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.     

Development of analytical methods for some penicillins in bovine milk by ion-paired chromatography and confirmation by thermospray mass spectrometry

Analytical methods for the determination of cloxacillin, ampicillin/hetacillin, and amoxicillin in bovine milk were developed. The methods involved ultrafiltration of milk diluted with methanol, acetonitrile, and water on a 10,000-dalton cut-off filter. Separation of penicillins from other milk components was accomplished by ion-paired chromatography using a microbore column. The penicillins were detected using ultraviolet photodiode array (UV-PDA) detection and confirmed by thermospray liquid chromatography-mass spectrometry (LC-MS). The thermospray spectra of these compounds exhibited [M + H]+ and [M + Na]+ ions along with several fragment ions. The limits of detection for these antibiotics were estimated to be 50 to 100 ppb for LC with UV-PDA detection and 100-200 ppb for thermospray LC-MS detection.     

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.     

Determination of pyrimethamine in human plasma after administration of fansidar of fansidar-mefloquine by means of high-performance liquid chromatography with fluorescence detection

A sensitive, rapid and selective high-performance liquid chromatographic (HPLC) method has been developed to measure plasma levels of pyrimethamine in human subjects dosed with the antimalarials Fansidar or Fansidar and mefloquine. The drug was extracted from plasma at basic pH with n-butyl chloride-dichloromethane (96:4, v/v) and quantified on a normal-phase HPLC column with fluorescence detection (excitation 290 nm, emission 345 nm). Pyrimethamine was almost quantitatively extracted from plasma in the concentration range 20-200 ng/ml. The sensitivity limit was about 10 ng/ml of plasma, using a 0.5-ml specimen. The method was shown to be specific with respect to the other two components in the antimalarial combinations, namely sulfadoxine and mefloquine, and their metabolites. The assay was applied to pharmacokinetic studies of pyrimethamine in man following the oral administration of Fansidar of Fansidar and mefloquine.     

Determination of nadolol in serum by high-performance liquid chromatography with fluorimetric detection.

A sensitive high-performance liquid chromatographic method for a routine assay of nadolol in serum is described. Serum samples spiked with atenolol (internal standard) were extracted with diethyl ether. After centrifugation, the organic layer was evaporated to dryness. The residue was redissolved in the mobile phase and injected onto an octadecyl silica column (150 mm x 4.6 mm I.D.). The mobile phase was 0.05 M ammonium acetate (pH 4.5)-acetonitrile (85:15, v/v). Fluorometric detection (excitation 230 nm, emission 300 nm) was used. The minimum detectable level of nadolol in serum was 1 ng/ml.     

Determination of 17-oxosteroid sulphates in serum by ion-pair extraction, prelabelling with dansylhydrazine and high-performance liquid chromatography with fluorescence detection

A new high-performance liquid chromatographic (HPLC) method with fluorescence detection is described for the direct determination of four serum 17-oxosteroid sulphates. Each serum sample was deproteinated with methanol, the methanol was evaporated and 17-oxosteroid sulphates in the residue were extracted with benzene as ion pairs in the presence of tetrapentylammonium ion. The ion pairs were labelled with dansylhydrazine and the hydrazones were separated by HPLC on a Capcell-Pak C8 (silicone polymer-coated silica gel modified with octyl groups) reversed-phase column using methanol-0.5% (w/v) sodium acetate-50% (v/v) acetic acid (57:42:1, v/v/v) as the mobile phase. The eluent was monitored with a fluorometric detector at an excitation wavelength of 330 nm and an emission wavelength of 540 nm.     

Determination of n-acetylmuramoyl-l-alanyl-d-isoglutamine in liposomes by HPLC

A method using reversed-phase high-pressure liquid chromatography (with spectrometric detection at 218nm) is described for the determination in new pharmaceutical preparations (liposomes) of a new immunostimulating agent (N-acetylmuramoyl-l-alanyl-d-isoglutamine). Separation was achieved with a mu-bondapak column and phosphate buffer (pH 2.5)-methanol mixture (93:7 v/v) as eluent, at a flow-rate of 2 ml min . Sodium acetate was used as an internal standard. The detector response at 218 nm was linear in the range 10-170 mug ml . The method is simple and accurate.     

Determination of sialic acids in human serum by reversed-phase liquid chromatography with fluorimetric detection.

A simple, rapid and highly sensitive reversed-phase liquid chromatographic method has been developed for the determination of sialic acids in human serum. The sialic acids, released by hydrolysis of serum, are converted in borate buffer with malononitrile to highly fluorescent compounds. The reaction mixture is separated isocratically within 5 min using an octadecyl-bonded silica column and a mobile phase of methanol and ammonium acetate buffer (15:85, v/v; pH 5.5). Measurement of the fluorescence intensity of the reaction mixture at 434 nm with irradiation at 357 nm allowed determination of 30-1000 ng/ml of sialic acids with high reproducibility. The limit of detection was 2 ng/ml. Intra-day and inter-day coefficients of variation for assaying 300 ng/ml N-acetylneuraminic acid (NANA) were 1.5% (n = 9) and 2.6% (n = 7), respectively. The recoveries of NANA were 98.5-101.1% for serum. The method has been used for clinical determinations.     

Determination of luxabendazole in biological fluids by high-performance liquid chromatography.

Luxabendazole, a new benzimidazole, is a highly potent broad-spectrum anthelmintic. A high-performance liquid chromatographic method has been developed for its determination in serum and urine samples. In order to optimize the clean-up of samples we compared two procedures: C18 Sep-Pak cartridges and ultrafiltration through a cellulose membrane with a 30,000 relative molecular mass cut-off. In order to obtain the most suitable mobile phase, we studied the influence of pH and acetonitrile content on the capacity factor (k'). Chromatographic separation and quantification were performed on a reversed-phase column packed with 5-microns Nucleosil C18. The mobile phase was acetonitrile-0.05 M phosphate buffer (pH 7.0), (40:60, v/v). The column effluent was monitored by ultraviolet-visible spectrophotometry at 290 nm. The method shows good recovery, precision and accuracy. The lower limit of detection for luxabendazole is 15 ng/ml in serum samples and 25 ng/ml in urine samples.     

Thermospray liquid chromatography-mass spectrometry of corticosteroids.

A high-performance liquid chromatographic method was developed for thermospray mass spectrometric analysis of steroidal hormones. Using a Nova-Pak C18 reversed-phase column and isocratic elution with a solvent comprised of 25 mM ammonium formate in 30% acetonitrile, corticosteroids were separated within 10 min. This solvent also permitted ultraviolet absorbance detection down to 220 nm with low-nanogram sensitivity. The use of acetonitrile was favourable for thermospray mass spectrometric analysis because mass spectra were obtained with a pseudomolecular ion as the base peak. A combination of liquid chromatography, ultraviolet absorbance detection and thermospray mass spectrometry provided a sensitive and reliable method for unequivocal confirmation of the presence of steroidal drugs in equine urine.     

Analysis of cannabinoids by liquid chromatography— Thermospray mass spectrometry and liquid chromatography— Tandem mass spectrometry

Summary A rapid method based on liquid chromatography and thermospray mass spectrometry without any derivatization or pre-purification steps has been developed for the identification and quantification of cannabinoids in drugs from cannabis plants. The extracts were separated on a C₁₈ reversed-phase column with an acidic acetonitrile-water gradient. Liquid chromatographymass spectrometry was performed with a thermospray interface and protonated molecular ions were obtained from the cannabinoids of interest. Liquid chromatography-tandem mass spectrometry experiments on the molecular ions gave additional structural information online. The sensitivity and selectivity of the method was sufficient to enable the detection of 100 pg of the cannabinoids.     

Determination of cefodizime in biological materials by high-performance liquid chromatography

Cefodizime (THR-221) is a new semi-synthetic cephalosporin. A high-performance liquid chromatographic method has been developed for the determination of cefodizime in biological materials. A plasma or serum sample was deproteinized with methanol and the resulting methanol eluate was concentrated to a volume of 0.5 ml. Urine and bile samples were diluted with buffer and each diluted sample was filtered. Faeces samples were homogenized and the supernate obtained after centrifugation was filtered. Visceral tissue samples were homogenized, the centrifuged supernate was deproteinized with methanol, and the methanol eluate was concentrated to a volume of 0.5 ml. Aliquots of each preparation were chromatographed on a reversed-phase column with an ion-pair chromatographic technique on a high-performance liquid chromatograph equipped with an UV detector set at 264 nm. The detection limits for cefodizime were 0.1 microgram/ml in plasma or serum, 0.3 microgram/ml in bile, and 0.5 microgram/ml in urine, 0.5 microgram/g in faeces and visceral tissue. This precise and sensitive assay for the determination of cefodizime is described, and its stability in several media is reported.     

Analytical procedure for the determination of rufloxacin, a new pyridobenzothiazine, in human serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantification of rufloxacin in human serum and urine has been developed and validated. The compounds, rufloxacin and internal standard, are extracted from buffered serum and urine using dichloromethane. They are then separated on an anion-exchange column using 0.05 M phosphate buffer-acetonitrile (80:20, v/v). The eluate is quantified by measuring the ultraviolet absorbance at 296 nm. The lower limit of detection for the analyte is 0.1 microgram/ml in serum and 0.05 micrograms/ml in urine. The method is linear from 0.3 to 10 micrograms/ml for serum and 0.1 to 10 micrograms/ml for urine. The method has been applied in a pharmacokinetic study in volunteers.     

Analysis of cephalexin from canine skin biopsy by liquid chromatography with ultraviolet-visible photodiode-array detection

A sensitive and selective ion-paired liquid chromatographic method with UV-VIS photodiode-array detection was developed to measure cephalexin in skin biopsy samples. The method involved a sonication of minced canine skin with ethanol-acetonitrile-water (30:20:50, v/v/v) and ultrafiltration of received extract through 10,000 daltons. Separation of cephalexin from other components was by liquid chromatography using a reversed-phase column which was eluted with an ion-paired acetonitrile-water solution. Detection was achieved with a UV-VIS photodiode-array detector scanning from 230 to 320 nm. Cephalexin in the eluate was quantitated at its wavelength maximum of 260 nm. The evaluation of chromatographic peak homogeneity was performed by absorbance ratios, contour maps, first-derivative spectra and a three-dimensional spectrochromatogram. Additionally, the cephalexin peak identity was confirmed by liquid chromatography-mass spectrometry.     

High-performance liquid chromatographic determination of forphenicine in mouse serum and muscle by pre-column fluorescence derivatization using 1,2-diamino-4,5-ethylenedioxybenzene as fluorogenic reagent

A simple and sensitive high-performance liquid chromatographic method has been developed for the determination of forphenicine in biological samples. Forphenicine in the deproteinized sample is converted by reaction with 1,2-diamino-4,5-ethylenedioxybenzene into a fluorescent derivative. The derivative is separated on a reversed-phase column (TSK gel ODS-120T) by isocratic elution with acetonitrile-30 mM phosphate buffer (pH 6.5) (5:1, v/v) and monitored fluorimetrically. The method allows the quantification of forphenicine in serum (100 microliters) and muscle (0.1 g) of mice dosed with forphenicine or forphenicinol. The limits of detection (signal-to-noise ratio of 3) are 7.35 pmol/ml in serum and 5.36 pmol/g in muscle. The distribution of forphenicine and forphenicinol in the mouse serum and muscle after oral administration of these compounds is also described.     

Pirmenol determination by high-performance liquid chromatography

A method for the determination of pirmenol in serum is presented in this paper. The method consists of extraction of pirmenol and chlorodisopyramide (internal standard) from serum at an alkaline pH using methylene chloride. The organic extract was analysed using high-performance liquid chromatography. The mobile phase consisted of 0.01 M K2HPO4 (pH 2.4)-acetonitrile (94:6, v/v) delivered at ambient temperature and 2 ml/min through a 25 cm x 0.4 mm C18 reversed-phase column. Detection of the compounds of interest was achieved at 210 nm. The analytical method demonstrated low intra- and inter-assay variation. During the analysis of patient samples and a therapeutic drug mixture test serum, no substances that interfered with pirmenol detection were found. The method is shown to be stable, accurate, selective and sensitive enough to be utilized for the analysis of multiple samples such as may be encountered in clinical or research situations.     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

高效液相色谱法测定头孢他啶的含量及杂质

采用高效液相色谱法测定了头孢他啶的含量及杂质。以Alltima C18色谱柱(250 mm×4.6 mm,5 μm)为分离柱,以乙腈和磷酸盐缓冲溶液(pH 3.9)分别为流动相A和流动相B进行梯度洗脱,流速1.3 mL/min,柱温35 ℃,紫外检测波长255 nm。从头孢他啶药物中共分出14个杂质,且14个杂质间具有良好的分离度。头孢他啶在0.267~1069 μg/mL范围内与峰面积具有良好的线性关系(r=1.0000);其定量限(S/N=10)和最低检出限(S/N=3)分别为3.1 ng和0.93 ng。3个浓度的日内测定值的相对标准偏差(RSD）为0.72%(n=3),日间测定值的RSD为0.91%(n=3)。头孢他啶溶液在4 ℃避光条件下放置24 h保持稳定。本方法与欧洲/英国药典和日本药局方的方法比较,具有分离杂质数量多、分离度好的优点。     

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.     

High-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of enrofloxacin and ciprofloxacin in canine serum and prostatic tissue. Sample preparation consisted of mixing canine serum with a 1:1 dilution of acetonitrile and 0.1 M sodium hydroxide followed by ultrafiltration through a 10,000 molecular mass cut-off filter. Prostatic tissue was sonicated with the same solution prior to ultrafiltration. Separation of these two quinolones in the ultrafiltrate was accomplished by ion-paired liquid chromatography using a reversed-phase analytical column eluted with an acetonitrile-methanol-water solution. Enrofloxacin and ciprofloxacin were detected by a photometric ultraviolet-visible detector set at 278.6 nm and confirmed by a photodiode array detector operating from 230 to 360 nm. The limits of detection for enrofloxacin and ciprofloxacin were 4 and 2 ng/ml, respectively.