Measurement and pharmacokinetics of unbound 20(S)-camptothecin in rat blood and brain by microdialysis coupled to microbore liquid chromatography with fluorescence detection

To characterize the pharmacokinetics of protein-free camptothecin in blood and brain we implanted microdialysis probes into the jugular vein and striatum of rats for unbound drug sampling and determination. Camptothecin (2 or 5 mg/kg, i.v., n=6) was then administered from the femoral vein, and microdialysates were collected from blood and brain of both sites and assayed by a validated microbore scale high-performance liquid chromatographic method. The mobile phase consisted of methanol–100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5) with a flow-rate 0.05 ml/min. The fluorescence response for camptothecin was observed at excitation and emission wavelengths of 360 and 440 nm, respectively. Pharmacokinetic parameters were calculated from the corrected data for dialysate concentrations of camptothecin versus time. The results suggest that the pharmacokinetics of unbound camptothecin in blood and brain can be fitted best to a two- and one-compartment model, respectively. Camptothecin rapidly entered the extracellular fluid of brain striatum at 10 min following camptothecin administration.     

On-line microdialysis coupled with microbore liquid chromatography with ultraviolet detection for continuous monitoring of free cefsulodin in rat blood

A microdialysis method followed by a microbore liquid chromatographic ultraviolet detection procedure has been performed for the assay of unbound cefsulodin in rat blood. A microdialysis probe was inserted into the jugular vein for blood sampling. This method involves an on-line design for submitting dialysate into the liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol-100 mM monosodium phosphoric acid (10:90, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 265 nm. Microdialysis probes, being laboratory-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity as well as intra- and inter-day variabilities. The method was then applied to pharmacokinetics profiling of cefsulodin in the blood following intravenous administration of cefsulodin (20 mg/kg) in rats. Pharmacokinetics were calculated from the corrected data for dialysate concentrations of cefsulodin versus time. Based on pharmacokinetic calculation, cefsulodin best fitted to a two-exponential disposition. This study provided specific pharmacokinetic information for protein-unbound cefsulodin and demonstrated the applicability of this continuous sampling method for pharmacokinetic study.     

Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by microdialysis-microbore liquid chromatography

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2–0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 μl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.     

Application of extraction disks in dissolution tests of clenbuterol and levothyroxine tablets by capillary electrophoresis

Sample preparation procedures using octadecyl (C₁₈) extraction disks were developed to obtain accurate and reproducible results for determinations of clenbuterol (20 μg per dose) and levothyroxine (100 μg per dose) in dissolution media of solid oral dosage forms. Preconcentration of samples allowed final concentrations of 1.1 μg/ml of clenbuterol and 4.0 μg/ml of levothyroxine to be reached prior to CE analysis. The results obtained by CE were in good agreement with those of HPLC. The precision of the migration time, peak area, peak height and accuracy were determined in both intea-day (n = 6) and inter-day (n =18) assays. Linearity was demonstrated over the ranges 0.5–80.0 μg/ml of clenbuterol and 1.0–30.0 μg/ml of levothyroxine. The mean recoveries were higher than 94.0%, ranging from 50 to 125% levels with respect to dose potencies. The proposed methodology may be generally applied to determine drugs at ng/ml concentrations.     

Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry

γ-Hydroxybutyric acid (GHB), a minor metabolite or precursor of γ-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid–liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 μm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations ≥2 μg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2–24 and 5–60 μg/ml, respectively. Data obtained suggest that LOD and LOQ (both in μg/ml) can be estimated with the relationships 0.83 κ and 2.1 κ, respectively, where κ is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1→m/z 85.1 precursor–product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 μg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in μg/ml) that are ≥ 4 κ.     

Critical study of fluorimetric determination of selenium in urine

Different steps for the fluorimetric determination of Se in urine have been investigated. A HNO₃---HClO₄ (4:1) mixture is useful for urine digestion, and reduction of Se(VI) to Se(IV) is effectively carried out with HCl (6M). Selenium(VI) present after the digestion process constitutes 14.5–36.6% of total Se. An optimum pH of 1.80±0.05 and the addition of 1 ml of 2,3-diaminonaphthalene (DAN) (0.1%, w/v) are established in the formation of Se—DAN complex. Heating to 60°C, a time of incubation of 15 min is recommended to assure the complete formation of Se—DAN complex. A volume of 5 ml of cyclohexane and vigorous shaking for 45 sec is necessary for the extraction process. With this optimized method, the detection limit of selenium was 0.82 μg/l., within-day precision for a 50.0 μg/l. standard solution and urine (27.3 μg/l.) were 2.4 and 2.7% and between-day for the urine was 3.9% (33.9 μg/l.). Analytical recovery of 0.5 ml of Se standard (250 μg/l.) added to 1 ml of urine was 99.9±2.9% (95.8–104.4, n = 12). Normal levels of selenium excretion in urine obtained from healthy people were 27.9±8.7 μg/day (13.2–44.1), not observing significant differences (P < 0.05) between sexes.     

Determination of unbound cefamandole in rat blood by microdialysis and microbore liquid chromatography

To analyze unbound cefamandole in rat blood, a method combing microdialysis with microbore liquid chromatography has been developed. A microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats to examine the unbound cefamandole level in the rat blood following cefamandole administration (50 mg/kg, i.v.). The dialysates were directly submitted to a liquid chromatographic system. Samples were eluted with a mobile phase containing acetonitrile-methanol-100 mM monosodium phosphate (pH 5.0; 15:20:65, v/v). The UV wavelength was set at 270 nm for monitoring the analyte. Using the retrograde method, at infusion concentrations of 1 microg/mL of cefamandole, the in vivo microdialysis recoveries were 55.44% for the rat blood (n = 6). Intra- and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.1-10 microg/mL. Pharmacokinetic parameters were calculated from the recovery-corrected dialysate concentrations of cefamandole vs time data. The elimination half-life (t1/2,beta) was 21.6 +/- 1.6 min. The results suggest that the pharmacokinetics of unbound cefamandole in blood following cefamandole administration (50 mg/kg, i.v., n = 5) fit best to the two-compartmental model.     

An on-line flow-injection microwave-assisted mineralization and a precipitation/dissolution system for the determination of molybdenum in blood serum and whole blood by electrothermal atomic absorption spectrometry

An on-line flow injection (FI) precipitation–dissolution system with microwave-assisted sample digestion has been developed for the electrothermal atomic absorption spectrometry (ETAAS) determination of trace or ultratrace amounts of molybdenum in human blood serum and whole blood samples. After the exposure of the sample to microwave radiation, the on-line precipitation of molybdenum was achieved by the merging-zone of a 0.5-ml plug of sample with a plug of potassium ferrocyanide, which were carried downstream with a solution of 0.5 mol l⁻¹ of HNO₃. The interfering effects of iron and copper were minimized by the introduction of a flow of a 5% (w/v) sodium potassium tartrate (for iron) and 2% (w/v) of thiourea (for copper and zinc) in a 5% (v/v) ammonia and 2% (v/v) ammonium chloride solution previous to the precipitation reaction. The reddish-brown precipitate of molybdenyl ferrocyanide was collected on the walls of a knotted reactor. The precipitate was dissolved with the introduction of 1 ml of a 3.0 mol l⁻¹ NaOH solution and the best performance in terms of detection limit and precision was achieved when a sub-sample of 140 μl was collected in a capillary of a sampling arm assembly, to introduce 20 μl volumes into the atomizer by means of positive displacement with air through a time-based injector. A detection limit (3σ) of 0.1 μg Mo l⁻¹ using an aqueous standard solution was obtained. The method is quantitative and is applied over the range 0.2–20.0 μg Mo l⁻¹. The precision of the method evaluated by ten replicate analyses of aqueous standard solutions containing 0.5 and 1.0 μg Mo l⁻¹ was 2.8 and 3.1% (relative standard deviation, RSD) (for n=5), respectively. Whereas, the precision evaluated by five replicate analysis of a serum and a whole blood sample were 3.3 and 3.8% RSD. An enrichment factor of ca. 3.5 was achieved with the introduction of 0.5 ml aqueous standard solutions at a sample flow rate of 1.0 ml min⁻¹. Recoveries of spiked molybdenum in blood serum and whole blood were in the ranges 96–102 and 94–98%, respectively. The results obtained for two human whole blood certified reference materials were in good agreement with the indicative values.     

Reverse flow injection spectrophotometric determination of iron(III) using norfloxacin

A reversed flow injection colorimetric procedure for determining iron(III) at the μg level was proposed. It is based on the reaction between iron(III) with norfloxacin (NRF) in 0.07 mol l⁻¹ ammonium sulfate solution, resulting in an intense yellow complex with a suitable absorption at 435 nm. Optimum conditions for determining iron(III) were investigated by univariate method. The method involved injection of a 150 μl of 0.04% w/v colorimetric reagent solution into a merged streams of sample and/or standard solution containing iron(III) and 0.07 mol l⁻¹ ammonium sulfate in sulfuric acid (pH 3.5) solution which was then passed through a single bead string reactor. Subsequently the absorbance as peak height was monitored at 435 nm. Beer's law obeyed over the range of 0.2–1.4 μg ml⁻¹ iron(III). The method has been applied to the determination of total iron in water samples digested with HNO₃–H₂O₂ (1:9 v/v). Detection limit (3σ) was 0.01 μg ml⁻¹ the sample through of 86 h⁻¹ and the coefficient of variation of 1.77% (n=12) for 1 μg ml⁻¹ Fe(III) were achieved with the recovery of the spiked Fe(III) of 92.6–99.8%.     

Multiple enzyme linked immunosorbent assay system on a capillary-assembled microchip integrating valving and immuno-reaction functions

Gastrodin is a bioactive constituent of rhizome in Gastrodia elata Blume (Orchidaceae) The aim of this study is to develop a rapid and sensitive liquid chromatographic method coupled to microdialysis sampling system to measure the unbound of gastrodin in rat blood, brain and bile. Microdialysis probes were simultaneously inserted into the jugular vein, brain striatum and bile duct of each anesthetized rat for sampling after the administration of gastrodin (100 or 300 mg kg⁻¹) through the femoral vein. Separation of unbound gastrodin from various biological fluids was applied to an RP-select B column (250 mm × 4.6 mm i.d., 5 μm). The mobile phase consisted of acetonitrile–50 mM potassium dihydrogen phosphate buffer–triethylamine (5:95:0.1, v/v/v, adjusted to pH 2.5 with orthophosphoric acid) with a flow rate of 1 mL min⁻¹. The UV detector wavelength was set at 221 nm. Fifteen minutes after the administration, the gastrodin reached the peak concentration in brain and bile. In addition, the results indicate that gastrodin penetrates the blood-brain barrier (BBB) and goes through hepatobiliary excretion.     

Comparative methodology in the determination of alpha-oxocarboxylates in aqueous solution ion chromatography versus gas chromatography after oximation, extraction and esterification

A direct, simple and rapid high-performance liquid chromatographic method has been developed for the determination of ketoprofen with ibuprofen as internal standard. Samples were chromatographed on a 5 μm Kromasil 100 C₁₈ column. The mobile phase was a mixture of acetonitrile–0.01 M KH₂PO₄ adjusted to pH 1.5 with orthophosphoric acid 85% (60:40, v/v). Detection was at 260 nm and the run time was 10 min. The detector response was found to be linear in the concentration range 0.02 to 40 μg/ml. This HPLC assay has been applied to measure the “in vitro” percutaneous penetration of ketoprofen through rat skin.     

Determination of fenpyroximate in apples by supercritical fluid extraction and packed capillary liquid chromatography with UV detection

A method using off-line supercritical fluid extraction (SFE) and micro liquid chromatography (μLC) with UV detection at 260 nm, was developed for selective determination of fenpyroximate in apple samples. The packed capillary liquid chromatography method utilises 20 μl injection volumes with on-column focusing. A 350×0.32 mm capillary column packed with Kromasil 100-C₁₈ of 5 μm particle size was used with a mobile phase of acetonitrile–10 mM ammonium acetate (85:15, v/v) at a flow of 5 μl/min. A two-step SFE procedure was used to extract fenpyroximate selectively in 2 g apple samples, with Hydromatrix (HMX) added as a water absorbent at a 1:1 (w:w) ratio. Fenpyroximate was extracted at 200 bar and 90°C for 15 min using carbon dioxide at a flow of 2 ml/min, and solvent trapping collection in 10 ml acetonitrile. The volume of the acetonitrile extract was reduced by evaporation and water was added to a final composition of acetonitrile–water (40:60, v/v). The resulting 2.0 ml solution was filtered using a 0.45 μm poly(vinylidene difluoride) syringe filter before μLC analysis. Validation of the method was accomplished with apple samples spiked with fenpyroximate, covering the range of 0.1 to 1.0 μg/kg. The within-day and between-day repeatabilities were in the range 4–18% relative standard deviation. Accuracy, measured as recovery, was found to be approximately 60%. Apple samples from a field treated with fenpyroximate were analysed. None of the samples contained fenpyroximate above the quantification level.     

Determination of silver in ores, concentrates, zinc process solutions, copper metal and copper-base alloys by atomic-absorption spectrophotometry after separation by extraction of the tribenzylamine-silver bromide complex

A method for determining 0.1 μg/g or more of silver in ores and concentrates and 0.001 μg/ml or more of silver in zinc process solutions is described. Silver is separated from the matrix elements by chloroform extraction of the tribenzylamine—silver bromide ion-association complex from 0.08M potassium bromide—2M sulphuric acid and stripped with 9M hydrobromic acid. This solution is evaporated to dryness and organic material is destroyed with nitric and perchloric acids. Silver is determined by atomic-absorption spectrophotometry in an air—acetylene flame, at 328.1 nm, in a 10% v/v hydrochloric acid—1% v/v diethylenetriamine medium. Cadmium, bismuth and molybdenum are partly co-extracted but do not interfere. The method is also applicable to copper metal and copper-base alloys. Results obtained by this method are compared with those obtained by a fire-assay/atomic-absorption method.     

Determination of Unbound Ampicillin in Rat Blood by Microdialysis and Liquid Chromatography

ABSTRACT

An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been applied for continuous monitoring of unbound ampicillin in rat blood. A microdialysis probe was inserted into the jugular vein/right atrium of Sprague-Dawley rats, and doses of 100 and 200 mg/kg ampicillin were then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Isocratic elution of ampicillin was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.5). The wavelength of the UV detector was set at 230 nm. The calibration curves from 0.25 to 50 μg/ml were linear with correlation coefficients of 0.995. The method provides a simple technique for rapid analysis of unbound ampicillin in rat blood for used in pharmacokinetic study.     

Determination of Lovastatin (mevinolin) and mevinolinic acid in fermentation liquids

A rapid and simple HPLC method for the determination of Lovastatin (mevinolin) and mevinolinic acid in fermentation fluids of Aspergillus terreus using a Separon SGX C₁₈ column and methanol-18 mM orthophosphoric acid (77.5:22.5, v/v) as mobile phase with detection at 238 nm is described. The detection limit of Lovastatin and mevinolinic acid was 20–30 ng/ml.     

Determination of trimethylselenonium iodide, selenomethionine, selenious acid, and selenic acid using high-performance liquid chromatography with on-line detection by inductively coupled plasma mass spectrometry or flame atomic absorption spectrometry

An analytical method has been developed for the determination of selenious acid, selenic acid, trimethylselenonium ion, and selenomethionine. The four selenium compounds were separated by HPLC on a column (25 cm×4 mm I.D.) of the anion-exchanger ESA Anion III with a mobile phase (1.5 ml/min) of 0.0055 M ammonium citrate (pH 5.5). Detection was carried out using an on-line inductively coupled plasma mass spectrometer (ICP-MS) or a flame atomic absorption spectrometer (FAAS) as the selenium-specific detector. The chromatographic parameters and the chemical factors affecting the separation of the selenium species were optimized. The four selenium compounds could be separated within 8 minutes. The detection limits of the coupled HPLC–FAAS system were approximately 1 mg Se/l for each compound (100 μl injection), estimated as three times the base-line noise of the chromatograms. More powerful selenium detection was achieved with an ICP-MS. Selenium was measured at m/z 78. To increase the nebulization efficiency, the Meinhard concentric glass nebulizer was replaced by an ultrasonic nebulizer. The ICP-MS signal intensity was increased with the ultrasonic nebulization by a factor of 7 times for selenious acid and 24 to 31 times for trimethylselenonium ion, selenomethionine, and selenic acid compared to that with the Meinhard nebulization. The detection limits achieved by the HPLC–ICP-MS with the ultrasonic nebulization were 0.08 μg Se/l for trimethylselenonium ion, 0.34 μg Se/l for selenious acid, 0.18 μg Se/l for selenomethionine, and 0.07 μg Se/l for selenic acid, respectively.     

Selective spectrophotometric determination of palladium(II) with 2(5-nitro-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)phenol(5-NO(2).PAPS) and tartaric acid with 5-NO(2).PAPS-niobium(V) complex

Spectrophotometric determinations of palladium(II) and tartaric acid were respectively investigated by using the color reactions between 2(5-nitro-2-pyridylazo)-5-(N-propyl-N-3-sulfopropylamino)phenol(5-NO₂.PAPS) and palladium(II) in strong acidic media, and between 5-NO₂.PAPS, niobium(V) tartaric acid in weak acidic media. The calibration graphs were linear in the range of 0–25 μg/10 ml palladium(II), with an apparent molecular coefficient () of 6.2×10⁴ l mol⁻¹ cm⁻¹ at 612 nm, and 0–23 μg/10 ml tartaric acid with =1.08×10⁶ l mol⁻¹ cm⁻¹ at 612 nm, respectively. The proposed methods were selective and sensitive in comparison with other chelating pyridylazo dyes–palladium(II) or metavanadic acid–tartaric acid method, and the effect of foreign ions such as copper(II) was negligible for the assay of palladium(II) with 5-NO₂.PAPS.     

Determination of lead in seawater by inductively coupled plasma optical emission spectrometry after separation and pre-concentration with cocrystallized naphthalene alizarin

An analytical method for separation and pre-concentration of lead in seawater for determination by inductively coupled plasma optical emission spectrometry has been investigated. Lead was retained in the solid phase (0.5 g) composed of co-precipitated naphthalene and alizarin red. The solid phase quantitatively sorbs Pb(II) at pH 8–9, and the metal was eluted using 5.0 ml of 2 mol l⁻¹ nitric acid. The effect of NaCl, KCl, BaCl₂, CaCl₂, Na₂SO₄, MgCl₂ and Na₃PO₄ on the sorption of Pb(II) in the solid phase was studied. A set of solutions containing varying amounts of electrolytes (0.5; 1.0; 3.0 and 5.0% m/v) with Pb (50 μg) was prepared and the recommended procedure applied. The Na₃PO₄ was found to interfere; the other electrolytes did not interfere up to 5% m/v. A pre-concentration factor of 40 was obtained in this analytical procedure. The limit of detection and limit of quantification for Pb(II) were 53 and 176 μg l⁻¹, respectively. Lead was determined in seawater samples collected in Salvador city, Bahia, Brazil. The precision, expressed as R.S.D., was 1.8–4.6%, and the recovery of lead added to seawater samples was 95–97%.     

Simultaneous determination of catecholamines by ion chromatography with direct conductivity detection

A simple method for simultaneous determination of three catecholamines using ion chromatography (IC) with direct conductivity detection (CD) based on the ionization of catecholamines in acidic medium without chemical suppression is developed in the present paper. The method could be used for the determination of these catecholamines in pharmaceutical preparations for the purpose of drug quality control. The recovery of catecholamines was more than 97% (n=3) and the relative standard deviation (R.S.D.) (n=11) was less than 2.1%. In a single chromatographic run, norepinephrine (NE), epinephrine (E) and dopamine (DA) can be determined in less than 10 min. The detection limits were found to be 0.001 μg/ml for NE, 0.01 μg/ml for E and DA respectively. Linear ranges were 0.01–50 μg/ml for NE (r²=0.9998), 0.1–50 μg/ml for E (r²=0.9995) and DA (r²=0.9999), respectively.     

Simultaneous determination of berberine in rat blood,liver and bile using microdialysis coupled to high-performance liquid chromatography

High-performance liquid chromatography coupled to microdialysis was used for the simultaneous determination of unbound berberine in rat blood, liver and bile for a pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward the right atrium, the median lobe of the liver, and the bile duct of male Sprague-Dawley rats for biological fluid sampling after administration of berberine (10 mg/kg) through the femoral vein. Berberine and dialysates were separated using a Zorbax SB-phenyl column and a mobile phase comprised of acetonitrile-methanol-20 mM monosodium phosphate (pH 3.0) (35:20:45, v/v) together with 0.1 mM 1-octanesulfonic acid. The detection limit for berberine was 10 ng/ml. The concentration-response relationship was linear (r2 > 0.995) over the concentration range 0.05-50 microg/ml; intra-assay and inter-assay precision and accuracy for berberine fell within predefined limits. The disposition of berberine in the blood, liver and bile fluid suggests that berberine might be metabolized in the liver and undergo hepatobiliary excretion.