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

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

High-performance liquid chromatographic determination of glipizide in human plasma and urine

A sensitive and selective high-performance liquid chromatographic method for determination of intact glipizide in human plasma or urine has been developed. The plasma and urine samples were acid-buffered, before tolbutamide was added as internal standard. The samples were extracted with benzene, and the organic layer was evaporated to dryness. The residue was dissolved in equilibrated mobile phase (acetonitrile-0.01 M phosphate buffer pH 3.5, 35:65), and an aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column. Quantitation was achieved by monitoring the ultraviolet absorbance at 275 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents were observed. The utility of the assay was demonstrated by determining glipizide in samples from a diabetic subject receiving a therapeutic dose of 5 mg of the drug.     

High-performance liquid chromatographic determination of nafazatrom in human plasma using fluorescence detection

A rapid, sensitive, and selective high-performance liquid chromatographic assay was developed for determination of the pyrazole derivative nafazatrom (Bay g 6575, NFZ) in human plasma. Separation was obtained using a normal-phase Si-60 column and a mobile phase of methylene chloride--methanol (90:10, v/v) containing 0.25% water. The fluorescence of NFZ was monitored at excitation and emission wavelengths of 232 and 362 nm, respectively. The recovery of NFZ extracted from plasma with methylene chloride was 109 +/- 5% (mean +/- S.D.) in the concentration range from 5.0 to 500 ng/ml. The assay was applied to the determination of plasma concentrations of NFZ following administration of the compound to patients in a Phase I clinical trial.     

Plasma analysis of celiprolol by HPTLC: a useful technique for pharmacokinetic studies

A rapid and sensitive high performance, thin-layer chromatographic (HPTLC) method has been developed for the measurement of celiprolol in human plasma and its use in pharmacokinetic studies has been evaluated. Detection and quantitation were performed without using an internal standard. A simple extraction procedure was followed for extracting celiprolol from plasma and a known amount of the extract was spotted on precoated silica gel 60 F254 plates using a Camag Linomat IV autosampler. Celiprolol was quantitated using a Camag TLC Scanner 3. The average recovery of authentic analytes (20 to 200 ng/mL) added to plasma was 72.06 +/- 2.8% and the lowest amount of celiprolol that could be detected was 10 ng/mL. The method provides a direct estimate of the amount of celiprolol present in plasma. Pharmacokinetic parameters of 2 marketed preparations have also been determined after oral administration to 12 healthy human volunteers.     

Gradient high-performance liquid chromatographic assay for the determination of the novel indoloquinone antitumour agent E09 in biological specimens

A high-performance liquid chromatographic method for the determination of the novel indoloquinone antitumour agent E09, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta-e n-alpha - ol, in mouse plasma and urine is described. Following protein precipitation by means of methanol (2 volumes), separation and quantification of parent drug, metabolites and internal standard E012 (5-morpholine substituted analogue) were achieved on a 5-microns Resolve C18 Rad-Pak with a 15-min linear gradient of 10-30% acetonitrile in a 0.02 M pH 7.4 sodium phosphate buffer with UV detection at 280 and 310 nm. The utility of the assay is also demonstrated for the aziridine ring-opened analogue E05A. 3-hydroxymethyl-5-beta-hydroxyethylamino-2-(1H-indole-4,7-dione)pr op-beta-en- alpha-ol. Plots of area ratios of analytes versus internal standard were linear in the range 50-15,000 ng/ml. The detection limit for indoloquinones in plasma was ca. 30 ng/ml. The within-assay and day-to-day variation were consistently lower than 12.5%. The assay was applied in preliminary pharmacokinetic investigations. One minor metabolite of E09 could be identified; further metabolites were characterized by ultraviolet-visible spectra.     

Simultaneous determination of the camptothecin analogue CPT-11 and its active metabolite SN-38 by high-performance liquid chromatography: application to plasma pharmacokinetic studies in cancer patients.

CPT-11 (I; 7-ethyl-10-[4-(1-piperidino)-1- piperidino]carbonyloxycamptothecin) is a new anticancer agent currently under clinical development. A sensitive high-performance liquid chromatographic assay suitable for the simultaneous determination of I and its active metabolite SN-38 (II) in human plasma, and their preliminary clinical pharmacokinetics, are described. Plasma samples were processed using a solid-phase (C18) extraction step allowing mean recoveries of I, II and the internal standard camptothecin (III) of 84, 99 and 72%, respectively. The extracts were chromatographed on a C18 reversed-phase column with a mobile phase composed of acetonitrile, phosphate buffer and heptanesulphonic acid, with fluorescence detection. The calibration graphs were linear over a wide range of concentrations (1 ng/ml-10 micrograms/ml), and the lower limit of determination was 1 ng/ml for both I and II. The method showed good precision: the within-day relative standard deviation (R.S.D.) (5-1000 ng/ml) was 13.0% (range 4.9-19.4%) for I and 12.8% (6.7-19.1%) for II; the between-day R.S.D. (5-10,000 ng/ml was 7.9% (5.4-17.5%) for I and 9.7% (3.5-15.1%) for II. Using this assay, plasma pharmacokinetics of both I and II were simultaneously determined in three patients receiving 100 mg/m2 I as a 30-min intravenous infusion. The mean peak plasma concentration of I at the end of the intravenous infusion was 2400 +/- 285 ng/ml (mean +/- standard error of the mean). Plasma decay was triphasic with half-lives alpha, beta and gamma of 5.4 +/- 1.8 min, 2.5 +/- 0.5 h and 20.2 +/- 4.6 h, respectively. The volume of distribution at steady state was 105 +/- 15 l/m2, and the total body clearance was 12.5 +/- 1.9 l/h.m2. The maximum concentrations of the active metabolite II reached 36 +/- 11 ng/ml.     

Determination of estazolam in plasma by high-performance liquid chromatography with solid-phase extraction.

A high-performance liquid chromatography (HPLC) assay was developed for the determination of estazolam in human plasma. Estazolam and alprazolam as an internal standard were detected by ultraviolet absorbance at 240 nm. Estazolam in plasma was extracted by a rapid and simple procedure based on cyanopropyl bonded-phase extraction. Chromatographic separation was achieved with a reversed-phase C8-5 column using a mobile phase of 0.5% potassium dihydrogenphosphate(pH 4.5)-acetonitrile (70:30, v/v). The determination of estazolam was possible in the concentration range of 1.0 - 200.0 ng/mL. The mean recovery of estazolam added to plasma was 96.1 +/- 1.5% with coefficients of variation of less than 5.5%. This method is applicable for accurately monitoring the plasma level of estazolam in healthy subjects participating in scientific research.     

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H- pyrido [2,1-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[ 2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with acetonitrile--methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido [2,1-b]-quinazoline-8-carboxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 +/- 8.6% and 107.0 +/- 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I . 2HCl, a 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.     

Determination of the anti-allergenic agent, 2-methoxy-11-oxo-11H-pyrido[2,1-b]quinazoline-8-carboxylic acid, in biological fluids by high-performance liquid chromatography.

A rapid, sensitive, and specific high performance liquid chromatographic (HPLC) assay was developed for the determination of 2-methoxy-11-oxo-11H-pyrido-[2,1-b]quinazoline-8-carboxylic acid (I) from biological fluids. The overall recovery from blood and plasma is 69 +/- 10% (S.D.) and 84 +/- 6% (S.D.), respectively, and the sensitivity limit of quantitation is 100 ng/ml by UV absorption and 5 ng/ml by fluorescence detection using a 1 ml specimen. The assay was used in the determination of blood levels of compound in the Rhesus monkey following intravenous administration of a 10 mg/kg dose, and of blood and urine levels of compound I in a dog following intravenous and oral administration of a 1 mg/kg dose.     

High-performance liquid chromatographic determination of glibenclamide in human plasma and urine

A selective and sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma or urine has been developed. With glibornuride as internal standard, acid-buffered plasma or urine was extracted with benzene. The organic layer was evaporated and the residue was dissolved in equilibrated mobile phase (acetonitrile-phosphate buffer 0.01 M pH 3.5, 50:50). An aliquot of 20 microliters was chromatographed on a Spherisorb ODS reversed-phase column, and quantitation was achieved by monitoring the ultraviolet absorbance at 225 nm. The response was linear (0-1000 ng/ml) and the detection limit was 5-10 ng/ml in plasma or urine. The within-assay variation was less than or equal to 10%. No interferences from metabolites or endogenous constituents could be noted. The utility of the method was demonstrated by analysing glibenclamide in samples from diabetic subjects on therapeutic doses of the drug.     

A sensitive and selective liquid chromatographic/electrospray ionization tandem mass spectrometric assay for the simultaneous quantification of alpha-,beta-arteether and its metabolite dihydroartemisinin in plasma,useful for pharmacokinetic studies

A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay method was developed and validated for the simultaneous quantitation of alpha-,beta-arteether (alpha-,beta-AE) and its metabolite alpha-dihydroartemisinin (DHA) in monkey plasma using the propyl ether analogue of beta-arteether (PE) as an internal standard. The method involves a simple two-step liquid-liquid extraction with hexane. The analytes were chromatographed on a C(18) reversed-phase chromatographic column by isocratic elution with methanol-ammonium acetate buffer (pH 4) (92 : 8, v/v) and analysed by mass spectrometry in the multiple reaction monitoring mode. The chromatographic run time was 7 min and the weighted (1/x(2)) calibration curves were linear over the range 0.78-200 ng ml(-1). The method was validated in terms of accuracy, precision, absolute recovery, freeze-thaw stability, bench-top stability and re-injection reproducibility. The limit of detection and lower limit of quantification in monkey plasma were 0.39 and 0.78 ng ml(-1) respectively for all the analytes. The intra- and inter-batch precision and accuracy were found to be well within acceptable limits (<15%). All three analytes were stable even after three freeze-thaw cycles (deviation < 15%). The average absolute recoveries of alpha-,beta-AE, DHA and PE, used as an internal standard, from spiked plasma samples were 85.85 +/- 6.56, 70.10 +/- 7.06, 54.37 +/- 3.39 and 93.90 +/- 6.9%, respectively. The assay method described here could be applied to study the pharmacokinetics of alpha-,beta-AE and DHA in rhesus monkeys.     

Stereoselective determination of demethyl- and didemethyl-citalopram in rat plasma and brain tissue by liquid chromatography with fluorescence detection using precolumn derivatization

A rapid and sensitive HPLC enantioselective method with fluorescence detection was developed to determine (-)-(R) and (+)-(S) enantiomers of the metabolites of citalopram, demethyl- and didemethyl-citalopram in plasma and brain tissue. This assay involves pre-column chiral derivatization with (-)-(R)-1-(1-naphthyl)ethyl isocyanate followed by separation on a normal-phase silica column. The developed liquid-liquid extraction procedure permits quantitative determination of analytes with recoveries ranged between 81 and 88% with intra- and inter-day relative standard deviations less than 10.5%. Linearity was obtained over the concentration range 5-1000 ng/mL and 100-10,000 ng/g for spiked drug-free plasma and brain tissue, respectively, with detection limits lower than 2.1 ng/mL and 42.8 ng/g.     

Simultaneous determination of midazolam and its three hydroxy metabolites in human plasma by electron-capture gas chromatography without derivatization

Electron-capture gas chromatography was carried out to determine midazolam and its three hydroxy metabolites (1-hydroxymethylmidazolam, 4-hydroxymidazolam and 1-hydroxymethyl-4-hydroxymidazolam) in human plasma. The assay involves extraction from plasma, buffered to pH 9.3, into cyclohexane-dichloromethane (6:4) and analysis by gas chromatography. The use of an HP-17 cross-linked, capillary column makes derivatization unnecessary. The sensitivity of the method was 2-3 ng/ml for midazolam, 1-hydroxymethylmidazolam and 4-hydroxymidazolam, and 20 ng/ml for 1-hydroxymethyl-4-hydroxymidazolam. The extraction recovery of midazolam, 1-hydroxymethylmidazolam, 4-hydroxymidazolam and 1-hydroxymethyl-4-hydroxymidazolam was 99.3 +/- 2.4, 67.0 +/- 4.6, 92.7 +/- 4.7 and 28.7 +/- 6.3%, respectively. This gas chromatographic assay was used to assess the concentration-time profiles of midazolam and its metabolites in human plasma after rectal and intravenous administration of midazolam.     

Second-derivative synchronous fluorescence spectroscopy for the simultaneous determination of cinnarizine and nicergoline in pharmaceutical preparations

A rapid, simple, and highly sensitive second-derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixtures of cinnarizine (CN) and nicergoline (NIC). The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Deltalambda) = 80 nm in aqueous methanol (50%, v/v). The different experimental parameters affecting the native fluorescence of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.025-1.5 and 0.25-5.5 microg/mL for CN and NIC, respectively, with lower detection limits of 0.58 and 0.82 ng/mL and quantitation limits of 1.93 and 2.73 ng/mL for CN and NIC, respectively. The proposed method was successfully applied for the determination of the studied compounds in synthetic mixtures and in commercial tablets. The results obtained were in good agreement with those obtained with reference methods. The high sensitivity attained by the proposed method allowed the determination of CN in real and spiked human plasma. The mean recovery in the case of spiked human plasma [number of trials (n) = 3] was 102.82 +/- 2.17%, while that in real human plasma (n = 3) was 105.25 +/- 2.05.     

Improved detection of steroids on NH2 layers

Summary Thin-layer chromatography (TLC) using NH₂-modified TLC plates is a reproducible method for the measurement of steroids. The sensitivity of determination of steroids (cortisol, cortisone, testosterone, progesterone) by measuring fluorescence in situ can be increased twofold if chromatograms are dipped into a mixture of hexane-paraffin. Fluorescence is further increased if filters are used, which cut emission wavelengths below 400 nm. Using the modifications described, steroid concentrations of about 5 ng per spot could be accurately determined. The increased sensitivity allows measurement of the plasma levels of cortisol in small laboratory animals such as guinea pigs.     

High-performance liquid chromatographic determination of pyridostigmine bromide, nicotine, and their metabolites in rat plasma and urine

This study reports on the development of a rapid and simple method for the determination of the antinerve agent drug pyridostigmine bromide (3-dimethylaminocarbonyloxy-N-methyl pyridinium bromide) (PB), its metabolite N-methyl-3-hydroxypyridinium bromide, nicotine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidine), and its metabolites nornicotine (2-(3-pyridyl)pyrrolidine) and cotinine (S-1-methyl-5-(3-pyridyl)-2-pyrrolidone) in rat plasma and urine. The compounds are extracted and eluted by methanol and acetonitrile using C18 Sep-Pak cartridges and separated using high-performance liquid chromatography by a gradient of methanol, acetonitrile, and water (pH 3.2) at a flow rate of 0.8 mL/min in a period of 14 min. UV detection was at 260 nm for nicotine and its metabolites and at 280 nm for PB and its metabolite. The limits of detection ranged between 20 and 70 ng/mL, and the limits of quantitation were 50-100 ng/mL. The average percent recovery of five spiked plasma samples were 85.7 +/- 7.3%, 80.4 +/- 5.8%, 78.9 +/- 5.4%, 76.7 +/- 6.4%, and 79.7 +/- 5.7% and for urine were 85.9 +/- 5.9%, 75.5 +/- 6.9%, 82.6 +/- 7.9%, 73.6 +/- 5.9%, and 77.7 +/- 6.3% for nicotine, nornicotine, cotinine, PB, and N-methyl-3-hydroxypyridinium bromide, respectively. The calibration curves for standard solutions of the compounds of peak areas and concentration are linear for a range between 100 and 1,000 ng/mL. This method is applied in order to analyze the previously mentioned chemicals and metabolites following their oral administration in rats.     

Rapid and sensitive determination of donepezil in human plasma by liquid chromatography/tandem mass spectrometry: application to a pharmacokinetic study

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the determination of donepezil in human plasma samples. Diphenhydramine was used as the internal standard. The collision-induced transition m/z 380 --> 91 was used to analyze donepezil in selected reaction monitoring mode. The signal intensity of the m/z 380 --> 91 transition was found to relate linearly with donepezil concentrations in plasma from 0.1-20.0 ng/mL. The lower limit of quantification of the LC/MS/MS method was 0.1 ng/mL. The intra- and inter-day precisions were below 10.2% and the accuracy was between -2.3% and +2.8%. The validated LC/MS/MS method was applied to a pharmacokinetic study in which healthy Chinese volunteers each received a single oral dose of 5 mg donepezil hydrochloride. The non-compartmental pharmacokinetic model was used to fit the donepezil plasma concentration-time curve. Maximum plasma concentration was 12.3 +/- 2.73 ng/mL which occurred at 3.50 +/- 1.61 h post-dosing. The apparent elimination half-life and the area under the curve were, respectively, 60.86 +/- 12.05 h and 609.3 +/- 122.2 ng . h/mL. LC/MS/MS is a rapid, sensitive and specific method for determining donepezil in human plasma samples.     

Determination of diazinon, chlorpyrifos, and their metabolites in rat plasma and urine by high-performance liquid chromatography

This study reports a simple and rapid high-performance liquid chromatographic (HPLC) method for the determination of the insecticide diazinon (O,O-diethyl-O[2-isopropyl-6-methylpyridimidinyl] phosphorothioate), its metabolites diazoxon (O,O-diethyl-O-2-isopropyl-6-methylpyridimidinyl phosphate) and 2-isopropyl-6-methyl-4-pyrimidinol, the insecticide chlorpyrifos (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphorothioate) and its metabolites chlorpyrifos-oxon (O,O-diethyl-O[3,5,6-trichloro-2-pyridinyl] phosphate), and TCP (3,5,6-trichloro-2-pyridinol) in rat plasma and urine samples. The method is based on using C18 Sep-Pak cartridges for solid-phase extraction and HPLC with a reversed-phase C18 column and programmed UV detection ranging between 254 and 280 nm. The compounds are separated using a gradient of 1% to 80% acetonitrile in water (pH 3.0) at a flow rate ranging between 1 and 1.5 mL/min in a period of 16 min. The limits of detection ranged between 50 and 150 ng/mL, and the limits of quantitation were 100 to 200 ng/mL. The average percentage recovery of five spiked plasma samples were 86.3 +/- 8.6, 77.4 +/- 7.0, 82.1 +/- 8.2, 81.8 +/- 8.7, 73.1 +/- 7.4, and 80.3 +/- 8.0 and from urine were 81.8 +/- 7.6, 76.6 +/- 7.1, 81.5 +/- 7.9, 81.8 +/- 7.1, 73.7 +/- 8.6, and 80.7 +/- 7.7 for diazinon, diazoxon, 2-isopropyl-6-methyl-4-pyrimidinol, chlorpyrifos, chlorpyrifos-oxon, and TCP, respectively. The relationship between the peak area and concentration was linear over a range of 200 to 2,000 ng/mL. This method was applied in order to analyze these chemicals and metabolites following dermal administration in rats.     

Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry

A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.     

Quick and Simple Determination of Dihydroergotamine by High-Performance Liquid Chromatography

Abstract

A simple and rapid liquid chromatographic assay method using a fluorescence detector for quantitation of dihydroergotamine in plasma without extraction was developed. After precipitating the protein with acetonitrile, the supernatant liquid was directly injected for analysis. Chromatographic separation was achieved on C₁₈ reversed phase column and the mobile phase was the isocratic mixture of methanol, acetonitrile and glycine buffer (0.5:3.5:6.0). With this eluting solvent the drug and its internal standard were well separated from the interference of the plasma sample. The average recovery of dihydroergotamine from 6 replicate samples of different concentrations (5-30 ng/ml) were 92.2 ± 3.37%. The minimum amount of dihydroergotamine detectable by this method was 2 ng/ml of sample.