High-performance liquid chromatographic analysis of the anticancer drug oxantrazole in rat whole blood and tissues.

A reversed-phase high-performance liquid chromatographic (HPLC) assay was developed for the antitumor anthrapyrazole analogue, oxantrazole (OX), in rat whole blood and tissues. Blood samples were mixed with equal volumes of a 25% (w/v) aqueous solution of L-ascorbic acid, whereas tissue samples were homogenized with 1.5-3 volumes of an L-ascorbic acid-methanol-water (1:10:1, w/v/v) mixture to prevent oxidative degradation of OX. Samples were then treated with 60% (v/v) perchloric acid (25-30 microliters/ml of stabilized sample) to precipitate proteins, and centrifuged, with the resultant supernatants analyzed on HPLC utilizing a C8 column. The mobile phase for blood and urine samples consisted of 8% (v/v) glacial acetic acid, 13% (v/v) acetonitrile, 79% (v/v) water, 0.16% (w/v) sodium acetate, and 0.05% (w/v) L-ascorbic acid (final pH 2.7), and was pumped at 1.8 ml/min. Tissue samples were eluted at 2 ml/min with a mobile phase consisting of 8% (v/v) glacial acetic acid, 12% (v/v) acetonitrile, 80% (v/v) water, 0.16% (w/v) sodium acetate, and 0.0;5% (w/v) L-ascorbic acid. OX and internal standard were detected at 514 nm and had retention times of 2.3 and 3.1 min, respectively. The limit of quantitation of OX was 25-50 ng/g. Recovery of OX from biological samples ranged from 50 +/- 0.9% in spleen to 102.8 +/- 1.8% in RG-2 glioma. The analytical method was applied to a pharmacokinetic study in rats.     

Determination of cyclosporin A in whole blood by high-performance liquid chromatography using automated column switching

A fully automated high-performance liquid chromatographic column-switching system is presented for the determination of cyclosporin A in whole blood. After blood proteins were precipitated with acetonitrile, the supernatant was automatically loaded on to a cyanopropyl column for initial separation, and then the fraction containing cyclosporin A was loaded on to a trimethylsilica column for final separation and quantitation. Cyclosporin A was detected by ultraviolet absorption at 205 nm. The minimum detectable concentration of cyclosporin A was 5 ng/ml in 100 microliter of blood. The coefficient of variation of the method was 1.755, 1.748 and 0.655% in whole blood when spiked at the 170, 425 and 850 ng/ml levels, respectively. One assay was completed in 15 min.     

Analysis of taurine in feline plasma and whole blood by liquid chromatography with fluorimetric detection and confirmation by thermospray mass spectrometry

A liquid chromatographic method with fluorimetric detection was developed to measure taurine (2-aminoethanesulfonic acid) in feline plasma and whole blood. Plasma or lysed whole blood was diluted with a mixture of acetonitrile-methanol-triethylamine-water (25:22:3:50, v/v), filtered through a 10,000 dalton exclusion filter and derivatized with dansyl chloride for 30 min at room temperature. Dansyl taurine was separated from other compounds by reversed-phase liquid chromatography using an octadecyl column and a methanol-acetic acid-triethylamine (30:0.5:0.025, v/v) aqueous mobile phase. The effluent was monitored fluorimetrically at an excitation wavelength of 329 nm and an emission wavelength of 530 nm. The presence of mono-dansylated taurine in feline plasma was confirmed by thermospray mass spectrometry. The limit of detection was 16 nmol/ml and the detector response was linear from 40 to 4000 nmol/ml taurine.     

Rapid determination of propyphenazone in plasma by high-performance liquid chromatography.

A rapid and simple high-performance liquid chromatographic assay for the determination of propyphenazone in plasma is described. Phenylbutazone was used as the internal standard. Plasma proteins were precipitated with acetonitrile before injection onto a 3-microns Supelcosil LC-18 column. The mobile phase, ethanol containing 0.2% (v/v) heptylamine-0.005 M potassium dihydrogenphosphate (30:70, v/v), was used at a flow-rate of 1.3 ml/min. The quantitation was performed by ultraviolet detection at a wavelength of 270 nm. The chromatographic time was 7 min. The within- and between-day coefficients of variation were less than 6% and the recoveries close to 100% for concentrations between 0.4 and 22 mumol/l. The limit of quantitation was 0.4 mumol/l (ca. 100 ng/ml).     

Simultaneous Determination of Flunixin,Phenylbutazone, Oxyphenbutazone and γ-Hydroxyphenylbutazone in Equine Plasma by High-Performance Liquid Chromatography: With Application to Pharmacokinetics

Abstract

A high performance liquid chromatographic method was developed for the simultaneous determination of flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone in equine plasma. Samples of plasma or sera were deproteinated by addition of acetonitrile containing the internal standard naproxen. The concentration step consisted of taking an aliquot of deproteinated plasma, evaporating under nitrogen to dryness and redissolving in mobile phase. The extracts were chromatographed on a Spherisorb 5 μm ODS column using an isocratic mobile phase of methanol (30% v/v), acetonitrile (20% v/v) and pH 3.0 1% acetate buffer (50% v/v) at a flow rate of 1.2 ml/min using naproxen as the internal standard. The detection limit for flunixin, phenylbutazone, oxyphenbutazone and γ-hydroxyphenylbutazone was 50 ng/ml.

The developed chromatographic method was applied to the determination of equine nonsteroidal anti-inflammatory treatment. Plasma samples from clinically treated horses administered flunixin and phenylbutazone simultaneously are reported. Effect of different anticoagulants used in sampling is reported.     

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.     

Improved and Rapid Liquid Chromatographic Determination of Indomethacin in Serum

Abstract

A rapid, specific and sensitive reversed-phase liquid chromatographic (LC) assay for the quantitative determination of indomethacin in serum without extraction was developed. Chromatographic separation using flunixin meglumine as the internal standard was achieved on octadecylsilane-coated particles with a mobile phase of 0.15 M acetate buffer pH 3.0 (50% v/v), acetonitrile (30% v/v) and methanol (20% v/v). The recovery of indomethacin from serum samples in the concentration range of 0.1-25 μg/ml was 95.5 ± 5.8% and as little as 100 ng/ml of indomethacin in serum samples can be quantitated by this procedure. A serum level versus time profile of dog with intravenously administered indomethacin demonstrated the applicability of the assay.     

Determination of methanol in whole blood by capillary gas chromatography with direct on-column injection.

A gas chromatographic procedure was developed for the determination of methanol in small-volume whole blood samples. Samples (100-200 microliters) were prepared by protein precipitation, with direct injection of the supernatant on a wide-bore capillary column. The recovery of methanol and acetonitrile (the internal standard) was approximately 90% and did not vary with sample volume. The assay was linear from 2 micrograms/ml (the limit of detection) through 1000 micrograms/ml and was highly reproducible (intra-day coefficient of variation less than 2.5%). Assay performance was assessed following exposure of rats to methanol. The results indicate that the present procedure is suitable for studies of methanol disposition in small rodent species.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.     

Concomitant determination of thiamin and its phosphate esters in human blood and serum by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous determination of thiamin and thiamin phosphate esters in human blood or serum has been developed. The eluent consists of acetonitrile and phosphate buffer, in the ratios 90:10 (v/v) for the elution of thiamine and 60:40 (v/v) for the phosphate esters. The four compounds are eluted within 15 min. The detection limit is 13-16 fmol. Between-assay variation is 5-11%. Samples of whole blood and serum from 30 healthy adults were analysed. The following reference values were obtained for 15 females 15 males (nM, mean +/- S.D.). In serum: thiamin, 10.9 +/- 2.9/16.9 +/- 3.3; thiamin monophosphate, 8.3 +/- 1.5/3.7 +/- 1.5. In whole blood: thiamin, 29.6 +/- 10.0/33.4 +/- 10.4; thiamin monophosphate, 9.7 +/- 2.3/10.9 +/- 5.1; thiamin diphosphate, 121 +/- 29.6/165 +/- 40.4.     

Quantification of the anticancer agent STI-571 in erythrocytes and plasma by measurement of sediment technology and liquid chromatography-tandem mass spectrometry

An isocratic high-performance liquid chromatographic method coupled to tandem mass spectrometry for the quantification of the revolutionary and promising anticancer agent STI-571 (tradenames Gleevec, Glivec, Imatinib) in blood plasma and red blood cells (RBCs) is described. The method involves measurement of sediment technology for RBCs and a subsequent single protein precipitation step by the addition of acetonitrile to both the RBC isolate and plasma. The sample mixture was centrifuged (10 min, 3600 g), and the supernatant filtered through a HPLC filter (0.45 microm). The analytes of interest, STI-571 and the internal standard [2H8]STI-571 were eluted on a Waters Symmetry C18 column (50x2.1 mm I.D., 3.5 microm particle size) using a methanol-0.05% ammonium acetate (72:28, v/v) mixture. STI-571 and [2H8]STI-571 were detected by electrospray tandem mass spectrometry in the positive mode, and monitored in the multiple reaction monitoring transitions 494>394 and 502<394, respectively. The lower limit of quantitation of STI-571 was 2.1 ng/ml in RBCs and 1.8 ng/ml in plasma. The recovery from both plasma and RBCs was between 65 and 70%. The method proved to be robust, allowing simultaneous quantification of STI-571 in RBCs and plasma with sufficient precision, accuracy and sensitivity and is useful in monitoring the fate of this signal transduction inhibitor in whole blood of cancer patients.     

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     

Application of point-to-point matching algorithms for background correction in on-line liquid chromatography-Fourier transform infrared spectrometry (LC-FTIR)

A new background correction method for the on-line coupling of gradient liquid chromatography and Fourier transform infrared spectrometry has been developed. It is based on the use of a point-to-point matching algorithm that compares the absorption spectra of the sample data set with those of a previously recorded reference data set in order to select an appropriate reference spectrum. The spectral range used for the point-to-point comparison is selected with minimal user-interaction, thus facilitating considerably the application of the whole method. The background correction method has been successfully tested on a chromatographic separation of four nitrophenols running acetonitrile (0.08%, v/v TFA):water (0.08%, v/v TFA) gradients with compositions ranging from 35 to 85% (v/v) acetonitrile, giving accurate results for both, baseline resolved and overlapped peaks.     

Semi-automated high-performance liquid chromatographic determination of cyclosporine A in whole blood using one-step sample purification and column-switching

A highly sensitive and semi-automated high-performance liquid chromatographic method, utilizing acetonitrile protein precipitation and column-switching, is described for the determination of cyclosporine A in whole blood. Following a rapid manual acetonitrile treatment of the blood samples, the supernatant is loaded automatically onto a 5-micron high-speed protein separation column without any further clean-up operations. The fraction containing cyclosporine A is switched to a 3-micron C18 reversed-phase high-speed column by a microprocessor-controlled column-switching unit for final separation and detection by absorption at 214 nm. Minimal sample handling and efficient separation resulted in a high recovery (75 +/- 3%) of cyclosporine A from blood and a detection limit as low as 2 micrograms/l with a highly reproducible and linear response up to 2500 micrograms/1 using 0.5 ml of sample. A separation cycle including regeneration of the first column is finished in 15 min, and this system was used continuously for ca. 1000 blood samples from heart, liver, kidney, pancreas and bone marrow recipients without change in separation parameters or material replacement. The method described allows accurate and very fast daily routine monitoring of cyclosporine A in large numbers of blood samples from transplant recipients.     

A Simple Isocratic High-Performance Liquid Chromatographic (HPLC) Determination of Naproxen in Human Plasma using a Microbore Column Technique

Abstract

A simple and sensitive HPLC method was developed for the determination of naproxen in human plasma. The assay employs a microbore column packed with a C18 reversed-phase material (5 μm ODS Hypersil) with an isocratic mixture of acetonitrile and 10 mM phosphate buffer, pH 2.5 (40:60, v/v) as the mobile phase. The mobile phase was pumped at a flow rate of 0.5 ml/min. For sample analysis 200 μl of acetonitrile containing internal standard (flurbiprofen) was added to 100 μl of plasma. After centrifugation 10 mM phosphate buffer, pH 7.4 (200 μl) was added to the tube, then vortexed and centrifuged. The supernatant (20 μl) was injected onto the HPLC column. The chromatographic separation was monitored by a fluorescence detector at an emission wavelength of 350 nm with an excitation wavelength of 225 nm. The direct precipitation of plasma protein using acetonitrile gave a good recovery for both naproxen and the internal standard. The detection limit was 0.1 μg/ml for naproxen. The intra- and inter-assay coefficients of variation at different concentrations evaluated were less than 10%.     

Liquid Chromatography–Electrospray Ionization–Mass Spectrometric Method for the Determination of Hydrochlorothiazide in Human Plasma: Application to a Pharmacokinetic Study

Abstract

A rapid, convenient, and sensitive liquid chromatography–electrospray ionization–mass spectrometry method was developed and validated for the quantification of hydrochlorothiazide in human plasma. The samples were first spiked with the internal standard, and the analyte was then extracted with ethyl acetate. The chromatographic separation was achieved on a C₁₈ column by using water–acetonitrile (68:32, v/v) as mobile phase. The method was linear within the range of 2.5–200 ng/ml. The lower limit of quantification was 1.0 ng/ml. Finally, the validated method was successfully applied for the evaluation of the pharmacokinetic profiles of hydrochlorothiazide in healthy male Chinese volunteers.     

Determination of hydroxy and peroxy acid derivatives of uroporphyrin in the plasma of patients with congenital erythropoietic porphyria by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatographic method is described for the determination of hydroxy and peroxy acid derivatives of uroporphyrin in the plasma of patients with congenital erythropoietic porphyria. The porphyrins were extracted from the plasma with 20% trichloroacetic acid-dimethyl sulphoxide (1:1, v/v). The supernatant after centrifugation was chromatographed on a Hypersil-ODS column by gradient elution with 9% (v/v) acetonitrile in 1 M ammonium acetate buffer (pH 5.16) (solvent A) and 10% (v/v) acetonitrile in methanol (solvent B) as the gradient mixture. The method was also suitable for the preparative isolation of the porphyrins.     

Mixture design optimization of extraction and mobile phase media for fingerprint analysis of Bauhinia variegata L

Two statistical mixture designs were used to optimize the proportions of solvents used in both the extraction medium and the reversed liquid chromatographic mobile phase to improve the quality of chromatographic fingerprints of Bauhinia variegata L extracts. For modeling, the number of peaks was used as a measure of fingerprint information. Three mobile phases, each with a chromatographic strength of two, gave good results. A methanol/water (77:23 v/v) mixture resulted in 17 peaks in the chromatographic fingerprint whereas acetonitrile/water (64.5:35.5 v/v) and methanol/acetonitrile/water (35:35:30 v/v/v) mixtures resulted in 18 and 20 peaks, respectively. The corresponding optimum solvent compositions to extract chemical substances for these three mobile phases were ethanol/acetone (25:75 v/v/v) and dichloromethane/acetone (70:30 v/v) mixtures, and pure dichloromethane, respectively. The mixture designs are useful for understanding the influence of different solvents on the strengths of the extraction medium and the mobile phase.     

Evaluation of astemizole purity by HPLC

A liquid chromatographic method is described for the evaluation of Astemizole purity. The method is also applicable for the analysis of pharmaceutical dosage forms of Astemizole using indole as internal standard. Separation was achieved with a micro bondapak C(18) column and phosphate buffer (pH 6.0):acetonitrile (50:50 v/v) as eluent, at a flow rate of 1.5 ml/min with UV detection at 280 nm. The method is suitable for estimating the possible impurities if any present in Astemizole up to 0.05% for 1a and 0.25% for 1b. The method is simple, rapid and accurate for the analysis of Astemizole.     

Determination of glycyrrhizin in rabbit plasma by high-performance liquid chromatography with photodiode-array ultraviolet detection and its pharmacokinetics application.

A simple and sensitive high-performance liquid chromatographic method for the determination of glycyrrhizin in rabbit plasma has been developed. Up to 0.1 ml of plasma containing glycyrrhizin was deproteinized by acetonitrile, which contained an internal standard (indomethacin). The supernatant was injected onto a LiChrospher RP-18 column using a methanol-water-ammonia solution (80:20:0.1, v/v, pH 3.0-3.2, adjusted with perchloric acid) as the mobile phase and ultraviolet detection at 254 nm, followed by ultraviolet spectrum identification (between 200 and 380 nm) with a photodiode-array detector. The method is rapid, easily reproduced, selective and sensitive. It was applied to pharmacokinetic studies of glycyrrhizin in rabbit, after a 2 mg/kg intravenous administration. A biphasic phenomenon with a rapid distribution followed by a slower elimination phase was observed from the plasma concentration-time curve. Compartmental analysis yielded a two-compartment model.