Determination of Reserpine in Plasma Using High-Performance Liquid Chromatography with Fluorescence Detection

Abstract

A high-performance liquid chromatographic method for determining reserpine in plasma has been developed. The procedure involves extraction of reserpine from buffered plasma into benzene, oxidation of reserpine to a fluorophor by treatment with vanadium pentoxide in phosphoric acid, and chromatographic separation of the reserpine fluorophor on an octadecylsilane column by ion-pairing with heptanesulfonate ions. Fluorescence monitoring of the column effluent provides high sensitivity of detection and increases the specificity of the procedure. A detection limit of approximately 100 pg of reserpine per ml of plasma was obtained following analysis of 2 ml samples. Analysis of a number of samples demonstrated the applicability of this method in confirming the presence of reserpine in equine plasma specimens collected at various horse shows and in evaluating the pharmacokinetic behavior of reserpine following intramuscular administration to horses.     

High Performance Liquid Chromatographic Determination of Diuretic-Antihypertensive Combination Products. II. Polythiazide and Reserpine

Abstract

A high performance liquid chromatographic method for the simultaneous determination of polythiazide and reserpine in tablets is described. Polythiazide, reserpine, and vanillin (an excipient) are separated isocratically on an octadecylsilane column using a methanol-water-acetic acid (55:44:1) mobile phase. The column effluent was monitored by ultraviolet ab-sorbance at 254 nm. Recoveries from synthetic formulations were 100.2 ± 1.7% for polythiazide and 98.7 ± 1.1% for reserpine.     

An Improved HPLC Method for the Determination of Furosemide in Plasma and Urine

Abstract

A sensitive HPLC method with minimal sample preparation and good reproducibility for the determination of furosemide in plasma and urine is described. Acidified plasma samples were extracted using CH₂Cl₂ containing desmethylnaproxen as internal standard (IS). Fresh urine samples were incubated with β-gluc-uronidase for 15 minutes and then treated with CH₃CN containing IS.

Chromatography was performed on a C18 column with 10 mcl sample injection, Mobile phases were: a) for plasma: 0.01 M NaH₂PO₄, pH 3.5 - CH₃OH (65:35), and b) for urine: acetic acid, pH 3.5 - CHS3OH (60:40) at 3 ml/min and fluorescence detection at Ex 235/Em 389 nm. The plasma standard curve was linear from 0.01 to 15.0 mcg/ml and the urine from 0.5 to 200.0 mcg/ml. The within run CV's were 3,2% at 0.74 mcg/ml plasma and 2.0% at 10.7 mcg/ml urine. Recovery from plasma was 69.9% at 2.0 mcg/ml and 98.6% from urine at 5.0 mcg/ml. The stability of furosemide and its glucuronide were studied. Both methods have been applied to the analysis of plasma and urine samples obtained from human volunteers.     

Automated Solid Phase Extraction and Hplc Analysis of Ibuprofen in Plasma

Abstract

Ibuprofen is a non-steroidal anti-inflammatory drug, widely used in arthritis and other disorders. We describe a high pressure liquid chromatographic (HPLC) method for the analysis of ibuprofen in plasma, using an automated solid phase extraction technique (the Varian AASP^R). In this method ibuprofen was extracted from 0.5 ml of plasma by application to a C₂ extraction cartridge followed by “on line” elution with the HPLC mobile phase (55% acetonitrile / 45% 0.02 H phosphate buffer; pH 3.0), at a flow rate of 1.5 ml/min. The analytical column was a Nucleosil C₁₈ column and the fluorescence detector was set at 253 nm (excitation wavelength) and 300 nm (emission wavelength). Chromatography was complete in less than 10 mins and the limit of detection was 1.3 /μg/ml. The method is linear through the range of 1.0 to 100.0 /μg/ml with a mean correlation coefficient of 0.9964. Absolute recovery of ibuprofen from the spiked plasma samples ranged from 77.8% to 86.5%. The method was shown to be precise within 11% C.V. and accurate to within 8% over the concentration range studied.     

High Performance Liquid Chromatographic Determination of Pentamidine in Plasma

Abstract

A high performance liquid chromatographic method for quantitating pentamidine in plasma has been developed. Sample clean-up involved precipitating plasma with acetonitrile containing the internal standard, hexamidine. The supernatant was passed through a C₈ Bond Elut column and eluted with a methanolic solution of sodium 1-heptanesulfonate. The eluate was then analyzed on an Altex C₈ column with a mobile phase consisting of 45% CH₈CN, 0.02% detramethylammonium chloride and 0.1% H₃PO₄. Using fluorescence detection (EX: 275 nm and EM: 340 nm), the detection limit was 1.25 ng/ml for 0.5 ml of plasma. The coefficients of variation for interday and intraday were around 10%.     

Rapid Determination of Bupropion in Human Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) technique has been developed for the determination of bupropion hydrocloride (Bup) in human plasma, using a reversed-phase method, with UV detection at 250 nm.

The internal standard 5-(P-methylphenyl)-5-phenylhydantoin (MPPH), was used as an aid to quantitation. The plasma was deprotemized with acetonitrile and the clear supernatant was directly injected in the chromatographic system. The lower limit of quantitation was 5.0 ng/ml using only 100 μl of plasma sample.

Linear regression analysis for the calibration plots obtained on five different days over a two-week period for the the two ranges used (10–250 ng/ml and 250–2000 ng/ml) in plasma indicated excellent linearity and reproducibility. The mean recovery of spiked Bup in plasma samples over the concentrations studied was found 96.5 ± 3.14%.

The method revealed that more than 30% of Bup was lost when the supernatant was stored at room temperature for 24 hrs.     

High Performance Liquid Chromatongraphy of ABBOTT-53385 in Dog,Rat, and Human Plasma

Abstract

A simple, sensitive and reproducible high-performance liquid chromatographic (HPLC) method was developed to monitor plasma ABBOTT-53385 (I) levels in dogs, rats and humans. The samples were first supplemented with the internal standard, then extracted on Bond Elut® extraction columns. They were analyzed by reverse-phase HPLC with UV detection at 240 nm. The calibration curve was rectilinear over the range of 0.05–2.0 μg/ml and the interday variance was less than 4%. The limit of detection for this method was about 0.03 μg/ml.     

An Isocratic HPLC Method for the Determination of Cephalosporins in Plasma

Abstract

An isocratic reversed-phase liquid chromatographic method for the determination of eight cephalosporins in human plasma using UV detection at 254 nm is described. Plasma proteins were precipitated using acetonitrile prior to injection of a 10 μl aliquot onto an octadecylsilane column. The mobile phases consisted of 6–11% acetonitrile in sodium dihydrogen phosphate (0.01M). The minimum detectable limit for each drug was less than 1 γg/ml of plasma. Possible interference from other drugs which might be administered concurrently is discussed. The reproducibility and precision of the method for cephalosporin assay are shown from the analysis of plasma containing 5–500 γg/ml of plasma. The chromatographic behavior of the eight cephalosporins was examined by varying mobile phase conditions.     

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.     

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.     

High Pressure Liquid Chromatographic Determination of Mecillinam in Human Plasma and Urine

Abstract

A simple, specific, rapid and sensitive method for the analysis of mecillinam in plasma and urine using high pressure liquid chromatography is described. The assay is performed by direct injection of a plasma protein free supernatant or a dilution of urine. A μBondapak phenyl column with an eluting solvent of 16% CH₃CN-0.2% H₃PO₄ was used, with UV detection of the effluent at 220 nm. Desacetyl-cephalothin was used as the internal standard and quantitation was based on peak height ratio of mecillinam to that of the internal standard. The lowest concentration detectable without extraction was 0.25 μg/ml for plasma and 8.9 μg/ml for urine. No interference from plasma and urine was noted.     

Determination of Reserpine in Pharmaceutical Formulations by High Performance Liquid Chromatography

Abstract

High performance liquid chromatography was employed in the assay of reserpine in tablet formulations. A reverse-phase RP-8 column was used and reserpine was separated from its major degradation products and quantitated by peak-height measurement using ultraviolet detection at 254 nm and fluorescence at 330 nm excitation. Tablets analysed were within the official limits even after more than ten years following manufacture.     

Determination of Pyridostigmine in Human Plasma by High-Performance Liquid Chromatography

Abstract

An easy to perform, specific, reproducible and sensitive high performance liquid chromatographic (HPLC) method to measure pyridostigmine concentration in human plasma was developed and validated. Sample clean-up consists of ion-pair extraction into dichloromethane in the presence of neostigmine as internal standard, followed by back extraction into an aqueous phase. Mean recovery of 110% (with a standard deviation of 10%) was determined for concentrations of 5 – 100 ng/ml. Chromatography on a 125·4 mm CN-propyl column using a mobile phase composed of 10% acetonitrile in 3.5×10^?4M NaH₂PO₄ and UV detection at 270 nm, yields clean chromatograms without any interferences from endogenous plasma components. Using 1 ml plasma samples the method has a limit of detection (LD) of 3 ng/ml, with %CV (precision) and bias (accuracy) ≥ 10% for concentrations in the range of 0–100 ng/ml. The method is being used in human pharmacokinetic studies of oral dosage forms of pyridostigmine.     

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

Abstract

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

High-Performance Liquid Chromatographic Determination of Acetazolamide in Human Plasma

Abstract

A simple, rapid and specific HPLC method has been developed to determine acetazolamide concentrations in human plasma. The assay procedure requires only 250 μl of sample with direct injection of the organic supernatant after protein precipitation with acetonitrile. Chlorothiazide was used as an internal standard. A reversed-phase C₁₈ μBondapak column was employed for the chromatographic separation. The eluent was monitored at 265 nm using a UV variable wavelength detector. The retention times for acetazolamide (ACZ) and chlorothiazide (CTZ) were 6 and 8 min respectively. A linear relationship (r).995) was obtained over the 1-20 μg/ml concentration range. The limit of sensitivity for ACZ was 0.5 μg/ml, with greater than 85% recovery of ACZ and internal standard. The method was applied to human plasma samples obtained after administration of a 250 mg acetazolamide tablet.     

Liquid Chromatographic Analysis of a New Antihypertensive Agent,PD 78,799, in Plasma on Silica Gel with Reversed-Phase Eluent

Abstract

A sensitive and selective HPLC assay has been developed for the analysis of a new antihypertensive agent in human plasma. The drug and internal standard were isolated from plasma by liquid-liquid extraction. Separation was accomplished on unmodified silica gel with a mobile phase of 60:40 acetonitrile:10 mM dibasic ammonium phosphate. Detection was by UV absorbance at 291 nm. The method is linear over a range of 20 to 4000 ng/ml. Relative error of calibration and control standards ranged from ?1.5 to 5.0% and precision, as indicated by relative standard deviation, ranged from 0.8 to 5.2%. The method has been successfully used for analysis of plasma samples from human volunteers following oral administration of PD 78,799.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

Rapid and Sensitive Determination of Furosemide in Human Plasma by High Pressure Liquid Chromatography

Abstract

A rapid and sensitive determination of furosemide in human plasma by high pressure liquid chromatography is described. The drug is extracted from plasma after addition of the internal standard (2,4-dinitrophenol), the extract is concentrated by means of evaporation and injected on to the liquid chromatograph. Separation is achieved on a reversed-phase column with 50 % methanol in water, containing 0.5 % glacial acetic acid; detection is carried out at 340 nm. The method is linear up to 5 μml and can determine concentrations down to 0.1 μg/ml in a 1ml plasma sample. It is much faster than existing methods and is at least as good with respect to accuracy and sensitivity.     

High Performance Liquid Chromatographic Determination of Oxamniquine in Plasma Samples

Abstract

A sensitive and reliable liquid chromatographic assay procedure for the quantitation of oxamniquine in plasma or urine was developed. Chromatographic separation was achieved on a reversed-phase phenyl colum using U.V. Detection at 254 nm. The eluting solvent was the mixture of 0.05 M acetate buffer pH 5 and acetonitrile (3:7). With this mobile phase the drug and its external standard were well separated from the interference of the blank samples. The average recovery of oxamniquine from 3 or more replicate dog plasma samples of different concentration (0.125 ? 4.00 μg/ml) was 95.5% and its coefficient of variation was 4.17%. The reproducibility of the assay was confirmed by the analysis of variance test for the slopes of the three standard plots obtained from plasma samples at three different occasions (F=4.2, p > 0.01). The detection limit for plasma samples was approximately 20 ng/ml. The method was applied to measure the plasma level vs, time profile of this drug following a single bolus intravenous dose of 16 mg/kg to a dog.     

Determination of Fleroxacin in Plasma by Direct Injection High Performance Liquid Chromatography with Column Switching

ABSTRACT

A high-performance liquid chromatography (HPLC) assay was developed for the determination of fleroxacin in plasma. The plasma samples were directly introduced onto a HPLC column after filtering through a MolcutII® membrane filter, which removes high molecular weight proteins. The fleroxacin in filtrate was separated from interfering substances and retained on a pre-column using an ODS stationary phase and then was introduced to an analytical column with an ODS stationary phase by column switching. Fleroxacin and lomefloxacin, as an internal standard, were detected by ultraviolet absorbance at 295 nm. Determination of fleroxacin was possible over the concentration range 50-4000 ng/ml; the limit of detection was 20 ng/ml. The recovery of fleroxacin added to plasma was 97.3-100.4% with a coefficient of variation of less than 2.2%. This method is applicable to drug level monitoring in the plasma of patients being treated with fleroxacin and of healthy volunteers participating in pharmacokinetic studies.