Determination of pharmacological levels of harmane, harmine and harmaline in mammalian brain tissue, cerebrospinal fluid and plasma by high-performance liquid chromatography with fluorimetric detection

Increased blood aldehyde levels, as occur in alcohol intoxication, could lead to the formation of beta-carbolines such as harmane by condensation with indoleamines. Endogenous beta-carbolines, therefore, should occur in specific brain areas where indoleamine concentrations are high, whilst exogenous beta-carbolines should exhibit an even distribution. The author presents direct and sensitive methods for assaying the beta-carbolines harmane, harmine and harmaline in brain tissue, cerebrospinal fluid and plasma at picogram sample concentrations using reversed-phase high-performance liquid chromatography with fluorimetric detection and minimal sample preparation. Using these assay methods, it was found that the distribution of beta-carbolines from a source exogenous to the brain results in a relatively even distribution within the brain tissue.     

High-performance liquid chromatographic determination of pyrazinamide in cerebrospinal fluid and plasma in the rabbit

A simple procedure for the determination of pyrazinamide in plasma and cerebrospinal fluid in the rabbit is described. The assay involves a preliminary extraction of the drug and an internal standard, paracetamol, from the acidified sample (pH 4.2). The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (50 microliters). A 25-microliters aliquot is injected into the liquid chromatograph and eluted with acetonitrile-10 mM phosphate buffer of pH 3.5 (10:90, v/v) on a 30-microns C8 pre-column linked to a 5-microns C8 reversed-phase column at ambient temperature (25 +/- 1 degree C). The eluate is detected at 215 nm. The method has been used to investigate the disposition of pyrazinamide in plasma and cerebrospinal fluid in six rabbits.     

Analysis of circulating immune complexes isolated from plasma, cerebrospinal fluid and urine.

The protein nature of soluble immune complexes (IC) from fresh plasma, cerebrospinal fluid (CSF), and urine was studied by combining several analytical and biochemical techniques. In plasma and CSF, free immunoglobulins G were separated from larger IC by gel filtration with a fast protein liquid chromatographic system. In urine, IC were separated by precipitation with polyethylene glycol. IC were further purified by protein-A and protein-G affinity chromatography and analyzed by two-dimensional gel electrophoresis. Apart from plasma samples from healthy donors, IC from cases with macrocreatine kinase type 1 and multiple sclerosis were analyzed. For CSF two cases of multiple sclerosis and for urine one case with urinary tract infection are shown. The method can be used for the examination of IC of unknown protein composition in body fluids.     

HPLC investigation of free and bound propofol in human plasma and cerebrospinal fluid

The paper compares the total propofol concentration in the cerebrospinal fluid (CSF) with the free drug concentration in plasma measured in 35 humans scheduled for elective neurosurgical procedures during propofol anaesthesia. The concentrations of total and free propofol in the blood and CSF samples were measured by means of HPLC using liquid-liquid extraction and ultrafiltration in the sample preparation procedure. The arterial blood and CSF samples (collected from intraventricular drainage) were taken at the same time. According to the obtained results, the usually expected equality between free drug concentration in plasma and its total concentration in CSF is not valid for propofol: the unbound propofol concentration in plasma is not equal to its total concentration in CSF (p < 0.05). This difference suggests a substantial contribution of active transport in propofol transfer from blood into CSF. Moreover, the paper shows the presence of bound propofol in CSF, which is a novel finding.     

Determination of cefepime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection

A simple micellar capillary electrokinetic chromatography (MEKC) with UV detection is described for analysis of cefepime in plasma and cerebrospinal fluid by direct injection without any sample pretreatment. The separation of cefepime from biological matrix was performed at 25 degrees C using a background electrolyte consisting of tris(hydroxymethyl)aminomethane (Tris) buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Several parameters affecting the separation of the drug were studied, including the pH and concentrations of the Tris buffer and SDS. Using cefazolin as an internal standard, the linear ranges of the method for the determination of cefepime in plasma and cerebrospinal fluid were 1-50 and 1-20 microg/mL, respectively; the detection limits of plasma (signal-to-noise ratio = 3; injection, 5 kV, 5 s) and cerebrospinal fluid (signal-to-noise ratio = 3; injection, 0.5 psi, 3 s) were 0.2 microg/mL and 0.3 microg/mL, respectively. Application of the proposed method for determination of cefepime in plasma and cerebrospinal fluid collected after intravenous administration of 2 g cefepime in patients with meningitis was demonstrated.     

Determination of ceftazidime in plasma and cerebrospinal fluid by micellar electrokinetic chromatography with direct sample injection

A simple micellar electrokinetic chromatography (MEKC) with UV detection at 254 nm for analysis of ceftazidime in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of ceftazidime from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Several parameters affecting the separation of the drug from biological matrix were studied, including pH and concentration of the Tris buffer and SDS. Using cefazolin as an internal standard (IS), the linear ranges of the method for the determination of ceftazidime in plasma and in CSF were all over the range of 3-90 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio = 3; injection 0.5 psi, 5 s) was 2.0 microg/mL. The applicability of the proposed method for determination of ceftazidime in plasma and CSF collected after intravenous administration of 2 g ceftazidime in patients with meningitis was demonstrated.     

Quantitative determination of melatonin in human plasma and cerebrospinal fluid with high-performance liquid chromatography and fluorescence detection

A validated new and precise reversed-phase high-performance liquid chromatographic method for the determination of melatonin in human plasma and cerebrospinal fluid, with 5-fluorotryptamine as internal standard, is described. Liquid-liquid extraction with dichloromethane was performed under alkaline conditions. After evaporation of the organic solvent, the extract was dissolved in eluent and chromatographed on a base-deactivated octadecyl column, using an eluent composed of 650 mL potassium dihydrogenphosphate solution (0.07 mol/L water), adjusted to a pH of 3.0 with a 43% phosphoric acid solution, mixed with 350 mL methanol. Fluorescence detection at an excitation wavelength of 224 nm and an emission wavelength of 348 nm was used for quantitation. Melatonin and 5-fluorotryptamine chromatographed with retention times of 5.3 and 9. 3 min, respectively. Mean recoveries of 96% (n = 10) and 95% (n = 5) were found for melatonin in plasma and cerebrospinal fluid respectively. 5-Fluorotryptamine was found to have a mean recovery of 90% (n = 10) and 82% (n = 5) in plasma and cerebrospinal fluid, respectively. The repeatability coefficients of variation for both melatonin and 5-fluorotryptamine in plasma were 4-5% [five different samples (r = 5) on two consecutive days (n = 2)], with reproducibility coefficients of 1.6-7% (n = 2, r = 5) and 0.9-4% (n = 2, r = 5) for melatonin and internal standard, respectively. In cerebrospinal fluid the repeatability coefficient of variation of the extraction procedure was 5% (n = 1, r = 5) for melatonin and 7% (n = 1, r = 5) for 5-fluorotryptamine. The correlation coefficients of the calibration curves were 0.9998 (n = 2) in plasma at a concentration range of 0.108-25.9 ng/mL and 0.9994 (n = 2) at a concentration range of 0.108-25.9 ng/mL in cerebrospinal fluid. The limit of detection was determined at 8 pg/mL which enables to measure melatonin concentrations at physiological concentrations reached during daytime.     

Combined thin-layer chromatography-photography-densitometry for the quantification of ifosfamide and its principal metabolites in urine, cerebrospinal fluid and plasma.

A method has been devised for the determination of the anticancer drug ifosfamide and its principal metabolites in urine, plasma and cerebrospinal fluid (CSF). The urine and CSF samples are absorbed onto Amberlite XAD-2 eluting the compounds of interest with methanol. Plasma is deproteinated using cold acetonitrile and centrifuged to yield a clear supernatant. The eluate and supernatant are analyzed by thin-layer chromatography, with spot visualization using 4-(4-nitrobenzyl)pyridine. The plates are photographed for subsequent densitometeric analysis. The intra-assay coefficient of variation for each compound in both urine and plasma was less than 10% and the lower limit of detection was 1 microgram/ml. The method provides a means of determining the full spectrum of metabolic products of ifosfamide in patients and will allow detailed investigation of variability in metabolism and pharmacokinetics of this drug.     

Determination of diclofenac and its metabolites in plasma and cerebrospinal fluid by high-performance liquid chromatography with electrochemical detection

A reversed-phase high-performance liquid chromatographic method with electrochemical detection for the quantitation of diclofenac and metabolites in plasma and cerebrospinal fluid has been developed. Pirprofen is employed as internal standard. Samples are extracted with C18 solid-phase extraction columns and eluted with methanol. Oxidation potentials for detection were established by constructing voltammograms for each compound. In the concentration range found in human studies, the intra-day coefficients of variation were always less than 6%. The procedure allows the simultaneous determination of diclofenac and its four major metabolites with very low detection limits (less than 1 ng/ml), which were sufficient even for kinetic studies in cerebrospinal fluid.     

Simple high-performance liquid chromatographic method for the measurement of toloxatone in rabbit cerebrospinal fluid and plasma

A simplified, rapid high-performance liquid chromatographic procedure has been developed for the measurement of toloxatone in rabbit plasma and cerebrospinal fluid. The method involves a single-step extraction of the alkalinized sample with diethyl ether and analysis of the evaporated extract on a C8 column. Detection was performed by ultraviolet absorbance monitored at 240 nm. The overall run-time of the assay was 8 min at a flow-rate of 1 ml min-1. The sensitivity limit of toloxatone was 70 ng ml-1 at a signal-to-noise ratio of 3:1 in rabbit cerebrospinal fluid and plasma. The assay has been used to define plasma toloxatone concentration-time profiles and to quantitate cerebrospinal fluid toloxatone levels after a single intravenous injection in rabbits.     

A simple LC-MS/MS method to determine plasma and cerebrospinal fluid levels of albendazole metabolites (albendazole sulfoxide and albendazole sulfone) in patients with neurocysticercosis

The development and validation of an LC-MS/MS method for the simultaneous determination of albendazole metabolites (albendazole sulfoxide and albendazole sulfone) in human plasma are described. Samples of 200 μL were extracted with ether-dichloromethane-chloroform (60:30:10, v/v/v). The chromatographic separation was performed using a C(18) column with methanol-formic acid 20 mmol/L (70:30) as the mobile phase. The method was linear in a range of 20-5000 ng/mL for albendazole sulfoxide and 10-1500 ng/mL for albendazole sulfone. For both analytes the method was precise (RSD < 12%) and accurate (RE <7%) with high recovery (>90%). The method was successfully applied to determine the plasma and cerebrospinal fluid levels of albendazole sulfoxide and albendazole sulfone in patients with subarachnoidal neurocysticercosis who received albendazole at 30 mg/kg per day for 7 days. This LC-MS/MS method yielded a quick, simple and reliable protocol for determining albendazole sulfoxide and albendazole sulfone concentrations in plasma and cerebrospinal fluid samples and is applicable to therapeutic monitoring.     

Rapid determination of piracetam in human plasma and cerebrospinal fluid by micellar electrokinetic chromatography with sample direct injection

A simple micellar electrokinetic chromatography (MEKC) method with UV detection at 200 nm for analysis of piracetam in plasma and in cerebrospinal fluid (CSF) by direct injection without any sample pretreatment is described. The separation of piracetam from biological matrix was performed at 25 degrees C using a background electrolyte consisting of Tris buffer with sodium dodecyl sulfate (SDS) as the electrolyte solution. Several parameters affecting the separation of the drug from biological matrix were studied, including the pH and concentrations of the Tris buffer and SDS. Under optimal MEKC condition, good separation with high efficiency and short analyses time is achieved. Using imidazole as an internal standard (IS), the linear ranges of the method for the determination of piracetam in plasma and in CSF were all between 5 and 500 microg/mL; the detection limit of the drug in plasma and in CSF (signal-to-noise ratio=3; injection 0.5 psi, 5s) was 1.0 microg/mL. The applicability of the proposed method for determination of piracetam in plasma and CSF collected after intravenous administration of 3g piracetam every 6h and oral administration 1.2g every 6h in encephalopathy patients with aphasia was demonstrated.     

HPLC with fluorescence detection assay of perampanel,a novel AMPA receptor antagonist,in human plasma for clinical pharmacokinetic studies

Perampanel (Fycompa®), a novel α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor antagonist, is registered for the adjunctive treatment of patients (aged ≥12 years) with refractory partial‐onset seizures. To support therapeutic drug monitoring, a simple high‐performance liquid chromatography (HPLC) assay with fluorescence detection was developed to determine perampanel concentrations in human plasma and validated to support clinical trials. Human plasma samples (1.0 mL) were processed by liquid extraction using diethyl ether, followed by chromatographic separation on a YMC Pack Pro C₁₈ column (150 × 4.6 mm i.d., 5 µm) with isocratic elution of acetonitrile–water–acetic acid–sodium acetate (840:560:3:1.8, v/v/v/w) at a flow rate of 1.0 mL/min. Column eluent was monitored at excitation and emission wavelengths of 290 and 430 nm, respectively. The assay was linear (range 1.0–500 ng/mL) and this could be extended to 25 µg/mL by 50‐fold dilution integrity. No endogenous peaks were detected in the elution of analytes in drug‐free blank human plasma from six individuals and no interference was observed with co‐medications tested. Intra‐ and inter‐batch reproducibility studies demonstrated accuracy and precision within the acceptance criteria of bioanalytical guidelines. Validation data demonstrated that our assay is simple, selective, reproducible and suitable for therapeutic drug monitoring of perampanel. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma

An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.     

Simultaneous high-performance liquid chromatographic determination of norepinephrine, serotonin, acetylcholine and their metabolites in the cerebrospinal fluid of anaesthetized normotensive rats.

A high-performance liquid chromatographic method with electrochemical detection was developed for the simultaneous determination of the levels of norepinephrine (NE), serotonin (5-HT), acetylcholine (ACh) and their metabolites in the cerebrospinal fluid (CSF) of anaesthetized rats. The response curve for each compound was linear for the concentration way of interest. The within- and between-day coefficients of variation (C.V.) for NE, 5-HT and their metabolites were less than 7.85% and 15.67%, respectively, and those for ACh and choline were less than 3.08% and 6.27%, respectively. This simultaneous determination should be useful for elucidating the noradrenergic, serotonergic and cholinergic nerve activity in the central nervous system.     

An LC-MS-MS method for quantitative determination of maraviroc (UK-427,857) in human plasma, urine and cerebrospinal fluid

Maraviroc is a first‐in‐class CCR5 antagonist that shows potent anti‐HIV‐1 activity in vitro and in vivo and is well tolerated in both healthy volunteers and HIV‐1‐infected patients. The method for determination of maraviroc (UK‐427,857) and its major metabolite (UK‐408,027) in human plasma consists of a protein‐precipitation procedure and analysis by liquid chromatography/tandem mass spectrometry using positive ion TurboIonSpray® ionization and multiple reaction monitoring. The assay has been validated over a concentration range of 0.500–500 ng/mL for both analytes. The determinations of maraviroc in human cerebrospinal fluid (0.500–500 ng/mL) and in urine (5.00–5000 ng/mL) have also been validated but do not include measurement of the metabolite. The validations included extraction recovery, intra‐assay and inter‐assay precision and accuracy, stability of stock and spiking solutions, freeze–thaw stability, matrix stability, processed‐extract stability, and evaluation of potential interferences from selected medications in plasma or urine. Copyright © 2010 John Wiley & Sons, Ltd.     

Application of microdialysis for study of caffeine distribution into brain and cerebrospinal fluid in rats.

The usefulness of microdialysis was examined for the chronological determination of caffeine concentration in the brain and cerebrospinal fluids (CSF) following intravenous administration of caffeine in rats. The recovery percent of caffeine by microdialysis, the concentration ratio of caffeine in the dialysate against that in the brain tissue or CSF was determined. The recovery percent was proved to be constant at 5 different steady-state plasma concentrations of caffeine (0.1-280 nmol/ml) and in different collecting periods of dialysate ranging from 30 s to 10 min. The mean recovery percent in the brain and CSF were 10.9 and 13.1%, respectively. Thus, microdialysis was proved useful for determination of drug concentration in the tissue and biological fluids with time resolution of more than 30 s. The microdialysis method was then applied for the chronological determination of caffeine concentration in the brain and CSF following intravenous bolus administration. The estimated caffeine concentration in the brain and CSF was the same as those obtained by direct determination in isolated brain and CSF, respectively. Transfer of caffeine from plasma to brain and CSF were further pharmacokinetically analyzed using a modified 2-compartment model. In this kinetic model, the transfer of caffeine between the CSF and brain was neglected, since the mutual transfer of caffeine was not detected in in vivo experiments. Calculated curves were well fitted on observed caffeine concentrations in the plasma, brain and CSF.