Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid-liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5-2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Determination of melanotan-II in rat plasma by liquid chromatography/tandem mass spectrometry: determination of pharmacokinetic parameters in rat following intravenous administration

A method is described for the determination of melanotan-II (MT-II), a synthetic cyclic heptapeptide analog of alpha-melanocyte-stimulating hormone (alpha-MSH), in rat plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The analyte is recovered from plasma by solid-phase extraction (SPE) and subsequently analyzed by LC/MS/MS. A SPE procedure using OASIS 96-well plates is used for extraction of MT-II from rat plasma. Using the described approach a limit of quantitation of 5 ng/mL was achieved in rat plasma. This level of sensitivity allowed the determination of pharmacokinetic parameters, following intravenous administration of MT-II in rat.     

A liquid chromatographic/tandem mass spectroscopic method for quantification of the cyclic peptide melanotan-II. Plasma and brain tissue concentrations following administration in mice

Melanotan-II (MT-II), a synthetic analogue of the natural melanocortin peptide, alpha-melanocyte-stimulating hormone (alpha-MSH), is well known for the anorexic effects it elicits in rodents. These effects are, at least partly, associated with agonistic action on the centrally located melanocortin receptors, MC3R and MC4R. Whether MT-II exerts this effect via brain penetration still remains unclear. In order to address this question we administered MT-II in rodents at efficacious doses and then employed a sensitive methodology for the determination of MT-II in plasma and brain samples. MT-II was extracted from mouse plasma and brain tissue by acetonitrile precipitation followed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. The described assay improved significantly previously reported MT-II levels of quantification in rat plasma and brain. The lower limits of quantification (LLOQs) of 0.5 ng/mL and 2.5 ng/g were obtained in 50 microL plasma and 100 microL brain homogenate, respectively. The calibration curve was linear over the concentration range of 0.5-500 ng/mL for plasma and 2.5-250 ng/g for brain tissue. The method was successfully applied in measuring levels of MT-II in plasma and brain tissue following intraperitoneal (ip) administration of 1 mg/kg of peptide in mice. Following administration of MT-II, clearance from plasma was rapid. The sensitivity of the assay allowed the determination of low concentrations of MT-II (11.4 +/- 5.5 ng/g) in brain homogenate at 30 min after dosing. However, the brain concentrations when compared with the high plasma levels of MT-II at the same time point confirmed the low penetrability of the peptide in mouse brain.     

Bioanalysis of the investigational anti-tumour drug 5,10-dideaza-5,6,7,8-tetrahydrofolic acid by high-performance liquid chromatography with ultraviolet detection.

A high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 278 nm is presented for the determination of 5,10-dideaza-5,6,7,8-tetrahydrofolic acid in plasma. Sample pretreatment was achieved by using cation-exchange solid-phase extraction columns with methotrexate as internal standard. Chromatographic separation was based on ion-pair HPLC with 1-octanesulphonic acid as the ion-pairing compound. The detection limit was 10 ng/ml using an 500-microliters sample volume. The assay was linear from the detection limit up to 5000 ng/ml with good reproducibility. The applicability of the assay was demonstrated in a study in the rat.     

Detection of oxilofrine in plasma and urine by high-performance liquid chromatography with electrochemical detection and comparison with gas chromatography-mass spectrometry

A high-performance liquid chromatographic (HPLC) method with electrochemical detection for the determination of oxilofrine [1-(4-hydroxyphenyl)-2-methylaminopropanol] in human plasma and urine (before and after cleavage of the metabolic conjugates) is described. Isolation from biological fluids is performed batchwise by weak acid cation exchange. Separation of plasma and urine components is achieved on a reversed-phase C18 column as an ion pair with heptanesulphonic acid. For amperometric detection the potential of the electrode was set at 0.95 V versus an Ag/AgCl reference electrode. The detection limit for oxilofrine in plasma is 1 ng/ml and in urine 12.5 ng/ml at a signal-to-noise ratio of 2.0 using 1.0 ml of plasma and 0.02 ml of urine. The method was compared with a gas chromatographic-mass spectrometric method and showed a good concordance for plasma (r = 0.996) and urine (r = 0.994). With the HPLC method it is also possible to determine related sympathomimetic drugs, e.g., etilefrine, norefenefrine or octopamine, after a slight modification of the mobile phase.     

Simultaneous determination of prednisolone acetate, prednisolone, prednisone, cortisone and hydrocortisone in swine plasma using solid-phase and liquid-liquid extraction techniques

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of prednisolone acetate (PA), prednisolone (PO), prednisone (PN), cortisone and hydrocortisone in swine plasma is described. Extraction of the steroid mixture from swine plasma with dexamethasone as internal standard was accomplished by solid-phase extraction (SPE) or the more traditional liquid-liquid extraction (LLE) techniques. These compounds were analyzed by normal-phase HPLC with ultraviolet detection. Although a detectable sensitivity of 5 ng/ml is achieved by the SPE technique, the practical sensitivity is established as 10 ng/ml. Conversely, the practical sensitivity is 5 ng/ml for all compounds by the LLE technique. Calibration curves were found to be linear between 10 and 500 ng/ml by the SPE technique and between 5 and 100 ng/ml by the LLE technique. The average recovery of the steroids PA, PO and PN at 20 ng/ml is between 70 and 90%. PA is stable for up to 3 h in swine plasma at room temperature (22 degrees C) but is completely converted to PO within 24 h. PA is stable in swine plasma in an ice bath for over 24 h. The usefulness of this analytical technique is demonstrated by the intraperitoneal administration of 125 mg of PA to swine and the quantitative determination of PA, PO and PN in the plasma as a function of time.     

Quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine: application of the plasma methodology to measure the plasma protein binding of remacemide.

Sensitive and selective methods have been developed for quantitation of the novel anticonvulsant remacemide in rat and dog plasma and urine. The methods employed liquid-liquid extraction (urine) or ion-exchange solid-phase extraction (plasma), with an internal standard, followed by high-performance liquid chromatography with ultraviolet detection. The detection limit for both methods was 10 ng/ml. Overall accuracy was 0.00% for plasma and -1.4% for urine with a precision of 6.04 and 3.87% for plasma and urine, respectively. The standard curves were linear for both plasma and urine over a wide concentration range (9.96-2490 ng/ml). The plasma method was also applied to measurement of in vitro plasma protein binding of remacemide in rat, dog and human plasma.     

A Sensitive High Performance Liquid Chromatographic Method for U-80, 278A,A Substituted Aminotetralin,in Rat Plasma,Whole Blood and Brain Tissue

Abstract

A sensitive analytical method for U-80, 278A, a substituted aminotetralin analogue in rat plasma, whole blood and brain tissue has been developed. The method involves solid phase extraction, efficient reversed phase HPLC and fluorescence detection, and can measure 1 ng/ml from 50 μl samples. During method development, many analogues were investigated and a wide range of extraction and HPLC conditions were explored. These experiments enabled rapid modification and revalidation of the method to support animal experiments with novel analogues.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Determination of Flecainide in Human Plasma by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A simple, rapid, selective, and sensitive high-performance liquid chromatographic (HPLC) method for the monitoring of plasma flecainide levels in a therapeutic or research environment is described. The drug is first separated from plasma by a single-step extraction with hexane and then quantitated by HPLC with fluorescence detection. Two linear ranges have been established; 100–2000 ng/ml for drug monitoring in clinical management of patients and 3–300 ng/ml for pharmacokinetic studies. The intra-day variation is less than 6%.     

An automated analyzer for vancomycin in plasma samples by column-switching high-performance liquid chromatography with UV detection

An automated analyzer for vancomycin in rat plasma by column-switching high-performance liquid chromatography (HPLC) with UV detection was developed. The method includes in-line extraction of vancomycin by ion-exchange cartridge column and a separation on a reversed-phase column with UV detection at 215 nm. Plasma samples were diluted by mobile phase solution and directly injected to HPLC. Vancomycin was quantitatively recovered from rat plasma samples. The separation was completed within 15 min. The calibration curve was linear over the range from 0.5 to 100 microg/mL with the detection and quantification limits of 0.5 microg/mL (2.5 ng on column; signal-to-noise ratio = 3). The values of precision in intra- and inter-day assays (n = 3) were less than 1.92 and 3.69%, respectively. This method does not require time-consuming pre-treatment and is suitable for the routine assay of plasma samples.     

High-performance liquid chromatographic assay for dilevalol in human plasma and urine using a PRP-1 column and fluorimetric detection

A single high-performance liquid chromatographic (HPLC) assay for the quantitative determination of dilevalol, the R,R isomer of labetalol, was developed for both plasma and urine. A significantly improved limit of detection for dilevalol in plasma was accomplished by extensive modification of an HPLC assay originally developed in our laboratory for labetalol. This simplified method is readily adaptable to urine and represents the first reported HPLC assay for the quantitative determination of dilevalol in this biofluid. Drug was recovered from plasma or urine by partition into diethyl ether under mildly alkaline conditions and back-extraction into dilute acid. Reversed-phase separation of dilevalol and the internal standard was accomplished on a 150 X 4.1 mm column commercially packed with a spherical (5 micron) macroporous copolymer (PRP-1). No interferences were observed in extracts obtained from drug-free plasma or urine. Selectivity for dilevalol in the presence of other beta-blockers was established. This method demonstrated a linear detector response to concentrations of unchanged drug typically observed in urine and plasma following once-a-day treatment with dilevalol hydrochloride (100-800 mg). The lowest limit of reliable quantitation was established at 1 ng/ml in plasma. The intra-assay precision (coefficient of variation) remained less than 6% at all concentrations evaluated from 1 to 800 ng/ml. In urine, the lowest limit of quantitation was validated to 20 ng/ml where the intra-assay precision (coefficient of variation) for unchanged drug was less than 4% at all concentrations evaluated up to 400 ng/ml. This method is suitable for routine quantitation of unchanged drug in human plasma and urine following the administration of therapeutically effective doses of dilevalol hydrochloride.     

High-performance liquid chromatographic evaluation of 2-(alpha-thenoylthio)propionylglycine and its two metabolites in biological fluids

A high-performance liquid chromatographic (HPLC) method for determining 2-(alpha-thenoylthio)propionylglycine (TTPG) and its two main metabolites, thiophenecarboxylic acid and thiopronine, in biological samples was developed. TTPG and its metabolites were extracted by solvent partition and then determined by reversed-phase HPLC with UV detection at 245, 295 and 360 nm. This procedure was validated in order to allow the assay of these compounds in plasma and urine samples with sufficiently low detection limits (50 ng/ml for TTPG and TCA and 100 ng/ml for thiopronine) and with good linearity within the concentration range investigated. It was applied to a comprehensive pharmacokinetic investigation of TTPG in healthy volunteers.     

Sensitive high-performance liquid chromatographic assay using 9-fluorenylmethylchloroformate for monitoring controlled-release lidocaine in plasma

A sensitive high-performance liquid chromatographic (HPLC) assay using fluorescence detection for quantifying lidocaine levels in plasma (in the ng/ml range) was developed. This novel HPLC assay has made possible the simultaneous monitoring of lidocaine levels in coronary and peripheral plasma obtained after myocardial controlled-release matrix administration (0.92 mg/kg during 4 h) in the arrhythmic dog. The method employed extracts the drug from plasma using 1-chlorobutane and a subsequent derivatization with 9-fluorenylmethylchloroformate in acetonitrile at 110 degrees C. The derivative was chromatographed on a C18 reversed-phase column and measured with fluorescence detection (excitation 254 nm, emission 313 nm). N-Methylephedrine was found to be suitable as an internal standard, post-derivatization. The derivatization product of lidocaine was identified and characterized by mass spectral analysis. It was found to have a unique and reproducible dicarbamate structure, which was stable for at least three days at room temperature. The method was tested with human plasma as well as on dog plasma. Analytical recoveries were 88.6 +/- 3.6 and 77.4 +/- 3.0% (mean +/- S.E.), respectively, at levels ranging from 25 to 200 ng/ml. The lower detection limit was 1 ng/ml lidocaine. In conclusion, this rapid and convenient analysis was found to be suitable for the bioavailability pharmacokinetic assessment of lidocaine following low-dose regional drug administration.     

High-performance liquid chromatographic method for the simultaneous measurement of remacemide (a novel anticonvulsant and cerebroprotectant) and an active metabolite in human plasma.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of both remacemide (a novel anticonvulsant and cerebroprotectant) and an active, major metabolite in human plasma. After the addition of an internal standard, the analytes were extracted from the plasma by ion-exchange solid-phase extraction and measured by an isocratic HPLC system with ultraviolet detection at 210 nm. The recovery of the analytes was > 90%. The standard curves were linear over the range of quantitation of approximately 10-500 ng/ml for remacemide itself and 15-250 ng/ml for the metabolite. Both intra-day and inter-day accuracy and precision data were excellent. Remacemide and its metabolite were shown to be stable in human plasma for at least a year when stored at -20 degrees C.     

High-performance liquid chromatographic analysis of trilostane and ketotrilostane in rat plasma

A simple high-performance liquid chromatographic (HPLC) method for assaying trilostane, a synthetic steroid, and one of its metabolites, ketotrilostane, in small volumes of rat plasma has been developed. A single liquid-liquid extraction was used to isolate the two compounds from acidified plasma prior to the quantitative analysis. The HPLC conditions involved the use of a Spherisorb ODS column (250 mm x 4.6 mm I.D.) and a mobile phase of 1,4-dioxan-Sorenson's buffer at pH 5.0 (52:48, v/v). Ethisterone was used as an internal standard. Trilostane and ketotrilostane were detected by their ultraviolet absorbance at 255 nm. Recoveries greater than 80% and detection limits of 50 ng/ml were obtained for both compounds. Inter-day coefficients of variation were less than 10%.     

Sensitive high-performance liquid chromatographic determination of famotidine in plasma. Application to pharmacokinetic study.

A sensitive high-performance liquid chromatographic (HPLC) method for the quantitation of famotidine in human plasma is described. Clopamide was used as the internal standard. Plasma samples were extracted with diethyl ether to eliminate endogenous interferences. Plasma samples were then extracted at alkaline pH with ethyl acetate. Famotidine and the internal standard were readily extracted into the organic solvent. After evaporation of ethyl acetate, the residue was analysed by HPLC. The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-water (12:88, v/v) containing 20 mM disodium hydrogenphosphate and 50 mM sodium dodecyl sulphate, adjusted to pH 3. The HPLC microbore column was packed with 5 microns ODS Hypersil. Using ultraviolet detection at 267 nm, the detection limit for plasma famotidine was 5 ng/ml. The calibration curve was linear over the concentration range 5-500 ng/ml. The inter- and intra-assay coefficients of variation were found to be less than 10%. Applicability of the method was demonstrated by a bioavailability/pharmacokinetic study in normal volunteers who received 80 mg famotidine orally.     

Development and validation of a high-performance liquid chromatographic method using fluorescence detection for the determination of vardenafil in small volumes of rat plasma and bile

A new, simple and sensitive high-performance liquid chromatography (HPLC) method with fluorescence detection was developed and validated for the determination of vardenafil in small volumes of rat plasma and bile. The absorbance and fluorescence characteristics of vardenafil were studied and factors that affect the HPLC resolution and fluorescence intensity were examined and optimized. Vardenafil and the internal standard cisapride were extracted using acetonitrile. The separation was achieved on a C18 column at 35 degrees C using acetonitrile-50 mM ammonium acetate aqueous solution (pH 6.8) (40:60) as mobile phase. At a flow rate of 1 ml/min, the total run time was 18 min. Fluorescence was measured with excitation and emission set at 280 and 470 nm, respectively. The calibration curves were linear from 10 to 1000 ng/ml and 0.2-100 microg/ml for plasma and bile samples, respectively. The intra- and inter-day imprecision did not exceed 10.8%, and the accuracy was within 9.6% deviation of the nominal concentration. The method was used successfully to investigate the disposition and biliary excretion of vardenafil in rats.     

Determination and purification of metallothioneins by high performance liquid chromatography.

A rapid, reproducible and sensitive high performance liquid chromatography (HPLC) method for the determination and purification of metallothionein-I (MT-I) and metallothionein-II (MT-II) in mouse and rabbit livers has been developed. Methallothioneins (MTs) were separated by an HPLC anion exchange column, eluted through a linear gradient of Tris buffer and the peak containing MTs was determined by atomic absorption spectrophotometry. Furthermore, the content of MT-I or MT-II was calculated by protein peak area in a short time (about 20 min). The sample to be tested was homogenized, centrifuged and saturated by cadmium. MT-I and MT-II were eluted at 15.9 and 19.3 min, respectively. The following mouse liver cytosols were tested: controls, Cd-injected samples and 60Co-irradiated samples. A detection limit of 5 micrograms/g liver was established for this method. We have analysed more than 100 biological samples and obtained satisfactory results.     

High-performance liquid chromatographic determination of plasma histamine after pre-column derivatisation with o-phthaldialdehyde

Summary A reversed-phase high-performance liquid chromatographic (HPLC) method has been developed for the sensitive and highly selective determination of histamine in plasma. This method includes selective extraction of histamine from plasma, pre-column derivatisation in aqueous phase with o-phthaldialdehyde (OPA) and HPLC analysis. The fluorescence of the histamine-OPA-complex was monitored at wavelengths of 350nm excitation and 450nm emission, after isocratic eluation with a mixture of 0.2 M NaCl and methanol. The reproducibility of this method including extraction, derivatisation and detection of histamine was >95% in a range of 0.35–17.6 pmol. The HPLC precision was 99±1% at 4 pmol of histamine. The lower limit of detection was 88fmol. A significantly increased concentration of plasma histamine was detected in patients (n=46) with various liver diseases (0.3–5.2 ng/ml). In comparison the plasma histamine levels of healthy blood donors were in the range of 0.0–0.4 ng/ml (p<0.01).