High Performance Liquid Chromatography With Fluorescence Detection Method Research Articles

Vasodilator effect of gaseous sulfur dioxide and regulation of its level by Ach in rat vascular tissues

To explore the toxicological and physiological role of gaseous SO2 on vascular contractility and its level in vascular tissues, a vasodilation study of isolated rat thoracic aortic rings by gaseous SO2 was carried out. The level of SO2 in vascular tissue was assayed using a modified high-performance liquid chromatographic method with fluorescence detection (HPLC-FD). The results show that gaseous SO2 (from 1 μM to 2000 μM) relaxed rat thoracic aortic rings in a dose-dependent manner. The physiological concentrations of SO2 in thoracic aortic tissues and plasma in rats were 127.76 ± 31.34 μM and 16.77±8.24 μM, respectively; The vasorelaxant effect of gaseous SO2 at physiological and low concentrations (<450 μM) was endothelium dependent, and at high concentrations (>500 μM) was endothelium independent. The results also show that SO2 could be endogenously generated in vascular tissues, and mainly in vascular endothelial cells; acetylcholine (Ach) increased the SO2 level in vascular tissue, and noradrenaline (NE) decreased the SO2 level. These findings demonstrate that gaseous SO2 is a vasorelaxant substance, and the vasorelaxant effect of gaseous SO2 is much stronger than that of its derivatives sulfite and bisulfite, which result from the inactivation process of SO2 gas transmitter by which SO2 is hydrated to form sulfite, and the latter is enzymatically oxidized to form sulfate. These findings also demonstrate that endogenous SO2 level in vascular tissue may be regulated by Ach and NE.

Determination of quinolone residues in tilapias (Orechromis niloticus) by HPLC-FLD and LC-MS/MS QToF

A method for the simultaneous determination of flumequine, oxolinic acid, sarafloxacin, danofloxacin, enrofloxacin, and ciprofloxacin in tilapia (Orechromis niloticus) fillet, using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) is presented. The quinolones were extracted from the food matrix with a solution of 10% trichloroacetic acid and methanol (80:20 v/v). Clean-up of the extract was performed using polymeric solid-phase extraction cartridges. Identification of the quinolones was confirmed by liquid chromatography-tandem mass spectrometry. The HPLC-FLD method was validated in-house and the following analytical parameters were obtained: linearity higher than 0.99 for all the quinolones; intra- and inter-assay precisions were lower than 3.5% and 10.9%, respectively; and recoveries ranged from 73% to 110%. The limit of quantification was below the maximum residue limit established by the Joint Expert Committee on Food Additives (JECFA), which indicates that the method is appropriate for the determination of quinolones in fish fillet.

Residue Depletion of Gentamicin in Swine Tissues after Intramuscular Administration

A sensitive and robust high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) was developed for the determination of gentamicin (GEN) residues in swine tissues. The limit of quantification (LOQ) of the method was 50 ng/g for muscle and 100 ng/g for liver and kidney. Mean recoveries at all fortification levels ranged from 82.34 to 93.20% with coefficient of variation (CV) below 5.39%. Residue depletion study of GEN in swine was performed after intramuscular injections twice daily at a dose of 4 mg/kg of bw with 12 h intervals for 5 consecutive days. The concentrations of GEN were determined in injection site, muscle, liver, and kidney by the HPLC-FLD method. The highest GEN concentration was measured in kidney, indicating that kidney was the primary target tissue for GEN residue. GEN concentrations in all examined tissues were below the maximum residue limit (MRL) recommended by the European Union (EU) at 50 days post-treatment.

A new approach for the sensitive determination of DNA adduct of aristolochic acid II by using high-performance liquid chromatography with fluorescence detection

A sensitive high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of DNA adducts induced by nephrotoxic and carcinogenic aristolochic acid (AA) is presented. The DNA adduct of AAII (dA-AAII) was synthesized by in vitro incubation, purified by preparative HPLC, characterized using fluorescence spectroscopy and high-resolution mass spectrometry, and was used as the biomarker for AA exposure in rats. The developed HPLC-FLD method was validated and applied for the determination of dA-AAII in rat kidney tissues. The method provided a detection limit of 18.3 fmol, which allowed the detection of dA-AAII in the rat kidney tissue samples collected after a single oral dose of AA. dA-AAII was detected in the kidney DNA digestion extracts of the rats that were dosed with AA at 5 mg/kg and 30 mg/kg at concentrations of 6.2 ± 1.1 and 41.3 ± 8.0 dA-AAII per 10 9 normal dA, respectively.

Study of the gastrointestinal biotransformation of zearalenone in a Caco‐2 cell culture system with liquid chromatographic methods

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) was developed and validated for the detection of zearalenone (ZON), alpha-zearalenol (alpha-ZOL) and beta-zearalenol (beta-ZOL) in in vitro biological samples. Furthermore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the detection of ZON, alpha-ZOL, beta-ZOL, alpha-zearalanol (alpha-ZAL) and beta-zearalanol (beta-ZAL) in in vitro biological samples. Zearalanone (ZAN) was used as internal standard in both methods. The limit of detection/limit of quantitation (LOD/LOQ) values for ZON, alpha-ZOL and beta-ZOL were 2/7, 2/7 and 4/13 microg l(-1), respectively, for the HPLC-FLD method. For the LC-MS/MS method LOD/LOQ values for ZON, alpha-ZOL, beta-ZOL, alpha-ZAL and beta-ZAL were 6/20, 5/17, 4/14, 9/30 and 6/19 microg l(-1), respectively. Within-day and between-day precision were less then 11 and 14%, respectively for the HPLC-FLD method, and both less then 20% for the LC-MS/MS method. The recovery of ZON and its metabolites ranged between 73 and 89% for the HPLC-FLD method and between 69 and 112% for the LC-MS/MS method. The methods were used for the detection of the compounds in in vitro biological samples, obtained with human intestinal Caco-2 cells culture experiments. The 8-days post-confluent Caco-2 cells were treated with ZON or a mixture of ZON and imazalil (IMA). After an incubation time of 24 h the samples were analysed with the HPLC-FLD method. Neither ZON nor its derivatives were detected in the samples. The disappearance of ZON could possibly point out the formation of phase II metabolites like glucuronide conjugates. Therefore, samples were pretreated with beta-glucuronidase before LC-MS/MS analysis. The LC-MS/MS results showed that ZON, alpha-ZOL and beta-ZOL could only be detected in the beta-glucuronidase pretreated samples. This confirmed the formation of glucuronide conjugates and the hydroxylation of ZON during the incubation with Caco-2 cells.

Liquid chromatographic methods for biotransformation studies of ochratoxin A

Liquid chromatographic methods were used for the detection of ochratoxin A (OTA) and its metabolites ochratoxin alpha (OTalpha), 10-hydroxy OTA (10-OHOTA), 4R-hydroxy OTA (4R-OHOTA) and the ethyl ester of OTA (OTC) in in vitro samples, obtained with Caco-2 cell culture experiments and in in vivo urine samples from sheep. A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed and validated for the detection of OTA and its metabolites OTalpha, 10-OHOTA, 4R-OHOTA and OTC, which was used as internal standard. The LOD/LOQ values for OTalpha, 4R-OHOTA and OTA were 0.63/2.11, 0.99/3.31 and 0.84/2.81 microg/L, respectively, for the HPLC-FLD method and 0.98/3.28, 1.11/3.72 and 0.88/2.96 microg/L, respectively for the LC-MS/MS method. Within-day and between-day precision were both <12% for the HPLC-FLD method, and <10% for the LC-MS/MS method. The recovery of OTA and its metabolites ranged between 71 and 111% for the HPLC-FLD method and between 79 and 110% for the LC-MS/MS method. In the first experiment only OTA was added to the Caco-2 cells while in the second experiment 3-methylcholanthrene (3MC) was also present in the cell culture systems. Besides OTA, which was recovered in all the samples, an unknown compound was also observed in the second experiment. When 3MC was added, the results showed that the OTA concentration in the basolateral samples was decreased by 50%. The methods were also implemented for the analysis of urine samples of sheep, fed increasing amounts of OTA. With the HPLC-FLD method it could be concluded that the concentration of OTA and OTalpha increased according to ingested amounts of OTA, with OTalpha being the most abundant compound. The results obtained with the LC-MS/MS method confirmed these results.

A rapid and simple high‐performance liquid chromatography method for the determination of human plasma levofloxacin concentration and its application to bioequivalence studies

A high-performance liquid chromatography method with fluorescence detection (HPLC-FLD) for the determination of levofloxacin in human plasma is described. Neutralized with phosphate buffer (pH 7.0), the sample (0.1 mL) was extracted with dichlormethane (1 mL). After voltex-mixing and centrifuged at 3000g for 6 min at 4 degrees C, the upper aqueous layer was aspirated using a micro vacuum pump and the organic layer was directly transferred to a clean test tube without pipetting. The organic solvent was evaporated and the residues were reconstituted with the mobile phase. Levofloxacin and terazosin (internal standard, IS) were chromatographically separated on a C(18) column with a mobile phase containing phosphate buffer (pH 3.0, 10 mm), acetonitrile and triethylamine (76:24:0.076, v/v/v) at a flow rate of 1 mL/min. The analytes were detected using fluorescence detection at an excitation and emission wavelength of 295 and 440 nm, respectively. The linear range of the calibration curves was 0.0521-5.213 microg/mL for levofloxacin with a lower limit of quantitation (0.0521 microg/mL). The retention times of levofloxacin and terazosin were 2.5 and 3.1 min, respectively. Within- and between-run precision was less than 12 and 11%, respectively. Accuracy ranged from -6.3 to 4.5%. The recovery ranged from 86 to 89% at the concentrations of 0.0521, 0.5213 and 5.213 microg/mL. The present HPLC-FLD method is sensitive, efficient and reliable. The method described herein has been successfully used for the pharmacokinetic and bioequivalence studies of a levofloxacin formulation product after oral administration to healthy Chinese volunteers.

Application of precolumn oxidation HPLC method with fluorescence detection to evaluate saxitoxin levels in discrete brain regions of rats

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning (PSP). This toxin blocks neuronal transmission by binding to the voltage-gated Na + channel and for this reason, it has been widely used in the study of Na + channel. The aim of this study was to analyze STX distribution in different rat brain regions after its acute intraperitoneal (i.p.) administration. Male rats (150–200 g) were injected i.p. with STX (5 and 10 μg/kg of body weight). After three time intervals of 30, 60, and 120 min (for 5 μg/kg STX dose) and 30 min (for 10 μg/kg STX dose) animals were sacrificed by cervical dislocation. Brains were removed and dissected in seven regions. STX concentration was measured using a precolumn oxidation high-performance liquid chromatographic method with fluorescence detection (HPLC/FLD). STX was found in all the regions evaluated at ppm levels meaning that STX peripherical administered across the blood–brain barrier and is distributed along the whole brain.