorption time, compositions of solvent for desorption, soaking periods and the flow rate during desorption period, extraction time, temperature, pH, and ionic strength of samples). Both CW/TPR and PDMS/DVB fibers are acceptable; a simple calibration-curve method based on simple aqueous standards can be used. The linearity of this method for analyzing standard solution has been investigated over the range 5-1000 ng mL⁻¹ for both PDMS/DVB and CW/TPR fibers. All the correlation coefficients in the range 5-1000 ng mL⁻¹ were better than 0.995 except Simazine and Atratone by CW/TPR fiber. The R.S.D.s range from 4.4% to 8.8 % (PDMS/DVB fiber) and from 2.4% to 7.2% (CW/TPR fiber). Method-detection limits (MDL) are in the range 1.2-2.6 and 2.8-3.4 ng mL⁻¹ for the two fibers. These methods were applied to the determination of trazines in environmental water samples (lake water).     

Determination of methoxsalen in dosage forms by high-performance liquid chromatography

Summary A new, rapid, sensitive, and specific method for the determination of methoxsalen in dosage forms using HPLC has been developed. methoxsalen is extracted in chloroform, evaporated on a water bath, and the residue is redissolved in ethanol. A standard solution of khellin (internal standard) in ethanol is added, and injected. A plot of peak height ratio (methoxsalen/internal standard) vs. concentration of methoxsalen gave a straight line (r=0.998). The column used was a stainless steel, 3.8 mm×30 cm, and the mobile phase was methanol: water (6040) at a flow rate of 2 cm³/min. Retention times for methoxsalen and khellin were 3.45 and 9.6 min, respectively. This method was found superior to the spectrophotometric assay in that no interference was encountered from structurally similar compounds or from coloring agents used in some commercial methoxsalen products.     

Solidified floating organic drop microextraction combined with high performance liquid chromatography for the determination of carbamazepine in human plasma and urine samples

Solidified floating organic drop microextraction (SFODME) in combination with high performance liquid chromatography was used for separation/preconcentration and determination of carbamazepine (CBZ) in human plasma and urine samples. Parameters that affect the extraction efficiency such as the type and volume of extraction solvent, ionic strength, sodium hydroxide concentration, stirring rate, sample volume and extraction time, were investigated and optimized. Under the optimum conditions (extraction solvent, 40 μL of 1-undecanol; sodium hydroxide concentration, 1 mol/L; temperature, 50 ℃; stirring speed, 400 r/min; sample volume, 8 mL; sodium chloride concentration, 3% (w/v) and extraction time, 60 min) the calibration curve was found to be linear in the mass concentration range of 0.4-700.0 μg/L. The limit of detection (LOD) was 0.1 μg/L and the relative standard deviation (RSD) for six replicate extraction and determination of carbamazepine at 100 μg/L level was found to be 4.1%. The method was successfully applied to the determination of CBZ in human plasma and urine samples.     

高效液相色谱法准确测定血清中的尿酸

建立了准确测定血清中尿酸含量的高效液相色谱方法,血清样品利用乙腈沉淀蛋白,过滤后直接进样测定。所采用的色谱柱为Inertsil ODS-SP(4.6mm i.d.×250 mm,5μm),柱温25℃,流动相体系为10 mmol/L乙酸铵(pH 4.5),流速为1.0 mL/min,紫外检测波长为280 nm。线性范围12.5~150μg/g,回收率为99.79%~100.5%。本文采用乙酸铵缓冲体系,对环境的污染小,同时也为建立液相色谱-质谱法测定尿酸奠定了良好的基础。