Determination of bufuralol and its major metabolites in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of bufaralol, a benzofuran analogue, in plasma is described.The unchanged drug, the major metabolites and an internal standard are extracted from plasma, purified by back-extraction steps and thereafter separated using a reversed-phase liquid chromatographic system. The detection is carried out by means of a fluorescence detector and an UV detector connected in series. The sensitivity of the assay for the unchanged drug and the major metabolite is about 1 ng/ml plasma using a 0.5 ml specimen per analysis and the relative standard deviation of the whole assay lies in the range ± 4–5%.The procedure was successfully used to determine plasma levels in volunteers following a single oral dose of 40 mg of bufaralol. The results obtained using the new high-performance liquid chromatographic method were compared with those determined by another method which combines gas chromatography with mass fragmentography, and it was found that these two sets of results coincided quite well.     

Rapid and simple determination of inulin in biological fluids by high-performance liquid chromatography with light-scattering detection

We report a new high-performance liquid chromatography method developed for measuring inulin in plasma and urine using ion moderated partition chromatography and evaporative light-scattering detection. Samples are deproteinized with a zinc acetate and phosphotungstic acid solution and added with melezitose as an internal standard. The chromatographic separation is carried out in 16 min at a flow-rate of 0.6 ml/min using deionized water as the mobile phase. Within-run precision, measured at four different concentrations (0.050 mg/ml, 0.150 mg/ml, 0.300 mg/ml and 1.200 mg/ml), ranges from 1.7 to 3.4% in plasma and from 1.5 to 3.5% in urine. Similarly, between-run precision is in plasma from 2.0 to 4.3% and in urine from 2.0 to 4.4%. Analytical recovery ranges from 97.9 to 100.1% in plasma and from 99.1 to 99.7% in urine, respectively. Detection limit (signal-to-noise ratio=3) is 5 μg/ml both in plasma and urine. The method is simple, sensitive, without interference due to hexoses or drugs commonly taken by patients with renal diseases, and offers the advantage of measuring inulin without previous hydrolysis of the molecule.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.     

Validation of a simple liquid chromatography-tandem mass spectrometric method for the determination of propiverine hydrochloride and its N-oxide metabolite in human plasma

A simple high-performance liquid chromatography (HPLC)-tandem mass spectrometric method has been developed for determination of propiverine hydrochloride and its metabolite, propiverine N-oxide (M-1) in human plasma using stable isotopes, propiverine hydrochloride-d10 and M-1-d10, as internal standards. The analytes were extracted with dichloromethane from 0.2 ml of plasma in neutral condition (pH 7.0) and separated by HPLC on a C18 reversed-phase column using methanol-1% acetic acid (50:50) as a mobile phase, and detected using positive electrospray ionization in selected reaction monitoring (SRM) mode. The method was validated over a concentration range of 2-500 ng/ml for propiverine hydrochloride and 4-1000 ng/ml for M-1 using 0.2 ml of human plasma per assay. The method developed was successfully applied to analysis of propiverine hydrochloride and M-1 in clinical studies.     

Determination of ractopamine in monkey plasma and swine serum by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic (HPLC) method is described for the determination of ractopamine (LY031537) in monkey plasma and swine serum. Plasma or serum (0.5 ml) was diluted with phosphate buffer pH 7.0. Ractopamine was isolated from the plasma matrix using ion exchange on a polymeric carboxylic acid solid-phase extraction cartridge followed by partitioning with ethyl acetate. An isocratic HPLC method using electrochemical detection at +700 mV was used to separate and measure ractopamine in the purified extract in 6.5 min of run time. Standard area response was linear with respect to concentration of ractopamine over the range of 0.5 to 40 ng/ml. Validation data were collected using rhesus monkey plasma and swine serum. The method precision and accuracy were evaluated in the range 1.0 to 20 ng/ml using fortified samples of monkey plasma. The method limit of quantitation was estimated at 2 ng/ml as determined in monkey plasma.     

Quantitative determination of naproxen in plasma by a simple high-performance liquid chromatographic method

A high-performance liquid chromatographic method for the determination of naproxen in plasma is described. The technique is based on the single extraction of the drug from acidified plasma with chloroform using 2-naphthalene acetic acid as internal standard. The chromatographic system consisted of a column packed with Spherisorb ODS (5 μm); the mobile phase was acetonitrile—phosphoric acid (pH 3) (45:55, v/v).The method can accurately measure plasma naproxen concentrations down to 1 μg/ml using 100 μl of sample, with no interference from endogenous compounds. The coefficients of variation of the method at 120 μg/ml and 1 μg/ml are 2.8 and 21.6%, respectively, and the calibration curve is linear. The method described is very suitable for routine clinical and pharmacokinetic studies.     

Quantification of pravastatin in human plasma and urine after solid phase extraction using high performance liquid chromatography with ultraviolet detection

A high performance liquid chromatography (HPLC) method for the estimation of pravastatin in human plasma and urine samples has been developed. The preparation of the samples was performed by automated solid phase extraction using clonazepam as internal standard. The compounds were separated by isocratic reversed-phase HPLC (C(18)) and detected at 239 nm. The method was linear up to concentrations of 200 ng/ml in plasma and 2000 ng/ml in urine. The intra-assay variability for pravastatin in plasma ranged from 0.9% to 3.5% and from 2.5% to 5.3% in urine. The inter-assay variability ranged from 9.1% to 10.2% in plasma and from 3.9% to 7.5% in urine. The validated limits of quantification were 1.9 ng/ml for plasma and 125 ng/ml for urine estimation. These method characteristics allowed the determination of the pharmacokinetic parameters of pravastatin after administration of therapeutic doses.     

Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of caffeic acid phenethyl ester in rat plasma and urine

Caffeic acid phenethyl ester (CAPE) is one of the most bioactive compounds of propolis, a resinous substance collected and elaborated by honeybees. A new liquid chromatography-electrospray ionisation tandem mass spectrometric method was developed and validated for its determination in rat plasma and urine, using taxifolin as internal standard. After sample preparation by liquid/liquid extraction with ethyl acetate, chromatographic separations were carried out with an ODS-RP column using a binary mobile phase gradient of acetonitrile in water. Detection was performed using a turboionspray source operated in negative ion mode and by multiple reaction monitoring. The method was validated, showing good selectivity, sensitivity (LOD = 1 ng/ml), linearity (5-1000 ng/ml; r > or = 0.9968), intra- and inter-batch precision and accuracy (< or =14.5%), and recoveries (94-106%) in both plasma and urine. Stability assays have shown that CAPE is rapidly hydrolysed by plasmatic esterases, which are however inhibited by sodium fluoride. The method was applied to the determination of CAPE levels in rat plasma and urine after oral administration, showing that CAPE is rapidly absorbed and excreted in urine both as unmodified molecule and as glucuronide conjugate.     

Liquid chromatographic analysis of penem antibiotic FCE22101 in biological fluids with a liquid-liquid extraction procedure

A sensitive high-performance liquid chromatographic method has been developed for the determination of penem antibiotic FCE22101 in plasma and urine. FCE22101 was extracted from plasma and urine with ethyl acetate. After evaporating, the sample solutions were analyzed by a reversed-phase high-performance liquid chromatographic system using a two-sided bracketing injection technique. The determination limit of FCE22101 was 5 ng/ml in plasma. Analysis of the spiked plasma samples demonstrated the good accuracy and precision of the method. The proposed method improved from five- to ten-fold on the analytical sensitivity in comparison with the most commonly ultrafiltration method.     

Quantitative analysis of exogenous peptides in plasma using immobilized enzyme cleavage and gas chromatography-mass spectrometry with negative ion chemical ionization

A method is presented for the analysis of peptides in plasma at picomole to femtomole levels. Peptides are isolated from plasma by solid-phase extraction, the peptide of interest is purified by reversed-phase high-performance liquid chromatography (HPLC) and selectively digested using immobilized trypsin or chymotrypsin to yield specific di- or tripeptides. These di- and tripeptides are esterified using heptafluorobutyric anhydride, alkylated with pentafluorobenzyl bromide, then quantified by gas chromatography-mass spectrometry with negative ion chemical ionization. This method has been evaluated for a model synthetic heptapeptide, using a deuterium labeled analog as an internal standard. The half-life of the heptapeptide in human plasma was found to be 2 min. Extraction efficiencies of a tritiated peptide of similar size to the heptapeptide, [³H]DSLET, from plasma using either C₁₈ or strong cation-exchange columns were 85±3 and 70±2%, respectively. Quantitation of fragments from the heptapeptide indicated that the analysis was linear from 1–50 ng of the heptapeptide per ml of plasma. This method was subsequently employed for pharmacokinetic studies of the biologically active peptide Met-enkephalin-Arg-Gly-Leu, where linearity was obtained from 50 to 1000 ng/ml in rat plasma. This method demonstrated negligible side reaction by-products due to autolysis, and has potential for extensive use given the wide availability of gas chromatography-mass spectrometry.     

Column-switching high-performance liquid chromatographic method for the determination of a thymidylate synthase inhibitor, LY231514, an investigational agent for the treatment of solid tumors, in human plasma

A reversed-phase, column-switching high-performance liquid chromatographic (HPLC) method is described for the determination of a new thymidylate synthase inhibitor in human plasma. The compound and an internal standard are extracted from plasma using a Certify II solid-phase cartridge. Extracts are evaporated to dryness and the residue is reconstituted with mobile phase buffer. The analytes are separated from polar interferences and buffer salts originating from the elution step on a 4-mm YMC Basic pre-column. The fraction containing the analytes is further separated on a 25-cm YMC Basic column. The analytes are detected by their absorbance at 250 nm. The limit of quantitation is 10 ng/ml. The method is linear from 10 ng/ml to 80 μg/ml using three standard curve ranges. Validation studies for all three ranges show the method to be reproducible. The method has been successfully used to support pharmacokinetic studies.     

Determination of a new bisphosphonate, YM175, in plasma, urine and bone by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the determination of disodium dihydrogen(cycloheptylamino)methylenebisphosphonate monohydrate (YM175) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is pretreated by Method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and removal of excess calcium ions by AG 50W-X8 resin. Plasma obtained in lower-dose clinical studies is treated by Method B, a more sensitive method using 10 ml of plasma, which is based on solid-phase extraction using a Sep-Pak C₁₈ cartridge coupled with Method A. Urine and bone are treated similarly to Method B. The chromatographic system consists of a mobile phase at pH 11, an alkali-stable column and an electrochemical detector operating in the oxidation mode. The determination limit is 5 ng/ml for Method A and 0.5 ng/ml for Method B in plasma, 1 ng/ml in urine, and 25 ng/g in bone.     

Determination of picogram levels of heptylphysostigmine in human plasma using high-performance liquid chromatography with fluorescence detection

A sensitive (50 pg/ml) method for the determination of heptylphysostigmine in human plasma is described. The procedure is based on liquid—liquid extraction of the drug from buffered plasma, and analysis of the concentrated organic extract using high-performance liquid chromatography on a silica column, under normal-phase chromatographic conditions, with fluorescence detection. Physostigmine was used as an internal standard. The assay has been fully validated in the concentration range 50–2000 pg/ml and utilized for the analysis of clinical samples from subjects dosed with heptylphysostigmine.     

Determination of catechins in human plasma after commercial canned green tea ingestion by high-performance liquid chromatography with electrochemical detection using a microbore column

Determination of catechins in human plasma was carried out by high-performance liquid chromatography with electrochemical detection using a microbore octadecylsilica column. Peak heights for catechins were found to be linearly related to the amount of each catechin injected, from 2 pmol/ml to 2 nmol/ml (r>0.999). Conjugated-form catechins in plasma were hydrolyzed enzymatically using beta-glucuronidase and sulfatase. Catechins in plasma and the hydrolyzed solution were extracted with ethyl acetate and determined by the present method. The time courses of concentrations of catechins in human plasma showed maxima at 1-2 h after ingestion of 340 ml of commercial canned green tea.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

Determination of physostigmine in plasma by high-performance liquid chromatography and fluorescence detection

Physostigmine (PHY) is an anticholinergic drug used in the treatment of neuromuscular disorders and organophosphate poisoning. We described a sensitive, accurate, and reproducible method for PHY determination in biological materials. The method utilized a liquid/liquid, ion pair extraction, normal phase HPLC separation, and fluorometric quantitation at 240 nm excitation and 360 nm emission wavelength. We used neostigmine as a stabilizing agent to protect PHY from degradation and dimethylphysostigmine as an internal standard. The peak-height ratio vs concentration was linear over a working range from 0.50 to 25.0 ng/ml of PHY in plasma. Sensitivity of the method was 100 pg/ml of plasma which was the limit of quantitative detection under the experimental conditions used. Precision of the method was evaluated using plasma spiked with two concentrations of PHY: 1.0 and 10.0 ng/ml. Intra-day coefficient of variation (CV) ranged from 3.8 to 5.3%, and inter-day CV ranged from 1.8 to 3.6% for the two levels. The average recovery was 92%. We applied the method to examine the stability of PHY in plasma stored at -15 and -80 degrees C. The data indicated that PHY can be stored at either temperature for 9 weeks without undergoing significant alterations.     

High-performance liquid chromatographic method for determination of gentamicin in biological fluids

A selective and sensitive method for the determination of gentamicin in plasma and urine by high-performance liquid chromatography has been developed. Following deproteinization, the gentamicin is reacted with fluorescamine to produce a fluorescent derivative. This reaction mixture is directly chromatographed on a cation-exchange column using as mobile phase acetonitrile—phosphoric acid (7:3). The gentamicin components elute as a single peak. Using 0.1 ml of plasma, quantitation of gentamicin concentrations as low as 1 mg/l are possible. Possible interference from other aminoglycosides and antibiotics is discussed.     

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of sildenafil in human plasma

A method to determine sildenafil in human plasma involving liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed. Sildenafil and the internal standard (I.S.), diazepam, are extracted from human plasma with ether-dichloromethane (3:2, v/v) at basic pH and analyzed by reversed-phase high-performance liquid chromatography (HPLC) using methanol-10mM ammonium acetate pH 7.0 (85:15, v/v) as the mobile phase. Detection by electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode was linear over the concentration range 0.125-40.0 ng/ml. Intra- and inter-day precision of the assay at four concentrations within this range were 2.5-8.0%. The method was used to evaluate plasma concentration-time profiles in healthy volunteers given an oral dose of 20mg sildenafil as a combination tablet also containing apomorphine.     

Improved method for the rapid determination of isosorbide dinitrate in human plasma and its application in pharmacokinetic studies

A rapid, accurate and highly sensitive method was developed for the determination of isosorbide dinitrate in human plasma. Concentrations in the lower nanogram and subnanogram range are determined by a one-step extraction of 2 ml plasma, containing 4 ng/ml nitroglycerine as internal standard, with 5.5 ml n-pentane. The extract is subjected to gas—liquid chromatography—electron capture detection analysis. The lower limit of quantitation is 200 pg/ml, but concentrations as low as 50 pg/ml are still detectable. The method allows the quantitative determination of isosorbide dinitrate plasma levels in man following a 5 mg sublingual administration up to four hours after application.