Quantitation of Cyproheptadine in Plasma and Urine by HPLC

Abstract

A sensitive, reliable and specific high performance liquid chromatographic procedure has been developed for the quantitation of cyproheptadine in plasma or urine. After extraction of the drug with ethyl acetate from alkalinized samples, the organic extract was evaporated to dryness, reconstituted with acetonitrile and chromatographed using a C₈ reversed-phase analytical column with UV detection at 254 nm. The average recoveries of cyproheptadine from spiked plasma and urine samples in the concentrations ranging from 0.2 – 3 mcg/ml were 95.7 and 100.3%, respectively and their respective CV was 4.1 and 3.9%. Regression analyses for the calibration plots for plasma and urine standards obtained on three different days for the drug concentrations between 0.2 – 3 mcg/ml indicated excellent linearity (r > 0.999) and reproducibility (CV < 2.0%, p > 0.01). The limit of sensitivity was 50 ng/ml for both plasma and urine samples. The method was applied to monitor the plasma concentration versus time profile of cyproheptadine following a single bolus IV dose of 1 mg/kg in a dog.

Urine samples taken from a human subject for the duration of 24 hours following a single oral dose of 8 mg showed that the cumulative amount excreted in urine as cyproheptadine was approximately 1% of the dose.     

High Performance Liquid Chromatographic Determination of Phenacemide in Biological Fluids

Abstract

A sensitive and reliable high performance liquid chromatographic procedure has been developed for the quantitation of phenacemide in plasma or urine. After simple extraction of the drug with ethylacetate from alkalinized samples and evaporation to dryness, the reconstituted extract was chromatographed using a C₈ reversed phase analytical column with UV detection at 254 nm. Regression analyses for the calibration plots obtained on 3 different days for the drug concentrations ranging 1–15 mcg/ml indicated excellent linearity (r >0.999) and reproducibility (CV< 4%, p >0.01). The mean recovery of spiked phenacemide in plasma and urine from the lower limit of quantitation (1 mcg/ml) to 15 mcg/ml was 97.9 and 96.3%, respectively and their respective CV was 3.53 and 2.58%. The method was applied to monitor the plasma vs. time profile of the drug following a single bolus IV dose of 12 mg/kg in a dog.     

High Performance Liquid Chromatographic Assay for Basic Amine Drugs in Plasma and Urine Using a Silica Gel Column and an Aqueous Mobile Phase. II. Chlorpheniramine

Abstract

A high performance liquid chromatographic [HPLC] method that involves the use of a silica gel column and an aqueous mobile phase for quantitation of chlorpheniramine in plasma and urine is presented. Alkalinized samples are cleaned by extraction with pentane [containing 1% CH₃CN], and the extraction is followed by evaporating the solvent and reconstituting the residue in a small amount of mobile phase. An aliquot of this solution is analyzed by an HPLC system with an Ultrasphere Si Column, an aqueous mobile phase at pH 7 containing 60% CH₃CN and 7.5 mM [NH₄]₂HPO₄, and UV detection at 200 nm. Although the average recovery of extraction is 58% ± SD 10%, the detection limit for the method is 0.7 ng/ml in plasma and 100 ng/ml in urine [s/n = 3] for 0.5 ml samples. The coefficients of variation [CV] on the results of samples run to measure interday and intraday precision and the bias on control samples were all 10% or less. We have used the method in a bioavailability study of a controlled release formulation involving over 1000 samples.     

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.     

Revised HPLC Determination of Atenolol in Plasma and Urine

Abstract

An HPLC method for analysis of atenolol in human plasma and urine is presented. Based on alkaline extraction, acid backextraction and reverse phase ion-pair chromatography this method is quite specific for atenolol. For a 0.5 ml plasma sample the sensitivity ranges from 20 ng/ml in fasted healthy volunteers to 50 ng/ml in various groups of patients. A sensitivity in urine of 1.0 mcg/ml was sufficient for all samples studied. As presented this method has been used in several clinical pharmacokinetic studies involving hundreds of samples.     

Ion-Pair Liquid Chromatographic Analysis of Phenylpropanolamine in Plasma and Urine by Post-Column Derivatization with O-Phthalaldehyde

Abstract

A high pressure Liquid chromatographic analysis of phenylpropano Lamine in plasma and urine by post-column derivatzaition with o-phthalaldehyde is described. Plasma samples are extracted with methylene chloride under alkaline conditions. Urine is diluted with mobile phase without extraction. Using fluorescence detection, the method is sufficiently sensitive (2 ng/ml in 0.5 ml of plasma and 0.5 mcg/ml in 0.2 ml of urine) so that phenylpropano lamine concentrations in plasma or urine may be measured for up to 24 hours following a 75 mg oral dose. Coefficients of variation for inter-day and intra-day precision are less than 10%.     

Quantitation of Creatinine in Urine and Plasma Samples by Reversed Phase HPLC

Abstract

Creatinine determination in urine and plasma affords an index of the renal function. Reversed-phase high pressure liquid chromatography was used for the separation and quantitation of creatinine in normal and arsenic exposed human urine samples. Acetonitrile/water (1:1) was the mobile phase. The method was compared with the Jaffé alkaline picrate reaction. Results show that the HPLC procedure has high reproducibility and samples are stable at the storage conditions. Plasma samples required depro-teinization and extraction with CH₃CN prior to HPLC analysis, while urine samples required only centrifugation.     

High Performance Liquid Chromatographic Assay of Methoclopramide in Plasma Using a Silica Gel Column and An Aqueous Meobile Phase

Abstract

A high performance liquid chromatographic method for quantitating matoclopramide in plasma is presented. Proteins ara precipitated from the plasma sample with acetonitri la containing the internal standard, procainamida. The treated samples ara than analyzad using an Ultrasphere Si column, an aqueous solution at pH 7 of 65% CH³CN and 5.0 mM (NH₄)₂HPO₄ as a mobile phase, and a fluorescence detector. The retention times for drug and intarna1 standard ara 11.2 and 13.2 min, respectively. The caibration curve is Linear from 0.89 to 44.5 ng/ml. The detection limit is 0.89 ng/ml [signaL/hoisa = 31] for 0.2 ml plasma samples Pracision is measured by intraday and intarday coefficients o f variation, which are less than 10%. This method is currently being used for pharmacokinetic studies of methoclopramide.     

A simple,rapid and sensitive liquid chromatography–tandem mass spectrometry method for the determination of dienogest in human plasma and its pharmacokinetic applications under fasting

A simple, rapid and sensitive analytical method using liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) detection with positive ion electrospray ionization was developed for the determination of dienogest in human K₂EDTA plasma using levonorgestrel d₆ as an internal standard (IS). Dienogest and IS were extracted from human plasma using simple liquid–liquid extraction. Chromatographic separation was achieved on a Zorbax XDB‐Phenyl column (4.6 × 75 mm, 3.5 µm) under isocratic conditions using acetonitrile–5 mm ammonium acetate (70:30, v/v) at a flow rate of 0.60 mL/min. The protonated precursor to product ion transitions monitored for dienogest and IS were at m/z 312.30 → 135.30 and 319.00 → 251.30, respectively. The method was validated with a linearity range of 1.003–200.896 ng/mL having a total analysis time for each chromatograph of 3.0 min. The method has shown tremendous reproducibility with intra‐ and inter‐day precision (coefficient of variation) <3.97 and 6.10%, respectively, and accuracy within ±4.0% of nominal values. The validated method was applied to a pharmacokinetic study in human plasma samples generated after administration of a single oral dose of 2.0 mg dienogest tablets to healthy female volunteers and was proved to be highly reliable for the analysis of clinical samples. Copyright © 2014 John Wiley & Sons, Ltd.     

Determination of Ciprofloxacin (Bay o 9867) in Biological Fluids by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method for the analyses of ciprofloxacin (BAY o 9867) (1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinoline carboxylic acid hydrochloride) in human serum, plasma and urine samples is described. Diluted serum, plasma, and urine samples are injected onto a RP-18 column without prior extraction or clean-up procedure. Ciprofloxacin is separated from the ballast by an eluent consisting of an 0.025M H₃PO₄ solution adjusted to pH=3 with tetrabutylammonium hydroxide and acetonitrile.

Ciprofloxacin is detected fluorimetrically giving a detection limit of 8ng/ml in plasma and serum and of 50ng/ml in urine. A statistical evaluation of the assay showed acceptable accuracy and precision for 10 to 500ng of BAY o 9867 per ml in serum and plasma and for 50ng to 600ng of BAY o 9867 per ml of diluted urine specimens. This method was used to monitor the concentrations of BAY o 9867 in serum, plasma and urine of volunteers after oral administration of ciprofloxacin.     

The hydroxyl radical reaction rate constant and atmospheric transformation products of 2-butanol and 2-pentanol

The relative rate technique has been used to measure the hydroxyl radical (OH) reaction rate constant of +2-butanol (2BU, CH₃CH₂CH(OH)CH₃) and 2-pentanol (2PE, CH₃CH₂CH₂CH(OH)CH₃). 2BU and 2PE react with OH yielding bimolecular rate constants of (8.1±2.0)×10⁻¹² cm³molecule⁻¹s⁻¹ and (11.9±3.0)×10⁻¹² cm³molecule⁻¹s⁻¹, respectively, at 297±3 K and 1 atmosphere total pressure. Both 2BU and 2PE OH rate constants reported here are in agreement with previously reported values [1–4]. In order to more clearly define these alcohols' atmospheric reaction mechanisms, an investigation into the OH+alcohol reaction products was also conducted. The OH+2BU reaction products and yields observed were: methyl ethyl ketone (MEK, (60±2)%, CH₃CH₂C((DOUBLEBOND)O)CH₃) and acetaldehyde ((29±4)% HC((DOUBLEBOND)O)CH₃). The OH+2PE reaction products and yields observed were: 2-pentanone (2PO, (41±4)%, CH₃C((DOUBLEBOND)O)CH₂CH₂CH₃), propionaldehyde ((14±2)% HC((DOUBLEBOND)O)CH₂CH₃), and acetaldehyde ((40±4)%, HC((DOUBLEBOND)O)CH₃). The alcohols' reaction mechanisms are discussed in light of current understanding of oxygenated hydrocarbon atmospheric chemistry. Labeled (¹⁸O) 2BU/OH reactions were conducted to investigate 2BU's atmospheric transformation mechanism details. The findings reported here can be related to other structurally similar alcohols and may impact regulatory tools such as ground level ozone-forming potential calculations (incremental reactivity) [5]. © 1998 John Wiley & Sons, Inc. Int J Chem Kinet 30: 745–752, 1998     

The structures and stability of pentacoordinate germylenoid PhCH<Subscript>2</Subscript>(OH)CH<Subscript>3</Subscript>GeLiF

The structures and stability of pentacoordinate germylenoid PhCH₂(OH)CH₃GeLiF were first theoretically studied by density functional theory. Two equilibrium structures, the three-membered ring (1a) and the p-complex (1b) structures, were located. Their energy are in the order of 1b > 1a. The Ge-O coordination energies at the B3LYP/6-311+G(d, p) level are 13.6 and 0.2 kJ/mol in 1a and 1b, respectively. The insertion reactions with CH₃F indicate that germylenoid PhCH₂(OH)CH₃GeLiF is more stable than germylene PhCH₂(OH)CH₃Ge. The insertion barrier of 1a with CH₃F is only 3.1 kJ/mol higher than that of PhCH₃CH₃GeLiF, indicating that the oxygen coordination PhCH₂(OH)CH₃GeLiF has the same stability as PhCH₃CH₃GeLiF.     

Determination of the Antineoplastic Agent Tiazofurin in Plasma by High Performance Liquid Chromatography

Abstract

A simple and sensitive sample clean up procedure and a reverse phase high performance liquid chromatographic assay for tiazofurin in dog plasma has been developed.

Thymidine was used as the internal standard and the elution solvent was 0.16 M acetic acid. The retention times of tiazofurin and thymidine at an elution rate of 2.0 ml/minute were 8.7 minutes and 16 minutes respectively. The assay sensitivity was 1.0 μG/ml. The within run coefficient of variation of 2.0 μG/ml and 20 μG/ml plasma samples were ±7.49% and ±3.37% respectively and the between run coefficient of variation of 2.0 μG/ml and 20.0 μG/ml plasma samples were ±10.46% and ±5.92% respectively.     

Determination of rimantadine in human urine by HPLC using a monolithic stationary phase and on‐line post‐column derivatization

In the present study, we propose the first HPLC method coupled to postcolumn derivatization for the determination of rimantadine in human urine samples. The analyte and amantadine (internal standard) were isocratically separated using an RP monolithic stationary phase (100 × 4.6 mm id) with a mobile phase consisting of CH₃OH/phosphate buffer (25 mmol/L, pH 3.0) at a volume ratio of 50:50. Postcolumn derivatization involved on‐line reaction with o‐phthalaldehyde (20 mmol/L) and N‐acetyl‐cysteine (5 mmol/L) at alkaline medium (100 mmol/L borate pH 11.0). Spectrofluorimetric detection at λ_ex/λ_em = 340/455 nm enabled the selective and sensitive determination of rimantadine in urine samples at a range of 50–500 ng/mL with an LOD of 5 ng/mL. Human urine samples were analyzed successfully after SPE using hydrophilic‐lipophilic balanced RP cartridges (30 mg/mL, Oasis HLB). Recoveries ranged between 89.7 and 102.7%.     

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%.     

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.     

Measurement of Nicotine in Plasma by High-Performance Liquid Chromatography

Abstract

A new procedure has been developed to measure nicotine in blood plasma by high-performance liquid chromatography (HPLC). Nicotine is extracted from plasma by elution with cholorform. Final determination is achieved by isocratic HPLC with ultraviolet detection. Twenty microliters of plasma extract is deluted over a silica column at a flow rate of 1.0 ml/min with a dioxane:isopropanol:NH₄OH (80:3.0:0.4) mobile phase. The procedure is sensitive to 0.05 ug of nicotine per ml of plasma and is linear within the range of 0.05 to 10.0 ug/ml of plasma. When a known amount of nicotine was added to plasma, the concentration of nicotine measured averaged 99.9 + 3.9 (S.D.)% of the known concentration. The within-sample coefficient of variation was 3.9%     

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.     

A facile and sensitive HPLC–MS/MS method for quantification of 3,3′-diindolylmethane in plasma samples

Indole-3-carbinol is the subject of ongoing biomedical research owing to its potential antiatherogenic, anticarcinogenic and antioxidant effects. The antitumor properties are mainly associated with its major metabolite, i.e. 3,3′-diindolylmethane (DIM). Typically, the biological activity of the chemical compound is manifested in the ng/ml concentration range. Consequently, the development of highly sensitive analytical methods to determine DIM in various biological samples is an urgent issue. In this study, an HPLC–MS/MS method was established for the quantification of DIM in human plasma. The developed method was validated according to the European Medicines Agency guidelines. Sensitivity, selectivity, accuracy and precision were good, allowing DIM quantification in the concentration range of 5–500 ng/ml. The limit of detection and the lower limit of quantification were 1 and 5 ng/ml, respectively. 4-Methoxy-1-methylindole was used as an internal standard (IS). The analytes were extracted from the human plasma by the acetonitrile-induced protein precipitation method with the addition of 3 mol/L ammonium sulfate as a salting-out agent, which is a facile and efficient approach for high-throughput bioanalysis. The chromatographic separation was performed on the Synergi Fusion-RP C₁₈ column (50 × 2.0 mm, 4 μm, 80 Å) under isocratic elution at 40°C. The mobile phase consisting of acetonitrile and water (0.1% formic acid; 85:15, v/v) was delivered at a flow rate of 0.20 ml/min. DIM and the IS were eluted at 2.36 ± 0.04 and 2.43 ± 0.03 min, respectively. The total analysis time was 3.20 min. Atmospheric pressure chemical ionization was carried out using multiple reaction monitoring in the positive polarity mode. The ion transitions were set to m/z 247.1 → 130.1 (DIM) and 162.1 → 147.1 (IS). The method was successfully applied to the analysis of plasma samples after a single oral administration of the Indinol® Forto drug (200 mg) to healthy female Russian volunteers. Also, the developed method was used for the analysis of rabbit plasma samples after a single oral dose of DIM (20 mg/kg).     

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.