Determination of Ibafloxacin,A New Quinolone Antibacterial,in Human and Dog Plasma and Urine by High Performance Liquid Chromatography

Abstract

A simple, selective and sensitive high performance liquid chromatographic method has been developed for quantifying plasma and urine ibafloxacin levels in humans and dogs.

Sample pretreatment is done by incorporation of an internal standard (IS) followed by a single step chloroform extraction. Samples are then chromatographed by reverse phase chromatography with UV detection. The lowest quantifiable concentration is 0.1 μg ibafloxacin/mL with a 1 mL sample. The assay was linear over the range of 0.1–50 μg/mL.     

A Direct and Sensitive Method for the Determination of Salicylamide in Microplasma Samples by High-Performance Liquid Chromatography Using Fluorescence Detection

Abstract

A quantitative analysis of salicylamide in microplasma volumes by high-performance 1iquid chromatography using fluorescence detection is reported. The procedure is extremely simple and very rapid, involving the direct introduction of the plasma sample on the HPLC column. The assay procedure is linear over the concentration range studied, 0–100 ng/ml with correlation coefficient for the linear regression, r = 0.998. This assay procedure enables the detection of salicylamide as low as 5.0 ng/ml in plasma, using sample volume of 100 μl.     

Quantitation of Meclizine Dihydrochloride in Serum by Reversed Phase Ion Pair High Performance Liquid Chromatography

Abstract

A specific and sensitive HPLC method has been developed for the assay of meclizine dihydrochloride in dog serum using an internal standard technique with a single step extraction. The extracts are injected into a reversed phase ion pair HPLC system using a solvent containing camphorsulfonate as paring anion. The detection limit is 5 ng/ml and the range of linearity is 5–250 ng/ml. The method has been used to quantitate meclizine dihydrochloride levels in bioavailability and pharmacokinetic studies in dogs.     

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.     

HPLC Determination of Xylazine in Equine Plasma by High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) method is described for the determination of xylazine in equine plasma. The drug and internal standard (pindolol) were separated on a 5 μm cyanopropyl-modified column (250 × 4.6 mm i.d.) using a buffer-acetonitrile mixture containing an ion pairing reagent. The drug and internal standard were isolated from plasma by liquid extraction into ethyl acetate. The method was validated over the concentration range 50–2000 ng/ml in plasma; the reproducibility, expressed as the mean co-efficient of variation was less than 5.0% for both between-day and within-day replicate determinations. The method was linear over the concentration range studied. No interferences were observed from endogenous plasma components and the limit of detection was 20 ng/ml. The method was successfully applied to the determination of xylazine in equine plasma in a crossover study design for pharmacokinetic measurements.     

Sodium Coumarin 6-Sulfonate,A Fluorometric Ion-Pairing Reagent for Tertiary Amines. Application to the Determination of Chlorpheniramine Maleate

Abstract

A study of sodium coumarin 6-sulfonate as a fluorescent ionpair reagent indicated that it could be useful in the analysis of tertiary amines such as chlorpheniramine maleate. The physical properties of the coumarin sulfonate that make it suitable as a fluorescent ion-pair reagent for basic drugs are its high relative quantum yield (0.76) and its acidity (pKa of ?7.66). Methylene chloride containing 5% n?pentanol was used to extract the coumarinchlorpheniramine ion-pair from aqueous solution. It was found that a 10^?2 M coumarin concentration yielded a 92% recovery of chlorpheniramine at pH 5. Following phase separation, the coumarin species was completely ionized by the addition of tetrabuty1 ammonium hydroxide to the organic phase. After irradiation for 30 min using long wavelength ultraviolet light (365 nm), the fluorescence intensity of the sample was measured using excitation and emission wavelengths of 400 and 540 nm, respectively. Comparison of fluorescence data of spiked aqueous samples to that of a chlorpheniramine maleate standard curve performed concurrently gave drug concentration in the samples. The calibration curve was linear in the 50–1000 ng/ml range (0.13 ? 2.6 × 10 ^?6 M). The procedure allowed the determination of chlorpheniramine maleate with an accuracy of 4–6% and a precision of 2–6% RSD (relative standard deviation). The minimum detectable concentration of drug (S/N = 2) that can be assayed by this method is 50 ng/ml of the maleate salt (35.3 ng/ml of chlorpheniramine free base).     

Development and Validation of High‐Performance Liquid Chromatographic Method for Estimation of Lercanidipine in Rabbit Serum

Abstract

A new, simple, and sensitive reverse‐phase liquid chromatographic method was developed and validated for the estimation of Lercanidipine hydrochloride in rabbit serum using UV detector under isocratic conditions. After subjecting serum to simple and efficient one‐step extraction procedure, 100 µl of sample was injected onto high‐performance liquid chromatography system. The detector response was linear in the concentration range of 25–1000 ng/ml. The developed method was validated as per standard guidelines. Validation demonstrated accuracy, precision, and selectivity of the proposed method. The drug was found to be stable under various processing and storage conditions.     

Assay of Yohimbine in Human Plasma Using High Performance Liquid Chromatography with Electrochemical Detection

Abstract

Yohimbine is a selective α₂ adrenoreceptor antagonist used in the study of α₂ adrenoreceptors in man. In order to better improve administration regimens for the study of yohimbine in man, we have developed an assay for the determination of yohimbine in plasma utilizing reverse phase high performance liquid chromatography with electrochemical detection. Using a C₁₈ column and a methanol:acetate (60:40) mobile phase, we detected yohimbine in plasma following a simple chloroform extraction. Reserpiline was used as an internal standard. The assay was linear over a concentration range of 50–250 ng/ml in spiked plasma and had a lower limit of sensitivity of 10 ng/ml. It was used to detect yohimbine in plasma sampled from 4 volunteers during an infusion of the alkaloid.     

Determination of bepridil in biological fluids by high-performance liquid chromatography

A rapid, selective and sensitive assay has been developed for the determination of the anti-anginal drug, bepridil, in biological samples. The lowest concentration of bepridil which can be measured accurately and precisely in a 2-ml plasma or urine sample is 10 ng/ml. The standard curve is linear in the concentration range 10-2000 ng/ml. Accuracy and precision of the assay, expressed as relative deviation and coefficient of variation (inter-run) are less than 6.5% at all concentrations in the linear range. No interfering peaks are observed. Using an automatic injector and a laboratory computer system, 48 samples are analyzed routinely in an 8-h day.     

HPLC Determination of Methylphenidate in Human Plasma

Abstract

A simple, rapid and sensitive method for measuring methylphenidate in human plasma by HPLC has been developed. After the addition of the internal standard, ethylphenidate, the two compounds are extracted under basic conditions. The residue obtained is resuspended in acetonitrile and analysed on an ODS reversed phase column with detection by UV absorbance at 192 nm. The limit of sensitivity is 5 ng/ml and the procedure is linear over the 5–50 ng/ml concentration range.     

An HPLC Assay for a Prostacyclin Analogue,Ciprostene Calcium,in Human Plasma

Abstract

A sensitive assay has been developed for the quantification of the prostacyclin analogue, ciprostene calcium, in human plasma. The method involves solid phase extraction of ciprostene calcium and internal standard, carbacyclin, from a small volume of human plasma. The extract is derivatized with 4-bramamethyl-7-acetoxycoumarin, and the derivatized product extracted with a polar solid phase cartridge and concentrated by evaporation. The final extract is separated by reversed phase HPIC and measured by a fluorimetric detector following post-column alkaline hydrolysis. The overall extraction efficiency is better than 75%, and the assay is linear over the concentration range studied (5–20 ng/ml). The limit of quantification was approximately 5 ng/ml. Ultimate sensitivity was limited by interfering peaks endogenous to the biological matrix. Coefficients of variation at mid-range concentrations are less than 10%.     

Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).     

Quantitative determination of tramadol in human serum by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the quantitative determination of tramadol in human serum, plasma or whole blood samples is described. The method involves the use of [2H2, 15N]tramadol hydrochloride as an internal standard and chemical ionization with isobutane, employing single-ion monitoring for quantification. It is specific, sensitive and precise, and has high accuracy. The within-run coefficient of variation is about 1% between 25 and 200 ng/ml and 1.8-2.9% at the lowest concentrations tested (6.25 and 12.5 ng/ml). The between-run coefficient of variation increases from 1.6% to 5.2% with decreasing concentration from 200 to 12.5 ng/ml. The calibration graphs were linear in the tested concentration range, and the accuracy of the assay was not dependent on the sample volume used. The detection limit was about 4 ng/ml for serum samples of 1 ml. The method proved suitable for pharmacokinetic studies. Its high sensitivity allows measurements of serum concentrations for at least 30 h after the single administration of therapeutic doses of tramadol hydrochloride.     

Development of a gas chromatographic/mass spectrometric method to quantify R(-)-apomorphine, R(-)-apocodeine and R(-)-norapomorphine in human plasma and urine

A method was developed and validated for the analysis of R(-)-apomorphine, (R-)-apocodeine and R(-)-norapomorphine in human plasma and urine with N-propylnorapomorphine as internal standard using gas chromatography/mass spectrometry (GC/MS) and single-ion monitoring after a single liquid-liquid extraction and silylation of compounds. The quantification limits were 1 ng/ml for apomorphine and apocodeine and 25 ng/ml for norapomorphine. Calibration curves were linear, within the range 1-100 ng/ml. Variation in intraday and interday precision was below 10%. This method was applied to study apomorphine bioavailability in nine patients with Parkinson's disease before and after coadministration of a catechol-O-methyl transferase inhibitor.     

Determination of the calcium entry blocker verapamil in plasma by capillary gas chromatography with on-column injection

A specific, sensitive, and accurate assay for quantitation of verapamil has been developed. The method involves a single extraction step with n-heptane, followed by evaporation at room temperature under nitrogen. 0.1 μl of the extract was injected into a capillary column coated with crosslinked 5% phenylmethylsilicone. The column separated verapamil, norverapamil, and its internal standard within 15 minutes using temperature programming from 90 to 290°C. The lower limit of detection was 1 ng/ml for verapamil. The calibration curve was linear in the concentration range (5–300 ng/ml). Plasma concentration data from dogs receiving intravenous verapamil infusion are presented.     

Determination of Ofloxacin in Pharmaceutical Forms by High - Performance Liquid Chromatography and Derivative Uvspectrophotometry

Abstract

A method for the determination of ofloxacin in pharmaceutical forms is described. The procedure is based on the use of the high-performance liquid chromatography, and of the second-derivative ultraviolet spectra, by utilizing the linear relationship between substance concentration and derivative peak amplitude. The minimum concentration detectable by derivative spectrophotometry was 20 ng/ml, and by HPLC 10 ng/ml. The proposed methods, which give thoroughly comparable data, are simple and rapid, and allow precise and accurate results.     

Triazolines. XXIII. High-performance liquid chromatographic assay in rat blood for a novel triazoline anticonvulsant (ADD17014)

A sensitive and specific high-performance liquid chromatographic (HPLC) method for the analysis of 1-(4-chlorophenyl)-5-(4-pyridyl)-delta 2-1,2,3-triazoline (ADD17014, I), a novel anticonvulsant agent, in rat blood is described. Compound I and the internal standard (dipyridamole) were extracted into diethyl ether (5 ml) from alkalinised blood (0.25 ml of blood plus 0.75 ml of pH 10.7 buffer), with extractability nearing 100% under these conditions. The assay is based on reversed-phase HPLC (25 cm x 0.46 cm I.D. Spherisorb 5-ODS) using a mobile phase of methanol-acetonitrile-McIlvaine's citric acid-phosphate buffer (pH 8.0, 0.005 M) (30:30:40, v/v) and ultraviolet detection at 290 nm. Calibration curves were linear and reproducible (correlation coefficient greater than 0.999). Measurement of I in rat blood (250 microliters sample size) was linear in the range 0-40 microgram/ml and the coefficient of variation was less than 5%. The minimum detectable level was about 0.1 microgram/ml; however, a larger blood sample size (1-2 ml) allowed measurement of levels as low as 10 ng/ml, especially for estimation of drug levels in samples withdrawn at later time points (24 h).     

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.     

Analysis of Bunaprolast,An Esterase Unstable Drug,with An Active Metabolite Subject to Oxidative Degradation,in Blood Plasma

Abstract

This report describes a sensitive, selective and robust assay for bunaprolast and an active metabolite in canine and human plasma. Method development was complicated in that bunaprolast is quickly hydrolysed by esterases even in vitro, and the metabolite is rapidly oxidised in dilute aqueous solutions. Effective measures to stabilize analytes in biological matrices and during sample extraction are described.

The method involves the selective solid phase extraction of analytes with a close analogue as internal standard followed by reversed phase HPLC and fluorescence detection. The limit of quantification was typically less than 1 ng/ml, and the assay was linear over the range 1 - 1,000 ng/ml.     

High-performance liquid chromatographic determination of galanthamine, a long-acting anticholinesterase drug, in serum, urine and bile

The anticholinesterase drug galanthamine is obtained from alkalinized serum by repeated liquid--liquid extraction. The resulting extract is approximately 100 times concentrated with respect to the original sample. Quantitative determination of galanthamine is performed with normal-phase liquid chromatography using a mixture of dichloromethane--n-hexane and ethanolamine as an eluent. Phenacetin is used as internal standard. The absorption of the column effluent is monitored at 235 nm. No endogenous sources of interference have been observed. A galanthamine serum level of 5 ng/ml is found as the minimum detectable concentration; the coefficient of variation at this level is 37.8% (n = 4). For the assay of galanthamine in the concentration range 10-100 ng/ml, standard deviations vary between 18.9 and 2.5% (n = 32).