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.     

Direct analysis of the dopamine agonist (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin hydrochloride in plasma by high-performance liquid chromatography using two-dimensional column switching.

A reversed-phase, two-dimensional, liquid chromatographic method incorporating column switching and electrochemical detection was used for the direct analysis of the dopamine (D2) agonist (-)-2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin hydrochloride in plasma. Sample work-up consisted of addition of internal standard, filtration, then direct injection of the plasma sample onto an internal surface reversed-phase (ISRP) guard column where the dopamine agonist and internal standard were separated from plasma proteins. An automated pneumatic valve was then used to switch to a stronger eluent which stripped the retained substances from the ISRP support onto a C18 analytical column where the analytes were separated from endogenous biological interferences. A dual-electrode electrochemical detector was used to minimize interferences and provide the desired sensitivity. The method has a detection limit of 1.5 ng/ml and requires a total assay time of 20 min per plasma sample. The method is linear from 1.5 to 1000 ng/ml and yielded greater than 80% drug recovery for plasma concentrations greater than 10 ng/ml. Precision for the method at 100 ng/ml yielded a relative standard deviation of 4.4%. Reproducibility was within 6.5% on a 20 ng/ml spiked plasma sample assayed on different days by different people. The method has successfully been applied to human plasma samples and for pharmacokinetic studies in rats and monkeys.     

Simultaneous determination of vinburnine and 6-hydroxyvinburnine in human plasma by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method has been developed to allow the simple and rapid determination of both vinburnine (I) and its main metabolite, 6-hydroxyvinburnine (II), in heparinized human plasma (0.5 ml). Compounds I and II and p-chlorodisopyramide (internal standard) were first extracted with alkalinized ethyl acetate and then with sulphuric acid. Separation was achieved on a reversed-phase muBondapak C18 column with a mobile phase of acetonitrile-water-0.1 M heptanesulphonate in acetic acid and with detection at 254 nm. Each run required 20 min. The within-day coefficients of variation for identical samples (20 ng/ml) were 7 and 6% and between-day coefficients of variation 8 and 26% for I and II, respectively. The detection limit was 5 ng/ml (normal therapeutic concentration, 10-300 ng/ml). The application of the method to drug monitoring was compared to that of a thin-layer chromatographic procedure.     

Kinetic spectrophotometric determination of josamycin in formulations and spiked human plasma using 3-methylbenzothiazolin-2-one hydrazone/Fe+3 system

A simple kinetic spectrophotometric method was developed for the determination of josamycin in its dosage forms and spiked human plasma. The method is based on reaction of the drug with 3-methylbenzothiazolin-2-one hydrazone/ferric chloride system for a fixed time of 20 min at 70 degrees C and measuring the produced color at 665 nm. The absorbance-concentration plot is rectilinear over the range of 5.0-30.0 microg/mL with detection limit of 1.0 microg/mL (1.2 x 10(-6) M). The determination of josamycin by the fixed concentration and the rate-constant methods is also feasible with the calibration equations obtained, but the fixed-time method proved to be more applicable. The procedure was successfully applied to commercial tablets. The results obtained were favorably compared with those given by reference methods. The method was further extended to the in vitro determination of josamycin in spiked human plasma. The recovery (n = 8) was 100.76 +/- 3.43%. The stoichiometry of the reaction between the drug and the reagent was studied by adopting the limiting logarithmic method, and a proposal of the reaction pathway was presented.     

High Performance Liquid Chromatographic Determination of Amiodarone in Plasma and Tissues

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of amiodarone (AD) in plasma and tissues was developed. The method involved deproteinization of plasma or homogenized tissue with acetonitrile containing an internal standard (N-Cetylpyridinium chloride) followed by reversed phase chromatography using μ bondapack C₁₈ column (10μm) with a mobile phase consisting of acetonitrile - methanol - sodium dihydrogen phosphate buffer (70:10:20%, v/v), the pH adjusted to 4.0 and pumped at flow rate of 1.0 ml/min. The column effluent was monitored at 242 nm. A linear relationship was obtained between peak height ratios (drug to internal standard) versus drug levels over the concentration range of 50–750 ng/ml. The detection limit of AD in plasma and tissues by this method was 20 ng/ml.     

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.     

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.     

Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction

A sensitive, enantioselective, high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-bisoprolol in human plasma. Baseline resolution was achieved using the teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100 : 0.02 : 0.025, v/v/v) at a flow rate of 1.5 ml/min and fluorescence detection set at 275 nm for excitation and 305 nm for emission. All analyses with S-(-)-atenolol as the internal standard were conducted at ambient temperature. The assay involved the use of a solid-phase extraction procedure for human plasma samples prior to HPLC analysis. The C18 cartridge gave good recovery rates for both enantiomers without any interference. The method was validated over the range of 20-200 ng/ml for each enantiomer concentration. Recovery rates for S-(-)- and R-(+)-bisoprolol enantiomers were in the range of 95-102%. The method proved to be precise (within-run precision expressed as % RSD ranged from 1.0-6.2% and between-run precision ranged from 0.9-6.7%) and accurate (within-run accuracies expressed as percentage error ranged from 0.2-4.8% and between-run accuracies ranged from 0.3-1.7%). The limit of quantitation and limit of detection for each enantiomer in human plasma were 20 and 5 ng/ml, respectively.     

Simultaneous determination of thioridazine and its S-oxidized and N-demethylated metabolites using high-performance liquid-chromatography on radially compressed silica

A method for simultaneously quantifying thioridazine, northioridazine, thioridazine-2-sulfoxide, thioridazine-2-sulfone and thioridazine-5-oxide in serum and plasma is described. Following solvent extraction these compounds were separated by high-performance liquid chromatography on radially compressed silica gel and detected by UV absorbance at 254 nm. Chromatography time is less than 7 min. The relative retention of these compounds as a function of the methanol and methylamine content of the mobile phase is discussed. Practical limits of detection, based upon on assayed plasma or serum volume of 1 ml, were 20 ng/ml for thioridazine-5-oxide and 10 ng/ml for the other compounds. The coefficient of variation for all compounds was less than 13%. The method is compared with more conventional high-performance liqiud chromatographic and gas chromatographic methodology.     

High Performance Liquid Chromatographic Separation of Tropatepine in Biological Fluids. Application to a Healthy Volunteer

Abstract

A high performance liquid chromatographic method for the determination of tropatepine in human plasma and urines is described here. After addition of an internal standard (2 chloro-11-(4-methyl piprazine 1-yl) dibenzo (b-f)(1–4) thiazepine) to the biological fluid and extraction at pH 12.0 in hexane, the analysis was performed on a reversed phase column (C18 microBondapak) with UV detection at 231 nm. The compound was eluted by a perchlorate buffer-acetonitrile mixture with a flow rate of 1.7 ml/min. The detection limit was about 25 ng/ml; reproducibility was around 7.5% for plasma concentrations below 50 ng/ml. Mass spectrometry by direct insertion probe had validated the chromatographic results. The method was successfully applied to plasma specimen collected from a healthy human volunteer following a single intravenous administration of 20 mg of tropatepine.     

Determination of tolperisone enantiomers in plasma and their disposition in rats.

A stereoselective assay for the determination of tolperisone enantiomers in plasma by high performance liquid chromatography was developed. Calibration curves obtained for the enantiomers were linear over plasma concentrations of 0.1-3.0 micrograms/ml with a detection limit of 20 ng/ml. Following intravenous bolus administration of 10 mg/kg of racemic tolperisone to rats, stereoselective disposition of tolperisone enantiomers was observed, and plasma concentrations were significantly higher for l-tolperisone than for d-tolperisone at 5, 15 and 30 min after administration. When either enantiomer was administered alone to rats, both enantiomers were found in plasma, indicating that a mutual chiral inversion occurs in the body.     

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.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

Determination of p-methylthiobenzamide and p-methylthiobenzamide-S-oxide from rat plasma using solid-phase extraction and high-performance liquid chromatography

p-Methylthiobenzamide (PMTB) is a thiocarbonyl compound exhibiting marked hepatotoxicity and nephrotoxicity. We describe a high-performance liquid chromatographic method for analyzing PMTB and a metabolite, p-methylthiobenzamide-S-oxide (PMTBSO), from rat plasma using a solid-phase extraction technique. In this way, PMTB and PMTBSO can be extracted from 0.5 ml of plasma and separation achieved by an ODS analytical column in as little as 9 min. The mobile phase used was methanol-water (55:45, v/v) and the wavelength for detection was 290 nm. The limits of detection in plasma were 15 ng/ml for PMTB and 33 ng/ml for PMTBSO; the absolute recovery from spiked plasma samples was greater than 84.4% for both compounds and the internal standard. The method was linear throughout the range used with correlation coefficients greater than 0.969. The intra-day accuracy ranged from 1.52 to 15.23% relative error for the PMTB concentration range 151-3025 ng/ml; accuracy of 4.97% or less was obtained for PMTBSO concentrations of 1672-20,068 ng/ml. The intra-day precision (coefficient of variation) of the procedure was found to be no greater than 5.28% for PMTB and 7.9% for PMTBSO. Inter-day accuracy and precision measurements were similar.     

Sensitive Determination of Piritramide in Human Plasma by High-Performance Liquid Chromatography

Abstract

A selective and sensitive method for the determination of piritramide in human plasma is described. After addition of 50 μl of 2 M ammonia and 20 μl of aqueous promethazine solution (100 ng/10 μ1) as an internal standard, 1 ml of plasma was extracted with 5 ml of toluene (extraction efficiency: 93.9 × 2.6%; mean × S. D.; n = 5). HPLC was performed with a phenyl hypersil NC-04 column, particle size 5 μm, 250 × 4 mm I. D.; mobile phase: 8 parts of acetonitrile and 2 parts of 10 mM potassium phosphate buffer (pH 3. 3). The flow rate was set to 2 ml/min and the column temperature was 22°C. The assay was linear in a concentration range of 3.75 ? 3000 ng/ml (r = 0.999), with a lower limit of detection of 3 ng/ml. The precision was determined using spiked plasma samples (15 ng/ml; 300 ng/ml), with coefficients of variation of 6.1 and 5.9% (intraday; n = 5) and 6.5 and 0.2% (interday; n = 3). In the range of 5.6 ? 1500 ng/ml, the accuracy of the assay was 2.82%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or postoperative analgesia.     

Direct determination of mitoxantrone and its mono- and dicarboxylic metabolites in plasma and urine by high-performance liquid chromatography

The simultaneous isolation and determination of mitoxantrone (Novantrone) and its two known metabolites (the mono- and dicarboxylic metabolites) were carried out using a high-performance liquid chromatographic (HPLC) system equipped with an automatic pre-column-switching system that permits drug analysis by direct injection of biological samples. Plasma or urine samples were injected directly on to an enrichment pre-column flushed with methanol-water (5:95, v/v) as the mobile phase. The maximum amount of endogenous water-soluble components was removed from biological samples within 9 min. Drugs specifically adsorbed on the pre-column were back-flushed on to an analytical column (Nucleosil C18, 250 X 4.6 mm I.D.) with 1.6 M ammonium formate buffer (pH 4.0) (2.5% formic acid) containing 20% acetonitrile. Detection was effected at 655 nm. Chromatographic analysis was performed within 12 min. The detection limit of the method was about 4 ng/ml for urine and 10 ng/ml for plasma samples. The precision ranged from 3 to 11% depending on the amount of compound studied. This technique was applied to the monitoring of mitoxantrone in plasma and to the quantification of the unchanged compound and its two metabolites in urine from patients receiving 14 mg/m2 of mitoxantrone by intravenous infusion for 10 min.     

High Performance Liquid Chromatographic Determination of (-)-N-Formylnorephedrine in Plasma

Abstract

An HPLC procedure for the detection and quantitative estimation of (-)-N-formylnorephedrine in rabbit plasma had been developed. The procedure involved the extraction of (-)-N-formylnorephedrine from plasma spiked with the internal standard (phenacetin), using ethyl acetate. The ethyl acetate extract is evaporated under nitrogen and the residue is reconstituted in water and injected onto the column. A u-Bondapak-C18 column 30 cm × 3.9 mm ID was used. The mobile phase is 20% acetonitrile in water; at a flow rate of 1.5 ml/min and uv detection at 256 nm. A linear relationship between concentration and peak height ratio (I/internal standard) was obtained (r = 1.00). The reported procedure allows the measurement of (-)-formylnorephedrine in concentrations as low as 150 ng/ml of plasma with total procedure time of about 10 min. The applicability of the procedure to pharmacokinetic studies is illustrated and metabolites are shown not to interfere with the assay procedure.     

Gas chromatographic assay for the new antitumor agent pyrazine-2-diazohydroxide (diazohydroxide) and its stability in buffer, blood and plasma

Diazohydroxide is a new antitumor agent being considered for clinical trial. A sensitive and specific assay for diazohydroxide in physiological media, plasma and blood has been developed based on conversion of diazohydroxide to 2-chloropyrazine in the presence of strong hydrochloric acid. The 2-chloropyrazine is extracted into the ethyl acetate and separated by capillary gas chromatography with nitrogen-phosphorus detection. Using 0.2 ml plasma the assay was linear up to 100 micrograms/ml diazohydroxide and had a lower limit of detectability for diazohydroxide of 50 ng/ml. The coefficient of variation of the assay at 1 micrograms/ml was 6.7%. Breakdown of diazohydroxide was rapid under mild acid conditions but slower under alkaline conditions,. The half-life of diazohydroxide in 0.1 M sodium phosphate buffer, pH 6.0, at room temperature was 5 min and at pH 8.0, 480 min. Breakdown of diazohydroxide in plasma was biphasic. In fresh mouse plasma diazohydroxide had a terminal half-life at 37 degrees C of 72 min while in fresh human plasma the terminal half-life was 23 min and in fresh blood 21 min. Diazohydroxide accumulated in red blood cells at 37 degrees C to a concentration 68% above the concentration in plasma. Diazohydroxide was 49% bound to human plasma proteins at room temperature.     

Ion-pair reversed-phase HPLC method for determination of sodium tanshinone IIA sulfonate in biological samples and its pharmacokinetics and biodistribution in mice

The ion-pair reversed-phase HPLC method for determination of sodium tanshinone IIA sulfonate (STS) in various biological samples was for the first time developed and validated, and was applied for pharmacokinetics and tissue distribution studies of intravenously administrated STS in mice. A linear relation was found between peak area and STS concentrations within the ranges of 0.1-5 micraog/ml for plasma, 0.1-5 microg/g of tissue for kidney homogenate, 0.1-20 microg/g of tissue for liver homogenate, 0.1-1 microg/g of tissue for heart, spleen and lung homogenates, respectively. In plasma and tissues, the limit of quantification (LOQ) and the limit of detection (LOD) for STS were 100 ng/ml and 20 ng/ml. In all biological specimens, the average inter- and intra-day precision of STS were within 4.9%. The recoveries were more than 92% at all concentration levels in each type of biological specimens. STS plasma concentration-time data were best fitted with a two-compartment model, characterized by an initial rapid phase of drug concentration decrease, and a slower terminal elimination phase. The pharmacokinetics of STS was characterized with a distribution half-life (t(1/2alpha)) of 1.2+/-0.18 min, a terminal half-life (t(1/2beta)) of 21.6+/-2.4 min, a distribution volume (V) of 0.057+/-0.011 l/kg, a plasma clearance (CL) of 0.86+/-0.12 l/h/kg and an AUC(0-infinity) of 58.41+/-6.21 microg x h/ml. STS was widely distributed into most tissues and was obviously accumulated in liver. This results indicated that STS may be promising to treat liver disease.     

Analysis of clomesone in plasma by gas chromatography-electrolytic conductivity detection

A sensitive and specific gas chromatographic (GC) method with electrolytic conductivity detection (ELD) for the analysis of clomesone (2-chloroethylmethylsulfonylmethane sulfonate), a new experimental antitumor alkylating agent, in plasma has been developed for the first time. Clomesone in plasma containing suitable internal standard was extracted with methylene chloride. After evaporation, the residue was analyzed by GC-ELD. Either a 15-m wide-bore DB-17 or a DB-1 column with the corresponding internal standards of propachlor or butachlor, respectively, was used. For the DB-1 column with butachlor as the internal standard, the routine assay limit was 20 ng/ml with linearity from 10 to 2000 ng/ml monitored. The within-run coefficient of variation of eight replicates at 50 ng/ml was 8.0% and the between-run coefficient of variation was 11% at 120 ng/ml. Using this assay procedure, the stability in several aqueous media and protein binding of clomesone were evaluated. In fresh mouse plasma, the half-life of clomesone was less than 1 h, although in aged pooled human plasma the drug was more stable. The mean protein binding value in mouse and human plasma was about 81-85%.