Determination of bisaramil and its metabolite in plasma using high-performance liquid chromatography with electrochemical detection.

A column liquid chromatographic method using electrochemical detection has been developed for determination of an antiarrhythmic agent, bisaramil, and its metabolite in plasma. The plasma was fractionated by extraction with chloroform and chloroform-ethanol, and each fraction was dried and dissolved in ethyl acetate. After back-extraction into an acidic buffer, bisaramil was chromatographed on a reversed-phase column, and the metabolite, which has a higher polarity, was analysed by ion-pairing chromatography. Calibration curves were linear over the concentration range 2-200 ng/ml with coefficients of variation, within-day or day-to-day, not exceeding 5% at any level. The limits of detection of bisaramil and its metabolite were 0.5 and 1 ng/ml, respectively, using 0.5 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, +0.55 V; electrode 2, +0.8 V), providing a greater specificity and reducing the background noise. This procedure was applied to a large number of samples in a pharmacokinetic study at the therapeutic dose.     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies.

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane-isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a muPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3-300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been successfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at -20 degrees C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.     

Enantioselective determination of felodipine and other chiral dihydropyridine calcium entry blockers in human plasma

A sensitive method for the enantioselective determination of felodipine in human plasma is described. Following alkaline extraction with dichloromethane-pentane, racemic felodipine and its primary pyridine metabolite are simultaneously assayed using capillary gas chromatography on a DB-1 column, with electron-capture detection. The enantiomers of felodipine are quantitatively separated by high-performance liquid chromatography on a Chiralcel OJ column, containing tris(4-methylbenzoate)-modified cellulose coated on silica, and off-line detection using the same gas chromatographic system is applied. The limits of determination in plasma (and the inter-assay coefficient of variation (C.V.) at levels below 1 ng/ml) were 0.1 ng/ml (C.V. 13%) for felodipine, 0.1 ng/ml (C.V. 15%) for the enantiomers of felodipine and 0.3 ng/ml (C.V. 7%) for its pyridine metabolite. The method has proved to be applicable to several other chiral dihydropyridine calcium entry blockers, including nitrendipine, with comparable sensitivities.     

Determination of a new 2-amino-2-oxazoline (COR 3224) in plasma and brain tissue of the rat by high-performance liquid chromatography with electrochemical detection

A reversed-phase (CN as stationary phase) liquid chromatographic method with electrochemical detection is described for the quantitation of COR 3224, a new 2-amino-2-oxazoline in plasma and brain tissue of the rat. Extraction was performed with dichloromethane and detection was achieved at a working electrode potential of +0.85 V versus an Ag/AgCl reference electrode. The recovery of the method is about 80 and 60% for plasma and brain, respectively. The limit of detection was less than 10 ng/ml for both plasma and brain, five times lower than that with ultraviolet detection.     

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.     

Determination of delta 9-tetrahydrocannabinol in human blood and saliva by high-performance liquid chromatography with amperometric detection

A rapid, sensitive and selective determination of delta 9-tetrahydrocannabinol (THC) in human plasma, serum and saliva was developed with high-performance liquid chromatography with electrochemical detection. Initially, samples were deproteinized, followed by a one step liquid-liquid extraction. Samples were measured by high-performance liquid chromatography with electrochemical detection with 4-dodecylresorcinol as the internal standard. The minimal detectable limit for THC in biological samples was ca. 1 ng/ml with a signal-to-noise ratio greater than 3, corresponding to an on-column sensitivity for THC of ca. 0.5 ng. The detector was operated at + 0.90 V vs. Ag/AgCl and exhibited linearity over a concentration range of 1-150 ng/ml with correlation coefficients of the standard curves greater than 0.99.     

High-performance liquid chromatography with electrochemical detection of gossypol in human plasma

A sensitive and selective high-performance liquid chromatographic method with electrochemical detection for the determination of gossypol in human plasma is described. Glutathione is used as a protective agent and gossypol dimethyl ether as an internal standard. Acetonitrile-treated protein-free plasma sample is first introduced on to a C18 pre-column for enrichment and clean-up. By using a column-switching technique, gossypol and the internal standard are subjected to further separation on a C8 analytical column, while the major interfering components are eliminated before entering the column. Methanol-0.1 M citrate buffer (pH 3.2) (80:20) is used as the mobile phase. The detector potential on the glassy carbon electrode is maintained at +0.6 V vs. an Ag-AgCl reference electrode. The linearity with human plasma ranged from 5 to 250 ng/ml. The absolute recoveries of gossypol and gossypol dimethyl ether were 91.3 and 97.5%, respectively, with a within-day precision of 2.5% and a day-to-day precision of 3.8%. The limit of detection is 5 ng/ml (signal-to-noise ratio = 3:1). The method is considered to be suitable for the clinical pharmacokinetic studies of gossypol.     

Sensitive determination of josamycin and rokitamycin in plasma by high-performance liquid chromatography with fluorescence detection.

Rokitamycin and josamycin were successfully derivatized with dansylhydrazine in 20 min at 60 degrees C. Rokitamycin and josamycin levels were determined in plasma after ion-pair extraction into hexane-isoamyl alcohol with lauryl sulphate and precolumn derivatization. Resolution was obtained by liquid chromatography with fluorescence detection (352/537 nm) in 12 min. The limit of detection was 20 ng/ml macrolide starting from 1 ml of plasma, and linearity was demonstrated between 50 and 400 ng/ml. Inter-run coefficients of variation were 10.2% at 100 ng/ml and 9.1% at 300 ng/ml. The system was reliably used for pharmacokinetic studies in plasma.     

High-performance liquid chromatographic assay for the alpha-melanotropin[4,10] fragment analogue (Melanotan-II) in rat plasma.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of Melanotan-II (MT-II), a cyclic heptapeptide which promotes rapid tanning of the skin, in rat plasma. The method involves precipitation of plasma proteins followed by direct-injection HPLC with ultraviolet detection. Calibration curves were linear over the range 100-1000 ng/ml for rat plasma. The method is reproducible and reliable with a detection limit of 50 ng/ml in plasma. Within- and between-day precision and accuracy reported as coefficient of variation and relative error, respectively, were < 7%. The application of the assay was successfully demonstrated by quantifying the concentration of MT-II in rat plasma samples following an intravenous dose of 0.3 mg/kg.     

Analysis of terbutaline in human plasma by high-performance liquid chromatography with electrochemical detection using a micro-electrochemical flow cell.

A high-performance liquid chromatographic method is described for the determination of terbutaline in human plasma in the range 1-35 ng/ml. Detection was achieved using a carbon fibre micro-electrochemical detector and a column-switching system. The microelectrode cell has advantages over conventional glassy carbon electrode-based detection systems in that it is easy to prepare, flexible in its operation and suffers less trouble from problems such as air bubbles and leaks. Furthermore, it has a better detection limit for terbutaline (0.8 ng/ml) to that obtained using a conventional glassy carbon electrode flow detector (2 ng/ml). Sample clean-up was by on-line solid-phase extraction with column switching, providing a method which was sensitive and reproducible, where the mean overall coefficient of variation was 5.60% and drug recovery in excess of 86% at the concentration levels studied.     

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.     

High-performance liquid chromatographic method for the quantitation of bupivacaine, 2,6-pipecoloxylidide and 4'-hydroxybupivacaine in plasma and urine.

A high-performance liquid chromatographic method with ultraviolet detection at 210 nm for quantitation of bupivacaine and two of its metabolites from plasma and urine is described. The compounds are extracted into n-hexane-isopropanol (5:1), evaporated and the reconstituted residue injected onto a reversed phase C18 column. Standard curves for all compounds were linear (r2 greater than 0.999) in the range 20-2000 ng/ml, with a limit of detection of 10 ng/ml. The inter-day coefficients of variation ranged between 2.7 and 12.2%. The method was applied to analyse bupivacaine and metabolite concentrations in patients on long-term epidural bupivacaine-fentanyl infusions.     

Analysis of ethambutol and methoxyphenamine by capillary electrophoresis with electrochemiluminescence detection

A capillary electrophoresis (CE) coupled with electrochemiluminescence (ECL) detection method for the analysis of ethambutol (EB) and methoxyphenamine (MP) has been investigated. Complete separation of EB and MP was achieved in 8 min using a background electrolyte of 20 mM sodium phosphate at pH 10.0 and a separation voltage of 9 kV. ECL detection was performed with an indium/tin oxide (ITO) working electrode biased at 1.4 V (versus a Pt wire reference) in a 200 mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)3(2+) (where bpy = 2,2'-bipyridyl). Linear correlation (r > or = 0.993) between ECL intensity and drug concentration was obtained in the range 2-50 ng/ml. The limits of detection (LODs) for EB and MP in water were 1.0 and 0.9 ng/ml, respectively. The relative standard deviation values on peak size (10 ng/ml level) and migration time for the two drugs were in the ranges 5-8 and 0.2-0.7% (n = 7), respectively. Applicability of the CE-ECL method to the analysis of human plasma spiked with EB and MP was examined. The LODs for EB and MP in plasma were 0.4 and 0.3 microg/ml, respectively.     

Determination of a potential antiasthmatic compound, tibenelast, and its application to phase I clinical trials

A sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of tibenelast, 5,6-diethoxybenzo[b]thiophene-2- carboxylic acid, in plasma and urine. The plasma assay involves protein precipitation with 4% trichloroacetic acid, while the urine assay is an automated solid-phase extraction procedure that utilizes the Waters Millilab workstation. The analysis was achieved by reversed-phase HPLC with ultraviolet detection at 313 nm. The quantitation limit of the assay was 50 ng/ml in plasma and 100 ng/ml in urine. The intra-day coefficient of variation for the plasma analysis was between 2.2 and 8.4%, while the overall inter-day coefficient of variation was 5.5 and 6.0% for the high and low calibration curves, respectively. The intra-day coefficient of variation for the urine analysis was between 0.3 and 3.0%, while the inter-day coefficient of variation was 2.1% for both the low and high validation samples. The assay methodology has been used in the evaluation of samples from pharmacokinetic and clinical safety studies.     

Determination of malotilate and its metabolites in plasma and urine

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.     

Improved high-performance liquid chromatographic determination with amperometric detection of alpha-amanitin in human plasma based on its voltammetric study

A sensitive and simple method is described for the selective determination in human plasma of alpha-amanitin, the most poisonous and prevalent toxin in the lethal fungi of species Amanita, using high-performance liquid chromatography with amperometric detection. After an extraction of plasma with disposable C18 silica cartridges, the extracts were separated by isocratic reversed-phase chromatography using a macroporous poly(styrene-divinylbenzene) column and a mobile phase of 0.05 M phosphate buffer-acetonitrile (91:9) at the apparent pH of 9.5. Amperometric detection was performed by applying an oxidation potential as low as +350 mV (vs. Ag/AgCl) to a glassy carbon electrode, in a thin-layer flow-cell. The linear range for alpha-amanitin was 3-200 ng/ml, and the relative limit of detection in plasma was 2 ng/ml at a signal-to-noise ratio of 2. The intra-assay precision was evaluated at levels of 10 and 200 ng/ml; the coefficients of variation were 4.5 and 2.6% (n = 5), respectively. Inter-assay coefficients of variation were 6.5 and 4.2% (n = 5) for the same concentrations of toxin. These analytical conditions have been chosen on the basis of a preliminary in batch cyclic voltammetric investigation of alpha-, beta- and gamma-amanitins, which has allowed their oxidation process to be clarified and the pH dependence of their oxidation potentials to be determined. All three amanitins are oxidized at the same potential values, and adsorption onto the electrode surface of both reactant and products was found in all cases. This adsorption did not affect the signal recorded for alpha- and gamma-amanitins at the amperometric detector, and for beta-amanitin a stronger adsorption for the anodic product was found, which leads to a marked positive shift of the potential required for the oxidation of this isomer in the amperometric detector cell.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

High-performance liquid chromatographic procedure for the determination of tiagabine concentrations in human plasma using electrochemical detection.

A sensitive and precise high-performance liquid chromatographic procedure has been developed for the measurement of tiagabine concentrations in human plasma. Isolation of tiagabine and the internal standard was achieved using solid-phase extraction on disposable C8 columns. Separation was performed on a C18 analytical column using a mobile phase containing sodium octanesulfonate. The effluent was monitored with coulometric electrochemical detection at ca. + 0.76 V. The workup procedure recovered more than 95% of tiagabine from plasma. Standard curves were linear over the concentration range 0-500 ng/ml. The precision of the method was good: coefficients of variation were typically less than 5% for concentrations as low as 8 ng/ml and although they were higher at concentrations less than 8 ng/ml, they remained within acceptable limits (less than 17%) for concentrations as low as the limit of quantitation (2 ng/ml using a l-ml plasma sample). The stability of tiagabine in plasma was excellent, with no evidence of degradation after 23 h at room temperature or 2 months at -20 degrees C.     

Rapid high-performance liquid chromatographic method for the determination of propranolol levels in canine and feline plasma.

A sensitive high-performance liquid chromatographic method that does not require organic extraction has been developed for the determination of propranolol levels in canine and feline plasma. Equal volumes of plasma and a mixture of methanol-acetonitrile-0.1 M sodium hydroxide (3:3:4, v/v/v) were added to a microseparation unit with a 10,000 molecular mass cut-off filter. The ultrafiltrate was analyzed by reversed-phase liquid chromatography with fluorimetric detection. The consistency of the recoveries obtained eliminated the need for an internal standard (coefficients of variation less than 4%). Linear regressions for the standard curves (2.5-100 ng/ml) gave correlation coefficients above 0.9955. The detection limit was 1 ng/ml. The assay retains high sensitivity while eliminating laborious sample preparation.     

Adsorptive stripping voltammetric assay of phenazopyridine hydrochloride in biological fluids and pharmaceutical preparations

A sensitive method for the measurement of phenazopyridine hydrochloride (PAP) by differential pulse polarography (DPP) based on adsorptive stripping technique, using a hanging mercury drop electrode (HMDE) is described. The voltammetric peak is obtained at -0.760 V, which corresponds to the reduction of the azo group in Britton-Robinson buffer. The redox behaviour is reversible. Optimum conditions were found to be: accumulation potential -50 mV (vs. Ag/AgCl), accumulation time 60 s, scan rate 5 mV s(-1), pulse amplitude -100 mV and supporting electrolyte Britton-Robinson buffer (0.04 M, pH=11). The relative standard deviation (at 20 ng ml(-1) level) was +/-0.6% for six measurements. The calculated detection limit was 0.0299 ng ml(-1) with a 60-s accumulation time. The applicability of such a method was evaluated through the assay of PAP in human plasma and urine samples after a simple extraction procedure and in pharmaceutical preparation. The mean recovery was 97+/-2 (100 ng ml(-1) plasma).