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We report a new high-performance liquid chromatography method developed for measuring inulin in plasma and urine using ion moderated partition chromatography and evaporative light-scattering detection. Samples are deproteinized with a zinc acetate and phosphotungstic acid solution and added with melezitose as an internal standard. The chromatographic separation is carried out in 16 min at a flow-rate of 0.6 ml/min using deionized water as the mobile phase. Within-run precision, measured at four different concentrations (0.050 mg/ml, 0.150 mg/ml, 0.300 mg/ml and 1.200 mg/ml), ranges from 1.7 to 3.4% in plasma and from 1.5 to 3.5% in urine. Similarly, between-run precision is in plasma from 2.0 to 4.3% and in urine from 2.0 to 4.4%. Analytical recovery ranges from 97.9 to 100.1% in plasma and from 99.1 to 99.7% in urine, respectively. Detection limit (signal-to-noise ratio=3) is 5 μg/ml both in plasma and urine. The method is simple, sensitive, without interference due to hexoses or drugs commonly taken by patients with renal diseases, and offers the advantage of measuring inulin without previous hydrolysis of the molecule.  相似文献   

3.
A rapid, simple and low-cost assay method of histamine-N-methyltransferase activity was developed. Methylhistamine, which was separated from the enzymatic reaction system on reversed-phase high-performance liquid chromatography using an ion-paired chromatographic technique, was detected spectrophotometrically at 226 nm. The mobile phase used for the separation of methylhistamine was 0.05M NH4H2PO4 (pH 3.0) containing 2 mM of sodium octanesulfonate. The new assay technique could detect methylhistamine as an enzyme activity product of histamine-N-methyltransferase in the brain and kidney of rats. Chloropheniramine maleate, an antihistamine, activated the histamine-N-methyltransferase. Whether neurotransmitter or neuromodulator, the role of histamine in the brain has not yet been made clear. Therefore, the present method could be applicable for the enzymatic investigation of histamine metabolism in central nervous system or inflammatory reactions.  相似文献   

4.
A rapid, sensitive and specific high-performance liquid chromatography (HPLC) procedure for the quantification of indinavir, a potent human immunodeficiency virus (HIV) protease inhibitor, in human plasma is described. Following C18 solid-phase extraction, indinavir was chromatographed on a reversed-phase C8 column using a simple binary mobile phase of phosphate buffer–acetonitrile (60:40, v/v). UV detection at 210 nm led to an adequate sensitivity without interference from endogenous matrix components. The limit of quantification was 25 ng/ml with a 0.1 ml plasma sample. The standard curve was linear across the range from 25 to 2500 ng/ml with an average recovery of 91.4%. The mean relative standard deviations for concentrations within the standard curve ranged between 1.4 and 9.7%. Quality control standards gave satisfactory intra- and inter-assay precision (R.S.D. from 3.5 to 15.8%) and accuracy within 15% of the nominal concentration. Sample handling experiments, including HIV heat inactivation, demonstrated analyte stability under expected handling processes. The assay is suitable for the analysis of samples from adult and pediatric patients infected with HIV.  相似文献   

5.
Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-μm reversed-phase C8 column (150×4.6 mm, I.D.) guarded by a 40-μm reversed-phase C18 column (50×4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.  相似文献   

6.
Here we describe a simple high-performance liquid chromatography (HPLC) procedure for the simultaneous detection and quantitation in standard solutions of 13 important metabolites of cellular energy metabolism, including 9 tricarboxylic acid (TCA) cycle components and 4 additional metabolites. The metabolites are detected by their absorbance at 210 nm. The procedure does not require prior derivatization, and an analysis can be carried out at ambient temperature within 15 min. The significance of the current work is that the current HPLC procedure should motivate the development of simplified TCA cycle enzyme assays, isotopomer analysis, and determination of selected TCA metabolite levels in plasma/tissues.  相似文献   

7.
A rapid and sensitive HPLC method was developed for the determination of ampicillin residues in muscle tissues of beef, pork, chicken and catfish. Muscle tissues were blended with a food processor into paste. A 5-g aliquot of the blended tissues was homogenized with 14 ml of 0.01 M phosphate buffer (pH 4.5) using a tissue homogenizer. Proteins were precipitated with the addition of 1 ml trichloroacetic acid (75%, w/v) followed by centrifugation. After filtration, 1 ml of the supernatant was reacted with formaldehyde under acidic and heating conditions. The ampicillin fluorescent derivative was then analyzed by reverse phase HPLC with fluorescence detection. Recoveries of spiked ampicillin at 5, 10 and 20 ng/g were >85%, with coefficients of variation <5%. The limit of detection and limit of quantitation for ampicillin in the tissues were 0.6 ng/g and 1.5 ng/g, respectively. The method is also applicable to the analysis of ampicillin residue in dry milk powder.  相似文献   

8.
When measuring fentanyl and midazolam simultaneously in the same plasma sample with standard high-performance liquid chromatography–ultraviolet (HPLC–UV) detection, overlap of the fentanyl peak by the midazolam peak occurs, which makes fentanyl determination impossible. We tested the hypothesis that by acidifying the methanol mobile phase with 0.02% perchloric acid, 70%, it would be possible to separate both peaks. The UV detector was set at 200 nm. Calibration curves for fentanyl (range 0–2000 pg/ml) and midazolam (range 0–400 ng/ml) were linear (r>0.99). The detection limits were 200 pg/ml (fentanyl) and 10 ng/ml (midazolam). Precision and accuracy for intra- and inter-assay variability as well as in-line validation with quality control samples (QCS) were acceptable (< 15 and 20%, respectively), except for fentanyl QCS of 200 pg/ml (17.8% precision). Although less sensitive than gas chromatography–mass spectrometry (GC–MS), reliable measurements of fentanyl, simultaneously with midazolam, can be performed with this HPLC–UV system.  相似文献   

9.
A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 μg/ml of succinylcholine. In a pilot study the plasma concentration—time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.  相似文献   

10.
We developed a method for the determination of serum 5α-cholestan-3β-ol (cholestanol). The sterols were derivatized to the 4′-bromobenzenesulfonyl esters and heated in isopropanol. The cholesterol-4′-bromobenzenesulfonate was solvolyzed to cholesteryl isopropyl ether, but the derivatized cholestanol did not change and could be measured in a high-performance liquid chromatographic system equipped with a UV detector at 235 nm. On the other hand, the resulting cholesteryl isopropyl ether, having different absorbance and chromatographic mobility was not detected. This method was used for measuring cholestanol levels in patients with cerebrotendinous xanthomatosis (CTX), liver cirrhosis and serum from healthy control subjects. Reproducibility, linearity and recovery tests were done on 0.3 ml of serum samples containing >2 μg/ml cholestanol, using stigmastanol as an internal standard (I.S.). Determining cholestenol by this method can be used for diagnosis and follow-up of patients with CTX and various liver diseases.  相似文献   

11.
A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-μl samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5–16 mg/l. The absolute recovery of tobramycin is 41%.The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.  相似文献   

12.
A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.  相似文献   

13.
Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects (n= 49), patients with inactive (n= 15), active (n= 28), and highly active SLE (n= 6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 micromol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2 +/- 165.38 micromol/mol creatinin) and highly active SLE (1753.8 +/- 453.9 micromol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3 +/- 121.3 micromol/mol creatinin) and healthy subjects (294.6 +/- 178.6 micromol/mol creatinin) (P< 0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.  相似文献   

14.
In this paper we develop an high-performance liquid chromatographic method with ultraviolet detection for the determination of verapamil and its primary metabolite norverapamil in biological samples. Both compounds, as well as the internal standard, imipramine, were extracted from alkalinised blood, with n-hexane–isobutyl alcohol, back-extracted into 0.01 M phosphoric acid and determined using a reversed-phase column and ultraviolet monitoring at 210 nm. The average coefficient of variation obtained over the concentration range of 1–1000 ng/ml is about 3%. The detection limit is below 5 ng/ml for both compounds, and extraction recoveries close to 80%. The method was applied to a pharmacokinetic study of the drug and its active metabolite and used to analyse blood samples from verapamil treated rabbits.  相似文献   

15.
A simple and rapid high-performance liquid chromatographic method with fluorescence detection was developed for the determination of loratadine in small volume plasma samples. Liquid-liquid extraction of loratadine and diazepam (as internal standard) from plasma samples was performed with n-butyl alcohol/n-hexane (2:98, v/v) in alkaline condition followed by back-extraction into diluted perchloric acid. Chromatography was carried out using a C8 column (250 x 4.6 mm, 5 microm) under isocratic elution with acetonitrile-20 mM sodium dihydrogen phosphate-triethylamine (43:57:0.02, v/v), pH 2.4. Analyses were run at a flow-rate of 1.0 ml/min at room temperature. The method was specific and sensitive with a quantitation limit of 0.62 ng/ml and a detection limit of 0.2 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery of loratadine from plasma was 84%, while the intra-and inter-day coefficient of variation and percent error values of the assay method were all less than 9.7%. Linearity was assessed in the range of 0.62-20 ng/ml in plasma with a correlation coefficient of greater than 0.999. The method has been used to analyze several hundred human plasma samples for bioavailability studies.  相似文献   

16.
A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20×3.9 mm I.D.) and analytical column (Symmetry C18, 75×4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile–water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40°C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r2>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.  相似文献   

17.
A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.  相似文献   

18.
A validated high-performance liquid chromatography method is described for the determination of scutellarin in rat plasma using a liquid-liquid extraction and ultraviolet (UV) absorbance detection. The separation used a Diamonsil ODS column (250 mm x 4.6mm i.d., 5 microm particle size) with an isocratic mobile phase consisting of methanol-acetonitrile-50mM dihydrogen ammonium phosphate buffer (22:15:63 (v/v/v), adjusted to pH 2.5 with 1M phosphoric acid). The ultraviolet detector operated at 335 nm. Plasma samples were extracted with ethyl acetate after acidification. The extraction recovery of scutellarin ranged from 68.1 to 80.5%. High selectivity and a low quantitation limit (0.050 microg/ml) were achieved. The linear range was 0.050-12.5 microg/ml, correlation coefficient r=0.9981. The method has a good reproducibility, R.S.D. values were below 7.9% for within-day and between-day precision. The method is simple, rapid, and applicable to preliminary pharmacokinetic studies of scutellarin in rats.  相似文献   

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20.
A high-performance liquid chromatography (HPLC) procedure for the separation of choline lysophospholids including 1-acyl-lysophosphatidylcholines and 1-O-alkyl-lysophosphatidyl-cholines, like the lysoform of the platelet activating factor (2-lysoPAF), is described. The lysophospholipids are derivatized at the sn-2 position of the hydroxyl group by 7-diethylaminocoumarin-3-carbonylazide, which converts them into the corresponding carbamoyl derivatives. The derivatized compounds were well separated by reversed-phase HPLC and quantified by fluorimetric detection. This method shows a high sensitivity and allows the separation and quantification of mixtures of lysophospholipids at picomolar level. The method was applied to assay enzyme activities, like phospholipase A2 and PAF-acetylhydrolase, on single phospholipids or their mixtures.  相似文献   

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