A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and plasma     

Rapid and sensitive liquid chromatographic determination of carbamazepine suitable for use in monitoring multiple-drug anticonvulsant therapy.

A rapid, sensitive and selective method for the determination of carbamazepine and its major metabolite in plasma has been developed. Other commonly used anticonvulsants can be determined in the same procedure without interference. After extraction with dichloromethane, the components are separated by high-pressure liquid chromatography without further clean-up or concentration on a column packed with small-particle silica gel. The mean recovery from plasma is 98.6% with a relative standard deviation of 1.6%. The detection limit for carbamazepine is approximately 2 ng/ml, requiring 1 ml of plasma.     

Gas chromatographic method for determination of a piperazine derivative (Trelibet) and its metabolites in human plasma and urine

A sensitive gas chromatographic method was developed for the determination of Trelibet, 1-benzyl-4-(2'-pyridinecarbonyl)piperazine, and of its major metabolites in biological fluids. The compounds were extracted as bases into dichloromethane, and the extracts were analysed by a dimethylsilicone capillary column with a nitrogen-phosphorus flame-ionization detector. The lower limit of detection was 1 ng/ml for Trelibet and 5 ng/ml for the metabolites. Peak-area ratios of the compounds and internal standard were linearly correlated to their plasma concentrations between 1 and 1000 ng/ml. The method was used for quantification of Trelibet and two of its metabolites in depressed patients after oral administration of a single dose of 200 mg of Trelibet. Concentration data measured in plasma and urine showed that the method is sensitive enough to monitor concentrations both for pharmacokinetic studies and for plasma steady-state levels daily.     

Determination of pirlindole in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for the analysis of pirlindole [2,3,3a,4,5,6-hexahydro-8-methyl-1H-pyrazino(3,2,1-jk)carbazole hydrochloride], a new antidepressive drug. The drug was extracted from plasma into dichloromethane, and the analysis was carried out on a reversed-phase column, the effluent being monitored by fluorescence detection. The method is selective and sensitive (limit of detection 1-2 ng/ml plasma). Urine analysis was done by direct injection of the diluted sample. The method was applied to the analysis of plasma and urine samples of eight healthy male volunteers who received a 75-mg oral dose of a tablet formulation of pirlindole. The method was also applied to a study in three beagle dogs which received pirlindole (1 mg/kg) by infusion (0.1 mg/kg/min) and orally (10 mg/kg) to estimate the absolute bioavailability of the drug.     

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.     

Determination of dioxopiperazine metabolites of quinapril in biological fluids by gas chromatography-mass spectrometry.

The dioxopiperazine metabolites of quinapril in plasma and urine were extracted with hexane-dichloroethane (1:1) under acidic conditions. Following derivatization with pentafluorobenzyl bromide and purification of the desired reaction products using a column packed with silica gel, the metabolites were analysed separately by capillary column gas chromatography-electron-impact mass spectrometry with selected-ion monitoring. The limits of quantitation for the metabolites were 0.2 ng/ml in plasma and 1 ng/ml in urine. The limits of detection were 0.1 ng/ml in plasma and 0.5 ng/ml in urine, at a single-to-noise ratio of greater than 3 and greater than 5, respectively. The proposed method is applicable to pharmacokinetic studies.     

Improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma using solid-phase extraction

An improved high-performance liquid chromatographic method for the simultaneous determination of caffeine and its N-demethylated metabolites in plasma is described. Excellent resolution of all components is provided by reversed-phase chromatography using a mobile phase consisting of 1% acetic acid-methanol (83:17) at a flow-rate of 2.7 ml/min, in conjunction with a Waters Assoc. Nova-Pak C18 column which was protected by a Waters Assoc. Guard-Pak precolumn module containing a Guard-Pak CN cartridge. Rapid extraction of caffeine and the dimethylxanthines from plasma was achieved using reversed-phase octadecylsilane bonded-silica columns (Bond-Elut C18). With only 100 microliters of sample, plasma levels in the region of 50 ng/ml for the dimethylxanthines and 100 ng/ml for caffeine can be determined using ultraviolet detection at 273 nm. The method has been used for measuring umbilical cord plasma samples to provide information regarding foetal exposure to caffeine and its metabolites and is also suitable for therapeutic drug monitoring of caffeine and theophylline levels in the treatment of neonatal apnoea.     

Quantitative analysis of veralipride in plasma and urine by gas chromatography-mass spectrometry and gas chromatography with flame-ionization detection

A highly sensitive and selective quantitative assay for unchanged veralipride has been developed. The compound is extracted from alkalized samples (plasma or urine) with dichloromethane and converted to its trimethylated derivative by reaction with trimethylanilinium hydroxide. The reaction mixture is then chromatographed on a 3% OV-1 column. Trimethylated derivatives of plasma samples were assayed by selected-ion monitoring in the chemical-ionization mode and quantified by comparing the intensity of the quasi-molecular ion m/z 426 (M + H) with the intensity of the corresponding ion from trideuterated internal standard, m/z 429 (M + H). Flame-ionization detection was used for the assay of urine samples. The peak height ratio of trimethylated veralipride over trimethylated sulpiride, the internal standard, was used for quantitation of urine samples. A relative standard deviation of less than 10% was found when quantifying 10 ng/ml veralipride in plasma or 1 microgram/ml in urine.     

Simple high-performance liquid chromatographic method for the determination of medroxyprogesterone acetate in human plasma

The high-performance liquid chromatographic (HPLC) method was developed as a simple, reliable alternative to available methods for measuring plasma concentrations of medroxyprogesterone acetate (MPA). The HPLC method has been successfully automated and is suitable for the rapid, inexpensive analysis of large batches of plasma samples. The best approach involves a solvent extraction followed by HPLC separation and analysis. MPA can be efficiently extracted, at all pH values, by nonpolar solvents. The Spherisorb 5-ODS2 HPLC column provides excellent separation of MPA from endogenous steroids of similar structure and from extraneous plasma blank peaks. A batch of 30-40 samples can be prepared by HPLC analysis in 2-3 hours, with a chromatographic run time of 10 minutes/sample. Calibration curves between 5-250 ng/ml show a good correlation between peak height ratio and MPA concentration, even at low levels. Plasma concentrations of MPA in patients receiving 1 g/day were between 12.6-270 ng/ml in this study, suggesting that the sensitivity of this method, 10 ng/ml, is sufficient for monitoring therapeutic concentrations of MPA. The results show a wide individual variation in plasma concentrations following similar dosing schedules--a finding reported by other workers.     

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.     

A quantitative gas-liquid chromatographic method for the determination of neostigmine and pyridostigmine in human plasma

A sensitive and selective analytical method was used to measure the concentration of neostigmine and pyridostigmine in human plasma. The procedure involved preliminary ion-pair extraction of the drugs into dichloromethane, followed by concentration and analysis of the ion-pair complex using a gas-liquid chromatographic system fitted with a nitrogen detector. Using the peak area ratio technique, pyri-dostigmine bromide was used as the internal standard for the quantitation of neo-stigmine in plasma; neostigmine bromide was the internal marker for the determination of pyridostigmine. The method depends on the thermal dequaternisation of the quaternary amines, and can be used to detect 5 ng/ml in a 3-ml plasma sample. Accurate measurement can be made at levels of 50–1000 ng/ml. This assay procedure has been applied to the separate determination of the plasma concentration of neostigmine and pyridostigmine after single administration of intravenous doses in aneasthetised patients.     

The Determination of a New Trifluorinated Quinolone,Fleroxacin, Its N-Demethyl,and N-Oxide Metabolites in Plasma and Urine by High Performance Liquid Chromatography with Fluorescence Detection

Abstract

A liquid chromatographic method is described for the determination of the new fluoroquinolone Ro 23–6240 and its N-demethyl and N-oxide metabolites in plasma and urine. The three substances were extracted from aqueous solution with dichloromethane/isopropanol containing sodium dodecyl sulphate. After evaporation and reconstitution, samples were analysed on a reversed-phase column using ion pair chromatography and fluorescence detection. The limit of quantification was 10–20 ng/ml (RSD 4%) using a 0.5 ml plasma sample, and the inter assay precision was 3–10% over the concentration range 50 ng/ml to 20 μg/ml. Recovery from plasma was 81% (RSD 10%) over the range 10 ng/ml to 5 μg/ml. The method has been applied successfully to the analysis of several thousand samples from human pharmacokinetic studies. Care has to be taken to avoid exposure of samples to direct sunlight, and the use of opaque vessels for sample storage and handling is recommended.     

Simultaneous determination of the anticonvulsants, cinromide (3-bromo-n-ethylcinnamamide), 3-bromocinnamamide, and carbamazepine in plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method is described for monitoring plasma concentrations of cinromide (3-bromo-N-ethylcinnamamide) and its de-ethylated metabolite. Carbamazepine levels can be easily measured by the same technique. The N-isopropyl analogue of cinromide is used as internal standard, and all compounds are easily separated on a reversed-phase column operated at 55 degrees with a small-diameter pre-column maintained at the same temperature. The extraction is rapid and generally applicable to plasma and urine samples that are to be analyzed by reversed-phase chromatography. Short- and long-term reproducibility studies show less than 4% relative standard deviation for replicate determinations for all drugs. Limits of quantitation are 10-20 ng/ml with an internal standard concentration of 3 micrograms/ml. Another metabolite of cinromide, 3-bromocinnamic acid, which may have some anticonvulsant effect, can be analyzed simultaneously by buffering the mobile phase and adding an ion-pairing reagent.     

Analysis of erythromycin in biological fluids by high-performance liquid chromatography with electrochemical detection

A simple and sensitive high-performance liquid chromatographic assay was developed for the quantitative determination of major erythromycin components and their potential metabolites or degradation products in plasma and urine. An ether extract of alkalized plasma sample was chromatographed on a reversed-phase column and the components in the column effluent were monitored by an electrochemical detector. The recovery of the drug from extraction was virtually 100%. The detection limits for erythromycin A in plasma were 5-10 ng/ml and 30 ng/ml using 1 and 0.2 ml of sample, respectively. For urine samples, a simple one-step deproteinization with two volumes of acetonitrile was satisfactory for analysis. The method has been evaluated in plasma and urine from dogs receiving oral or intravenous erythromycin A. The standard curves for potential metabolites or degradation products were not constructed due to the lack of sufficient samples.     

Determination of cefquinome in pig plasma and bronchoalveolar lavage fluid by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5-2500 ng ml(-1) for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4-1000 ng ml(-1). The limit of quantification (LOQ) was set at 5.00 ng ml(-1) for plasma and at 4.00 ng ml(-1) for BAL fluid. The limit of detection (LOD) was 3.12 ng ml(-1) and 0.41 ng ml(-1) for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml(-1) for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied.     

Determination of tenoxicam in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of tenoxicam in plasma has been developed. Tenoxicam was extracted from buffered plasma (pH 3 or 4, respectively) with dichloromethane and the evaporated extracts were analysed on a C18 reversed-phase column using a methanol-phosphate buffer mobile phase and with UV detection at 371 nm. The detection limit was 20 ng/ml using a 0.5-ml sample. The method is selective with respect to the 5'-hydroxy metabolite, which is present in plasma after multiple administration of tenoxicam; this metabolite may also be determined using this procedure.     

Gas chromatographic assay with pharmacokinetic applications for monitoring T-2 and HT-2 toxins in plasma

A gas-liquid chromatographic (GLC) method for monitoring T-2 and HT-2 toxins in plasma was developed. The procedure involved extraction of the toxins with ethyl acetate, chromatography on a C18 reversed-phase column and derivatization with heptafluorobutyric anhydride (HFBA). The T-2 and HT-2 HFBA derivatives were chromatographed on OV-17 at various temperatures and measured with an electron-capture detector. Iso-T-2 toxin and iso-HT-2 toxin were used as internal standards. Recoveries averaged 95.1 +/- 8.6% for T-2 toxin and 102.1 +/- 5.2% for HT-2 toxin at levels ranging from 40 to 120 ng/ml. The limits of detection were 30 and 5 ng/ml of T-2 and HT-2 toxin, respectively. The range of the assay covers plasma concentrations at which toxicity becomes manifest. The pharmacokinetic application of this GLC method is illustrated by simultaneous monitoring of T-2 and HT-2 toxins levels in plasma obtained after intravenous administration of T-2 toxin to a dog.     

A rapid and sensitive liquid chromatography/positive ion tandem mass spectrometry method for the determination of cimetropium in human plasma by liquid-liquid extraction

We have developed and validated a simple detection system with high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) for determining cimetropium levels in human plasma using scopolamine butyl bromide as an internal standard (I.S.). The acquisition was performed in the multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 357.9 > 103.1 for cimetropium and m/z 359.9 > 103.1 for butyl-scopolamine. The method involves a simple single-step liquid-liquid extraction with dichloromethane. The analyte was chromatographed on an YMC C18 reversed-phase chromatographic column by isocratic elution with 10 mM ammonium formate buffer-methanol (19:81, v/v; adjusted to pH 4.0 with formic acid). The results were linear over the studied range (0.2-100 ng ml(-1)), with r2 = 1.0000, and the total analysis time for each run was 2 min. Intra- and interassay precisions were 0.70-8.54% and 1.08-4.85%, respectively, and intra- and interassay accuracies were 97.56-108.23% and 97.48-103.91%, respectively. The lower limit of quantification (LLOQ) was 0.2 ng ml(-1). At this concentration, mean intra- and interassay precisions were 8.54% and 4.85%, respectively, and mean intra- and interassay accuracies were 97.56% and 98.91%, respectively. The mean recovery ranged from 62.71 +/- 4.06 to 64.23 +/- 2.32%. Cimetropium was found to be stable in plasma samples under typical storage and processing conditions. The devised assay was successfully applied to a pharmacokinetic study of cimetropium bromide administered as a single oral dose (150 mg) to healthy volunteers.     

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.     

A Rapid Liquid Chromatographic Method for Determination of Flecainide in Human Blood Plasma Using Ultraviolet Detection

Abstract

A liquid chromatographic method for the assay of the antiarrhythmic drug flecainide in plasma has been developed. The method is rapid, simple and with sufficient detection sensitivity to render it suitable for therapeutic drug monitoring. Flecainide and added internal standard, a non-fluorinated analogue, were extracted by a single ether extraction from alkalinized plasma followed by a back-extraction of the ether with dilute phosphoric acid. A portion of the acid extract was then applied directly to a 30 cm ODS column eluting isocratically with 30% acetonitrile in water containing 0.01M dibutylamine phosphate. Monitoring was by ultraviolet detection at 214 nm and the total run time was 8 min. This method is specific and can quantitate plasma levels to less than 30 ng/ml (free base) from 0.5 ml of plasma without interference from antiarrhythmic drugs commonly used in therapy.