Rapid and specific method for the determination of vancomycin in plasma by high-performance liquid chromatography on an aminopropyl column

A high-performance liquid chromatographic method has been developed for the quantitative analysis of vancomycin in plasma. The method involves protein precipitation with acetonitrile, followed by normal-phase chromatography on an aminopropyl column. The clear supernatant was injected after centrifugation, and the eluent was monitored at 240 nm. No interference was found either with endogenous substances or with many currently used drugs, indicating a good selectivity for the procedure. The standard curve was linear between 0.1 and 100 micrograms/ml, and the detection limit was 0.01 microgram/ml of plasma. The mean intra- and inter-assay coefficients of variation were 2.4 and 4.0%, respectively, in the 10-50 micrograms/ml range. Application of the method to the study of vancomycin pharmacokinetics in a rabbit after a single intravenous dose is also reported.     

Determination of psychotropic phenylalkylamine derivatives in biological matrices by high-performance liquid chromatography with photodiode-array detection.

Several procedures using high-performance liquid chromatography with photodiode-array detection have been developed to create phytochemical and toxicological profiles of phenylalkylamine derivatives in biological samples (e.g. plant materials and urine). Mescaline-containing cactus samples were extracted with basic methanol, using methoxamine as internal standard; the extraction and clean-up of urine samples were performed on cation-exchange solid-phase extraction columns. The extracts were separated on a 3-micron ODS column with acetonitrile-water-phosphoric acid-hexylamine as the mobile phase. Peak detection was performed at 198 or 205 nm; peak identity and homogeneity were ascertained by on-line scanning of the UV spectra from 190 to 300 nm. The detection limit of phenylalkylamine derivatives in urine and cactus material was 0.026-0.056 micrograms/ml and 0.04 micrograms/mg, respectively. Following a single oral dose of 1.7 mg/kg methylenedioxymethylamphetamine (MDMA) the concentrations found in urine ranged from 1.48 to 5.05 micrograms/ml MDMA and 0.07-0.90 micrograms/ml methylenedioxyamphetamine (a metabolite of MDMA). The mescaline content of the cactus Trichocereus pachanoi varied between 1.09 and 23.75 micrograms/mg.     

Determination of ceftriaxone, a novel cephalosporin, in plasma, urine and saliva by high-performance liquid chromatography on an NH2 bonded-phase column

A high-performance liquid chromatographic method has been developed for the determination of a new cephalosporin antibiotic in plasma, urine and saliva (mixed saliva) using normal-phase technique and an NH2 bonded-phase column. The eluent mixture was a combination of acetonitrile and an aqueous solution of ammonium carbonate. The rapid method involved precipitation of protein from fluids by means of acetonitrile followed by automatic injection of the supernatant. The detection limit was 0.4 micrograms/ml for plasma, 3 micrograms/ml for urine and 0.03 micrograms/ml for saliva using UV detection.     

Simultaneous analysis of flunixin, naproxen, ethacrynic acid, indomethacin, phenylbutazone, mefenamic acid and thiosalicylic acid in plasma and urine by high-performance liquid chromatography and gas chromatography-mass spectrometry.

Simple and reproducible high-performance liquid chromatographic (HPLC) and gas chromatographic-mass spectrometric (GC-MS) methods have been developed for the simultaneous analysis of several acidic drugs in horse plasma and urine. Although the capillary GC-MS column provided better separation of the drugs than the reversed-phase C8 (3 microns, 75 mm) HPLC column, the total analysis time with HPLC was shorter than the total analysis time with GC-MS. The HPLC system equipped with a diode-array detector provided simultaneous screening (limit of detection 100-500 ng/ml) and confirmation (limit 1.0 micrograms/ml) of the drugs. The HPLC system equipped with fixed-wavelength ultraviolet and fluorescence detectors provided a relatively sensitive screening [limit of detection 50-150 ng/ml for ultraviolet and 10 ng/ml for fluorescence (naproxen only) detectors] of the drugs. However, the positive samples had to be confirmed by using either the diode-array detector or the GC-MS system. The GC-MS system provided simultaneous screening and confirmation of the drugs at very low concentrations (20-50 ng/ml).     

Determination of cholesterol and cortisone absorption in polyurethane. I. Methodology using size-exclusion chromatography and dual detection.

A size-exclusion chromatographic method is described for measuring the absorption of the steroid-based lipids cholesterol and cortisone into Pellethane 2363, a polyurethane used in biomedical implants. The method uses refractometry and ultraviolet diode-array detection, with tetrahydrofuran as the mobile phase. Using an injection volume of 150 microliters, the lower limit of accurate measurement for cholesterol (refractive index detection) was 6 micrograms/ml with a lower limit of detection, based on a 2:1 signal-to-noise ratio, of 0.15 micrograms (1 microgram/ml). For cortisone (ultraviolet detection), the lower accurate limit was 0.6 micrograms/ml with a lower limit of 0.015 micrograms (0.1 micrograms/ml). The results show that after 44 h, 2037 micrograms/g cholesterol and 3131 micrograms/g cortisone were absorbed by the polyurethane. The method eliminates extensive sample manipulation and is sensitive to low levels of lipid in the presence of a high-molecular-mass synthetic polymer.     

Determination of oxytetracycline in blood serum by high-performance liquid chromatography with direct injection.

A direct injection analysis by high-performance liquid chromatography has been developed for oxytetracycline in serum of animals and fish. A Hisep shielded hydrophobic phase column (15 cm x 4.6 mm I.D.) and a mobile phase of methanol-0.2 M oxalic acid (10:90, v/v, pH 7.0) with ultraviolet detection at 360 nm were used. The standard calibration curves in serum of chicken, hog, cattle and rainbow trout were linear over the range 0.1-20 micrograms/ml. The recoveries of oxytetracycline from all serum samples determined at two different concentrations (0.5 and 2.0 micrograms/ml) were 88-103%. The detection limit was 0.05 micrograms/ml for every serum sample.     

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.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

Plasma determination of the novel anticonvulsant D,L-3-hydroxy-3-ethyl-3-phenylpropionamide and preliminary pharmacokinetic studies in the rat.

A sensitive gas chromatographic method with flame ionization detection was developed for the analysis in plasma of the novel anticonvulsant D,L-3-hydroxy-3-ethyl-3-phenylpropionamide (HEPP), using D,L-2-hydroxy-2-ethyl-2-phenylacetamide as the internal standard. HEPP was extracted from alkalinized plasma into dichloromethane and quantified after derivatization with bis(trimethylsilyl)-trifluoroacetamide: Standard curves were linear from 0.5 to 50 and from 2 to 100 micrograms/ml of plasma, using 1.5 and 5 micrograms of the internal standard, respectively. The lower limit of detection at a signal-to-noise ratio of 3 standard deviations was 0.33 micrograms/ml of sample. The sensitivity, accuracy and reproducibility of the method were shown to be satisfactory for pharmacokinetic studies of HEPP. After intraperitoneal administration of 50 mg/kg to Wistar rats, the principal kinetic parameters were: absorption half-life = 0.04 h; volume of distribution = 1.32 l/kg; clearance = 4.40 ml/min; peak concentration = 50 micrograms/ml; peak time = 0.25 h; mean residence time = 4.55 h.     

High-performance liquid chromatographic methods for the determination of the penems SCH 29482 and FCE 22101 in human serum and urine.

High-performance liquid chromatographic methods have been developed for the determination of two 6-(1-hydroxyethyl)penems, SCH 29482 (I) and FCE 22101 (II), in serum and urine. Serum samples were combined with an equal volume of methanol to remove proteins and, after centrifugation, an aliquot of the supernatant was analysed by ion-pair chromatography on a reversed-phase C18 column with hexadecyltrimethylammonium bromide as the ion-pairing agent. The compounds were detected by their ultraviolet absorbance at 305 nm for II and 322 nm for I. Urine samples were diluted, filtered and analysed by the same chromatographic procedure. At concentrations of 1-500 micrograms/ml of each compound, the within- and between-day precisions were 1.8-3.6 and 2.6-5.1%, respectively. The detection limit was 0.2 micrograms/ml for I and 0.3 micrograms/ml for II.     

Quantitation of N,N,N',N'-tetrakis (2-hydroxypropyl)-ethylenediamine in plasma by gas chromatography

A wide-bore capillary gas chromatographic method with nitrogen-selective thermionic detection is described for the quantitative analysis of N,N,N',N'-tetrakis (2-hydroxypropyl)ethylenediamine (Quadrol) in plasma. N,N,N',N'-tetrakis (2-hydroxybutyl)ethylenediamine is used as an internal standard. Rat or human plasma samples (0.5 ml) are mixed with internal standard, adjusted to alkaline pH and subjected to a single extraction with dichloromethane. Quadrol recovery from plasma typically exceeds 90%. The method is linear over the range 1.0-50 micrograms/ml. The working detection limit is 0.5 microgram/ml and the analysis time is under 7 min. The procedure has been used to obtain plasma concentration versus time data for the evaluation of Quadrol pharmacokinetics in rats.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.     

Comparison of pyrolysis-mass spectrometry with high performance liquid chromatography for the analysis of vancomycin in serum

Mass spectrometry coupled with a pyrolysis inlet system (Pyr-ms) is compared with high performance liquid chromatography (HPLC) for the determination of vancomycin and its crystal degradation products (CDP-Is) in human serum. Quantitative analysis of Pyr-ms was performed by selected ion monitoring (SIM) method at 108 mass:charge (m/z) of pyrolysis product of vancomycin. 3-Nitroaniline (138 m/z) was used as an internal standard. A mu-Bondapak C(18) column and the gradient mobile phase of triethylamine buffer (pH 6.2), acetonitrile and tetrahydrofuran and a photometric detection at 205 nm are found to be the optimum conditions for the HPLC determination of vancomycin and CDP-Is. The limit of detection (LOD=1 ng ml(-1)), linearity (1 ng ml(-1)-10 mg ml(-1)), relative standard deviation (R.S.D.=1%), time analysis (1/2 h) and sample volume (50 mul) of Pyr-ms are far better than of the HPLC method. However, the HPLC method can individually determine the concentration of vancomycin and its degradation products.     

Simple high-performance liquid chromatographic method for the determination of a new phytochemical drug, fellavine, and its metabolites in human and rat plasma and urine.

The use of a small precolumn instead of an injection loop for the determination of a new phytochemical drug, fellavine, and its metabolites is described. The method combines the direct injection of plasma and urine into the reversed-phase precolumn with separation on a Spheri-5 RP-18 analytical column. Different sorbents in the precolumn were compared. A recovery of fellavine and its metabolites from biological fluids except rat plasma of almost 100% was achieved on Chrompack RP (30-40 microns) and LiChrosorb RP-18 (7 microns). For rat plasma only the last sorbent gave 80% fellavine recovery. The influence of the protein binding on the fellavine recovery was examined. The limit of detection was equal to 0.05 micrograms/ml fellavine for plasma and 0.02 micrograms/ml for urine. To enhance the limit of detection longer precolumns were perferred.     

Rapid and accurate high-performance liquid chromatographic method for the determination of 3-methylindole (skatole) in faeces of various species

A rapid method for the determination of skatole (3-methylindole) in faeces by reversed-phase high-performance liquid chromatography is described. Samples of 0.5 g were extracted with 2 ml of methanol. The extract was purified on Amberlite XAD-8. The lower limit of detection was 2.5 ng per injection (0.2 microgram/g faeces). The mean recovery of skatole was 95%, and the mean coefficients of variation were 7.0% (intra-assay) and 11.8% (inter-assay). Skatole concentrations were clearly lower in faeces from ruminants (average 2.6 micrograms/g for goat, sheep and cattle) than in those from monogastrics. Mean concentrations in human samples were 15.5 micrograms/g, and 10 micrograms/g in mature domestic pigs. An effect of the anabolic status on skatole concentrations in faeces of pigs is likely.     

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

A high-performance liquid chromatographic method is described for the analysis of the anti-bacterial agent cefotaxime and desacetylcefotaxime in physiological fluids. Plasma or serum samples were mixed with chloroform--acetone to remove proteins and most lipid material. The aqueous phase was then freeze-dried, reconstituted in mobile phase and chromatographed on a reversed-phase column using UV detection at 262 nm. Urine was analysed directly after centrifugation to remove particulate matter. The detection limit was 0.5--1.0 micrograms/ml for serum and 5 micrograms/ml for urine. The method has been applied to the analyses of cefotaxime and desacetylcefotaxime in plasma, serum, urine, cerebrospinal fluid, saliva, and pus from infected wound secretions. Two additional metabolites, which are lactones in which the beta-lactam ring has been opened, could be separated by this method.     

High-performance liquid chromatographic method for simultaneous determination of enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2-dihydrobenzofuran-2, carboxylic acid and its N-monodemethyl metabolite in monkey plasma and urine after chiral derivatization

A quantitative method for the simultaneous high-performance liquid chromatographic (HPLC) resolution and determination of the enantiomers of 5-dimethylsulphamoyl-6,7-dichloro-2,3-dihydrobenzofuran-2-carboxyl ic acid, a new diuretic, and its N-monodemethylated metabolite in monkey plasma and urine is described. The method includes diethyl ether extraction of the samples and S-(-)-alpha-methylbenzylamide derivatization of the extract, followed by reversed-phase solid-phase extraction and injection of the resulting diastereoisomers onto a reversed-phase HPLC column. Baseline separation was obtained. The assay showed linearity over the range 0.1-50 micrograms/ml of plasma and 0.25-500 microliters of urine, with a lower limit of detection of ca. 0.01 micrograms/ml for each of the enantiomers. The method is adequate for pharmacokinetic and enantioselective disposition studies of both the diuretic and its metabolite.     

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.     

High-performance liquid chromatographic determination of vinblastine, 4-O-deacetylvinblastine and the potential metabolite 4-O-deacetylvinblastine-3-oic acid in biological fluids.

Procedures for the determination of vinblastine (VBL), 4-O-deacetylvinblastine (DVBL) and 4-O-deacetylvinblastine-3-oic acid (DVBLA) in biological samples using high-performance liquid chromatography (HPLC) combined with selective sample clean-up are presented. VBL and DVBL were determined in plasma and urine using ion-exchange normal-phase HPLC with fluorescence detection. The limit of detection was 1 microgram/l for both compounds using a 500-microliter sample. Successful chromatographic analyses of DVBLA were achieved by using a glass column packed with 5-microns Hypersil ODS and acetonitrile-0.05 M phosphate buffer (pH 2.7) (23:77, v/v). Positive identification was supported by the use of diode-array detection. The limit of detection (at 270 nm) was 10 micrograms/l using 1-ml samples.