High-performance liquid chromatographic determination of oxodipine enantiomers, a new 1,4-dihydropyridine, applied to stereoselectivity studies in man and dog

A specific and reproducible HPLC method using a Chiral-AGP column and UV detection was developed for the evaluation of the pharmacokinetic profile of oxodipine enantiomers in dog and man. Each enantiomer was determined in plasma in the concentration range 1–400 ng/ml using the internal standard calibration method with linear regression analysis. After extraction of oxodipine and the internal standard at alkaline pH with diethyl ether—n-hexane (50:50, v/v), this method permitted the determination of each enantiomer at levels down to 10 ng/ml in dog plasma and 25 ng/ml in human plasma with sufficient accuracy (relative error <11%, n = 6) and precision (coefficient of variation <16%, n = 6). The extracted plasma volume was 500 μl and after evaporation of the organic phase, the dry residue was dissolved in 100 μl of water—2-propanol; an aliquot of 80 μl was injected into the HPLC system.     

Modified method of nalbuphine determination in plasma: validation and application to pharmacokinetics of the rectal route

A solid-phase extraction (SPE) procedure was developed for the quantification of nalbuphine in a small volume (500 μl) of human plasma with subsequent assay by high-performance liquid chromatography (HPLC) and electrochemical detection using 6-monoacetylmorphine as internal standard. Plasma was extracted using Bond Elute certified extraction columns (LCR: 10 ml, 130 mg) after conditioning with methanol and 0.2 M Tris buffer (pH 8). Elution was performed with a CH₂Cl₂-isopropanol-NH₄OH (79:20:, v/v). The organic phase was evaporated to dryness and resuspended in HPLC mobile phase containing 2% isopropanol. Linearity was assessed over the 5–100 ng/ml concentration range and a straight line passing through the origin was obtained. Experiments with spiked plasma samples resulted in recoveries of 95±5.4% and 98±6.2% for nalbuphine and 6-monoacetylmorphine, respectively. The optimal pH conditions for the SPE were found at pH 8. The intra-day coefficients of variation (C.V.) for 5, 40, and 100 ng/ml were 5.3, 3.0 and 2.3% (n=8) and the inter-day C.V.s were 7.7, 3.2 and 3.5% (n=10), respectively. The detection limit for 500 μl plasma sample was 0.02 ng/ml and the limit of quantification 0.1 ng/ml (C.V.=12.4%). The ease of the proposed method of analysis, as well as its high accuracy and sensitivity allow its application to pharmacokinetic studies. A preliminary kinetic profile of nalbuphine after rectal administration in a pediatric patient is presented.     

Determination of pyronaridine in blood plasma by high-performance liquid chromatography for application in clinical pharmacological studies

A method is described for the determination of pyronaridine in plasma using high-performance liquid chromatography with fluorescence detection. The method involves liquid-liquid extraction with phosphate buffer (pH 6.0, 0.05 M) and diethyl ether-hexane (70:30%, v/v) and chromatographic separation on a C₁₈ column (Nucleosil, 250 × 4.6 mm I.D., 5 μm particle size) with acetonitrile-0.05 M phosphate buffer pH 6.0 (60:40%, v/v) as the mobile phase (1 ml/min) and detection by fluorescence (λ_ex = 267 nm, λ_em = 443 nm). The detector response is linear up to 1000 ng and the overall recoveries pyronaridine and quinine were 90.0 and 60.3%, respectively. The assay procedure was adequately sensitive to measure 10 ng/ml pyronaridine in plasma samples with acceptable precision (< 15% C.V.). The method was found to be suitable for use in clinical pharmacological studies.     

Interaction of amitriptyline with muscarinic receptor subtypes in the rat brain

The affinity of amitriptyline for muscarinic receptors in rat brain areas was studied using autoradiographic techniques including image analysis. As shown by competitive inhibition of [³H]-l-quinuclidinyl benzilate binding, amitriptyline was found to be a potent inhibitor of muscarinic receptors throughout the rat brain. Muscarinic receptors in the external layers of the cortex displayed a high affinity for amitriptyline (IC₅₀ = 65.8 ± 2.1 nM), while the hippocampal regions had somewhat lower affinities (e.g. IC₅₀ = 96.3 ± 3.4 nM). Amitriptyline bound with lower affinity in the thalamus and various midbrain regions, such as the paraventricular nucleus of the thalamus and the superior colliculus, which had IC₅₀ values of 112 ± 6.8 and 117 ± 32.6 nM, respectively. Other midbrain regions displayed higher affinities, for example, the substantia nigra had an IC₅₀ value of 62.8 ± 0.9 nM. The data show that amitriptyline binds with high affinity to muscarinic receptors with a modest subtype selectivity that is unlike that of either pirenzepine or AF-DX 116. In addition, amitriptyline at concentrations of 10 nM-1 μM antagonized the oxotremorine-induced inhibition of acetylcholine release in cortical nerve endings, demonstrating activity at M₂ autoreceptors.     

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), in human plasma. Didanosine (2′,3′-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a μBondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)—methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.     

High-performance liquid chromatographic determination of bupivacaine in plasma samples for biopharmaceutical studies and application to seven other local anaesthetics

A sensitive analytical procedure for bupivacaine dosing in plasma samples by reversed-phase high-performance liquid chromatography is described. After a two-step extraction, the analysis was performed using a C₁₈ column and a mobile phase of 0.01 M sodium dihydrogen-phosphate (pH 2.1)—acetonitrile (80:20, v/v). The extraction yield of bupivacaine from plasma was 73.5 ± 5.1% (mean ± S.D., n = 10). The within-day and between-day reproducibilities at a concentration of 100 ng/ml were 2.1% and 5.6%, respectively (n = 10). Calibration curves were linear (r² = 0.9996) between 5 and 1000 ng/ml. The limit of detection, defined by a signal-to-noise ratio of 3:1, was 2 ng/ml. The accuracy at a concentration of 100 ng/ml was 2.3%. This method could be applied to the plasma analysis of seven other local anaesthetics (articaine, etidocaine, lidocaine, mepivacaine, pramocaine, procaine and tetracaine). The procedure was used in bioavailability studies of bupivacaine-loaded poly( -lactide) (i.e. PLA) and poly( -lactide-co-glycolide) (i.e. PLGA) microspheres after subcutaneous and intrathecal administrations in rabbits.     

Determination of sodium cromoglycate in human plasma by liquid chromatography–mass spectrometry in the turbo ion spray mode

A highly sensitivity liquid chromatography–tandem mass spectrometry method has been developed for the quantitation of sodium cromoglycate (SCG) in human plasma. The method was validated over a linear range of 0.100–50.0 ng/ml, using ¹³C₄ sodium cromoglycate as the internal standard. Compounds were extracted from 1.0 ml of lithium heparin plasma by methanol elution of C₁₈ solid-phase extraction cartridges. The dried residue was reconstituted with 100 μl of 0.01 N HCl, and 30 μl was injected onto the LC–MS–MS system. Chromatographic separation was achieved on a C8 (3.5 μm) column with an isocratic mobile phase of methanol–water–0.5 M ammonium acetate (35:64.8:0.2, v/v/v). The analytes were detected with a PE Sciex API 3000 mass spectrometer using turbo ion spray with positive ionization. Ions monitored in the multiple reaction monitoring (MRM) mode were m/z 469.2 (precursor ion) to m/z 245.1 (product ion) for SCG and m/z 473.2 (precursor ion) to m/z 247.1 (product ion) for ¹³C₄ SCG (I.S.). The average recoveries of SCG and the I.S. from human plasma were 91 and 87%, respectively. The low limit of quantitation was 0.100 ng/ml. Results from a 4-day validation study demonstrated excellent precision (C.V.% values were between 1.9 and 6.5%) and accuracy (−5.4 to −1.2%) across the calibration range of 0.100–50.0 ng/ml.     

Capillary gas chromatographic determination of permethrin insecticide by transesterification

A sensitive method was developed to determine permethrin extracted from phosphate buffer and cattle plasma by potassium cyanide catalyzed transesterification of this insecticide with refluxing ethanol and detection of the resulting ethyl esters by capillary gas chromatography with an electron capture detector. With a reflux time of 2 h and with 3-phenoxybenzyl 2-chlorobenzoate as an internal standard, linear calibration curves from buffer (5–250 ng) and plasma (5–100 ng) were obtained. Precision and accuracy of the method were 15%. The limit of detection was approximately 2.5 ng/ml (cis) and 1 ng/ml (trans) from buffer. In cattle sprayed along the back at 2 mg/kg, the concentration of cis- and trans-permethrin in plasma was below the detection limit (5 ng/ml).     

Stereoselective determination of mepivacaine in human serum using a brush-type chiral stationary phase and solid-phase extraction

A stereoselective high-performance liquid chromatography assay method was developed for the quantitation of R-(+)- and S_-(−)-mepivacaine in human serum. The assay uses a Pirkle brush-type. ((S)-tert.-leucine, (R)-(-naphthyl)ethylamine stationary phase (Sumichiral OA-4700, 250×4 mm I.D.) at ambient temperature with a mobile phase of hexane-ethylenedichloride-absolutte methanol (85:10:5, v/v) for the separation of R-(+) and (S)-(−)-mepivacaine. The eluents were monitored using UV detection at 220 nm. Isolation of the analytes from serum was performed using a 1-ml C₁₈ solid-phase extraction cartridge with high recovery and selectivity. The detection limits were 100 ng/ml for each enantiomer and the limits of quantitation were 150 ng/ml for both enantiomers. Linear calibration curves in the 150–2400 ng/ml range showed good correlation coefficients (r>0.9994, N=3). Precision and accuracy of the method were within 2.1–5.3 and 2.0–3.6%, respectively, for (R)-(+)-mepivacaine and 2.7–5.7% and 1.7–4.2%, respectively, for S-(−)-mepivacaine.     

Quantification of ketoprofen enantiomers in human plasma based on solid-phase extraction and enantioselective column chromatography

An HPLC method for the quantification of ketoprofen enantiomers in human plasma is described. Following extraction with a disposable C₁₈ solid-phase extraction column, separation of ketoprofen enantiomers and I.S. (3,4-dimethoxy benzoic acid) was achieved using a chiral column [Chirex 3005; (R)-1-naphthylglycine 3,5-dinitrobenzoic acid] with the mobile phase, 0.02 M ammonium acetate in methanol, set at a flow-rate of 1.2 ml/min. Baseline separation of ketoprofen enantiomers and I.S., free from interferences, was achieved in less than 20 min. The calibration curves (n = 14) were linear over the concentration range of 0.16 to 5.00 μg/ml per enantiomer [mean r² of 0.999 for both enantiomers, root mean square error were 0.015 for R(−) and 0.013 for S(+)]. The inter-day coefficient of variation for duplicate analysis of spiked samples was less than 7% and the accuracy was more than 93% over the concentration range of 0.2 to 4.0 μg/ml for individual enantiomer using 1 ml of plasma sample. This method has been applied to a pharmacokinetic study from healthy human volunteers following the administration of a ketoprofen extended release product (200 mg). This method is simple, fast and should find wide application in monitoring pharmacokinetic studies of ketoprofen.     

Measurement of nitrite and nitrate levels in biological samples by capillary electrophoresis

Nitrite is one of the products of NO-synthase in biological media. It is slowly oxidized in animals to nitrate. We developed a simple and rapid method to determine simultaneously nitrite and nitrate in biological samples. Capillary ion electrophoresis with direct UV detection at 214 nm was used employing a carrier electrolyte consisting of 10 mM sodium sulfate and an electroosmotic flow modifier. The detection limit in ultrafilates of plasma, urine and brain tissue extracts was 25 ng/ml for both compounds. Nitrate levels in human plasma and urine were in the μg/ml range. Nitrite could not be detected. Rat brain tissue extracts contained detectable amounts of nitrite and nitrate.     

Stereoselective determination of R-(+)- and S-(−)-remoxipride, a dopamine D2-receptor antagonist, in human plasma by chiral high-performance liquid chromatography

A stereoselective high-performance liquid chromatographic (HPLC) method is described for the selective and sensitive quantitation in human plasma of R-(+)- and S-(−)-enantiomers of remoxipride. Remoxipride was extracted from basified plasma into hexane-methyl-tert.-butyl ether (20:80, v/v), washed with sodium hydroxide (1.0 M), then back-extracted into phosphoric acid (0.1 M). A structural analog of remoxipride was used as an internal standard. The sample extracts were chromatographed using a silica-based derivatized cellulose chiral column, Chiralcel OD-R, and a reversed-phase eluent containing 30–32% acetonitrile in 0.1 M potassium hexafluorophosphate. Ultraviolet (UV) absorbance detection was performed at 214 nm. Using 0.5-ml plasma aliquots, the method was validated in the concentration range 0.02-2.0 μg/ml and was applied in the investigation of systemic inversion of remoxipride enantiomers in man.     

Determination of amphotericin B, liposomal amphotericin B, and amphotericin B colloidal dispersion in plasma by high-performance liquid chromatography

Amphotericin B is a potent polyene antifungal drug for intravenous treatment of severe infections. It is used as amphotericin B-deoxycholate and in order to reduce amphotericin B toxicity as lipid-formulated complex (liposomal or colloidal dispersion). A sensitive and specific analytical method is presented for the separation of lipid-complexed and plasma protein-bound amphotericin B in human heparinized plasma. This separation, which is required for pharmacokinetic studies, is achieved by solid-phase extraction (SPE) via Bond Elut C₁₈. The protein-bound amphotericin B has a higher affinity to the SPE material and is therefore retained, whereas the lipid-complexed amphotericin B is eluted in the first step. The recovery of the SPE was >75% for high concentrations and >95% for low concentrations. Quantification was performed by reversed-phase HPLC using a LiChrosorb-RP-8 column, UV detection (λ=405 nm) and a mixture of acetonitrile–methanol–0.010 M NaH₂PO₄ buffer (41:10:49, v/v) as mobile phase. The retention time for amphotericin B under the given conditions was 6.7 min. The calibration curves were found to be linear (r≥0.999) in two different ranges (5.0–0.50 μg/ml and 0.50–0.005 μg/ml). Intra- and inter-day precision and accuracy fulfilled the international requirements. No interference from other drugs (typical broad medication for intensive-care patients) or common plasma components was detected in >400 samples analyzed.     

Sensitive and rapid method for the simultaneous quantification of five antidepressants with their respective metabolites in plasma using high-performance liquid chromatography with diode-array detection

A high-performance liquid chromatographic method with diode array detection (HPLC-DAD) has been developed for the simultaneous separation and quantification of imipramine, amitriptyline, maprotiline, fluoxetine, clomipramine and their respective metabolites using a 500-μl plasma sample and clovoxamine as the internal standard. The substances were eluted on a Symmetry C₁₈ 5-μm column (250×4.6 mm, I.D.). Full UV spectra from 200 to 450 nm were recorded on-line during the entire analysis and were automatically compared to spectra stored in a library. The quantification was performed at 226, 254, and 400 nm. Peak height ratios were linear over a concentration range of 10–3000 ng/ml: the correlation coefficient (r) was better than 0.998 for all substances at each wavelength. Acceptable coefficients of variation are demonstrated for both within-run and day-to-day assays. The method is simple, highly specific and currently being used for drug monitoring and toxicological studies in children and adult patients.     

High-performance liquid chromatographic separation of ondansetron enantiomers in serum using a cellulose-derivatized stationary phase and solid-phase extraction

R(−)-Ondansetron and S(+)-ondansetron in human serum were resolved and quantified using a stereospecific HPLC method. Each enantiomer and the internal standard prazosin were isolated from serum using a solid-phase extraction procedure on a cyanopropyl column. Recoveries of 97, 96 and 88% were obtained for the R(−)-enantiomer, the S(+)-enantiomer, and the internal standard, respectively. A cellulose-based chiral analytical column (Chiralcel OD) was used with a mobile phase consisting of hexane—95% ethanol—2-propanol—acetonitrile (65:25:10:1, v/v). Linear calibration curves were obtained for each enantiomer in serum in the concentration range 10–200 ng/ml. The limit of quantitation of each enantiomer was 10 ng/ml. The detection limit for each enantiomer in serum using UV detection at 216 nm was 2.5 ng/ml (signal-to-noise ratio of 3).     

Determination of metoclopramide and two of its metabolites using a sensitive and selective gas chromatographic—mass spectrometric assay

A modified gas chromatographic—mass spectrometric (GC—MS) assay has been developed to quantitate metoclopramide (MCP) and two of its metabolites [monodeethylated-MCP (mdMCP), dideethylated-MCP (ddMCP)] in the plasma, bile and urine of sheep. The heptafluorobutyryl derivatives of the compounds were formed and quantitated using electron-impact ionization in the selected-ion monitoring mode (MCP, m/z 86, 380; mdMCP, m/z 380 and ddMCP, m/z 380). No interference was observed from endogenous compounds following the extraction of various biological fluids obtained from non-pregnant sheep. Sample preparation has been simplified and the method is more selective and sensitive (2 fold) than our previous assay using electron-capture detection. The limit of quantitation for MCP, mdMCP and ddMCP was 1 ng/ml in plasma, urine and bile, requiring 0.5 ml of sample. This represents 2.5 pg of the analytes at the detector. The standard curves were linear over a working range of 1–40 ng/ml. Absolute recoveries in plasma ranged from 76.5–94.7%, 79.2–96.8%, 80.3–102.2% for MCP, mdMCP and ddMCP, respectively. In urine, recoveries ranged from 56.5–87.8%, 61.5–87.5%, 62.6–90.2% for MCP, mdMCP and ddMCP, respectively. Recoveries in bile ranged from 83.5–100.9%, 78.5–90.5%, 66.9–79.2% for MCP, mdMCP and ddMCP, respectively. Overall intra-day precision ranged from 2.9% for MCP in plasma to 12.6% for mdMCP in bile. Overall inter-day precision ranged from 5.9% for MCP in urine to 14.9% for ddMCP in bile. Bias was the greatest at the 1 ng/ml concentration in all biological fluids ranging from a low of 2.4% for mdMCP in plasma to a high of 11.9% for ddMCP in urine. Applicability of the assay for pharmacokinetic studies of MCP, mdMCP and ddMCP in the plasma and urine of a non-pregnant ewe is demonstrated.     

Determination of Guan-Fu Base A, a new anti-arrhythmic, in human plasma by gas chromatography and electron–capture detection

A gas chromatographic–electron capture detection (GC–ECD) method has been developed for determining Guan-Fu Base A (GFA), an experimental anti-arrhythmic, in human plasma. The method was based on one-step liquid–liquid extraction with toluene and chemical derivatization with pentafluoropropionic anhydride followed by GC–ECD. The derivatives of GFA and metoprolol (Met, internal standard) were confirmed by gas chromatography–mass spectrometry (GC–MS) to be dipentafluoropropionyl-GFA and dipentafluoropropionyl-Met. The method was linear over the concentration ranges of 0.1–20.0 and 1.0–30.0 μg/ml with the detection limit of 0.05 μg/ml at S/N=5. The intra- and inter-assay precisions were less than 6 and 10%, and accuracy 99.70±3.30 and 97.60±5.99%, respectively. The absolute recoveries were 81.88, 77.35, 80.79 and 83.85% for GFA at concentrations of 0.5, 1.0, 5.0 and 14.0 μg/ml and 88.24% for Met at 3.0 μg/ml, respectively.     

High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C₁₈ column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.     

Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection

A rapid, selective, sensitive and reproducible HPLC with recutive electrochemical detection for quantitatvie determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: and β isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a μBondapak CN column. The method was capable of separating the two isomeric forms of DHA (, β). The retention times of -DHA, β-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80–640 ng/ml were 86–93%. The coefficients of variation were below 10% for all three drugs (ART, -DHA, ARN). The minimum detectable concentrations for ART and -DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.     

Determination of amitriptyline-n-oxide,amitriptyline and nortriptyline in serum and plasma by high-performance liquid chromatography

A method for the determination of amitriptyline-N-oxide, amitriptyline and nortriptyline in serum and plasma has been developed. After extraction from serum or plasma the drugs were analysed by high-performance liquid chromatography.The detection limit was 10 ng/ml (2 ml serum or plasma actually used). The coefficient of variation for all three compounds was below 10%.Amitriptyline-N-oxide was found in rat plasma after an oral dose (10 mg/kg) of amitriptyline-N-oxide.