硝呋烯腙的纯化及其在饲料中的HPLC及LC-MS检测技术研究

硝呋烯腙(nitrovin),化学名称1,5-双(5-硝基-2-呋喃基)戊二烯-3酮咪基腙,是20世纪70年代中期发现的一种新型抗生素,作为生长促进剂和抗菌剂广泛用于猪、禽和牛上,一般的添加剂量为10～30 mg/kg. 近年来随着人们对动物食品安全的关心,尤其是对动物产品中抗生素残留的关注,我国农业部于2002年的193号文件明令规定禁止使用硝呋烯腙于食品源性动物.     

Determination of Tiopronin in Human Plasma by SPE then Reversed-Phase HPLC–UV

An accurate, simple and sensitive method based on reversed-phase high-performance liquid chromatography with UV detection has been developed for determination of tiopronin (TP) in human plasma. TP in plasma was reacted with p-bromophenacyl bromide (p-BPB) to give the TP-p-BPB adduct and this derivative was then extracted from the plasma on a silica gel cartridge. Potential interfering compounds were removed by washing with water, and the TP-p-BPB adduct was then eluted with acetonitrile. The organic phase obtained was evaporated to complete dryness under a stream of nitrogen. The residue was dissolved in acetonitrile and this solution was injected on to a reversed-phase ODS HPLC column. The mobile phase was usually the ternary mixture acetonitrile–water–trifluoroacetic acid, 40:59.88:0.12 (v/v). The retention times of TP-p-BPB and the internal standard adduct were 14.4 and 17.9 min, respectively. No interfering peaks were encountered in several blank plasma samples examined. The limit of detection for TP was 12 ng mL^?1. Extraction recovery exceeded 70%. The calibration plot for the TP derivative was linear in the range 40?4000 ng mL^?1, regression coefficient 0.9989, and the coefficient of the variation of the points of the calibration plot was below 10%. The method was validated appropriately and successfully applied to the determination of TP in human plasma.     

高效液相色谱-质谱法对饲料及食品添加剂中三聚氰胺的测定

建立了饲料中三聚氰胺的高效液相色谱-质谱测定方法.色谱条件:Kromasil C18柱(4.6 mm×250mm,5 μm),流动相:乙腈-0.1%(体积分数)甲酸(体积比5:95),流速0.4 mL/min.采用正离子模式的电喷雾质谱检测,以一级质谱得到的准分子离子m/z 127作为母离子,进行碰撞诱导解离(CID)二级质谱(MS2)分析,选择母离子和MS2的碎片离子m/z 85、109定性确证,提取m/z 85、109、127三个离子质量色谱峰面积定量.实验优化了质谱条件.线性范围为0.01～0.5 mg/L,检出限0.01 mg/L(S/N=3),回收率为80%～99%.     

Silica Microspheres for SPE and Determination of Fungicides in Water by LC

A new method has been developed for the determination of metalaxyl, myclobutanil, and tebuconazole in environmental water samples with preconcentration by cartridges packed with SiO₂ microspheres prior to LC. Several parameters such as the volume and composition of eluent, sample flow rate, sample pH, and sample volume were optimized. Under the optimal conditions, excellent detection limits (S/N = 3) and precision (RSD, n = 6) were 0.02 ng mL^?1, 1.3% for metalaxyl, 0.02 ng mL^?1, and 2.4% for myclobutanil and 0.08 ng mL^?1 and 4.3% for tebuconazole, respectively. The method was applied to the analysis of real-water samples, and satisfactory results were obtained. The average spiked recoveries were in the range of 86.3–97.5%. These results indicate that SiO₂ microspheres have great potential to be used as a novel solid phase extraction adsorbent that could have wide applications in the environmental field.     

Determination of Flavonoid Markers in Honey with SPE and LC using Experimental Design

Determination of flavonoid markers quercetin, hesperetin, and chrysin, found in north Iranian citrus honey samples, was carried out by solid phase extraction (SPE) and isocratic liquid chromatographic separation using central composite design. Optimum conditions for SPE were achieved using 10 mL methanol/water (13:87, v/v, pH = 7) as the washing solvent and 4 mL methanol for elution. Good clean-up and high recovery >90% were observed for all analytes. The use of water/ACN/THF/AcOH (54:36:5:5, v/v) was found to serve as the optimum mobile phase composition and allowed for the separation of analytes from endogenous compounds present in honey. SPE parameters, such as maximum loading capacity and breakthrough volume, were also determined for each analyte. Limit of detection, linear range, recovery, repeatability of retention times, and peak heights were 3.11 × 10^?8–4.44 × 10^?8 g g^?1, 0.50–50.0 μg mL^?1 (R ² > 0.99), 90.7–96.9%, 3.0–3.6%, and 1.0–2.6%, respectively. Precision of the overall analytical procedure, estimated by five replicate measurements for quercetin, hesperetin and chrysin in citrus honey, as well as the relative standard deviations were 4.3%, 3.8%, and 5.5%, respectively.     

Determination of Flavonoid Markers in Honey with SPE and LC using Experimental Design

Determination of flavonoid markers quercetin, hesperetin, and chrysin, found in north Iranian citrus honey samples, was carried out by solid phase extraction (SPE) and isocratic liquid chromatographic separation using central composite design. Optimum conditions for SPE were achieved using 10 mL methanol/water (13:87, v/v, pH = 7) as the washing solvent and 4 mL methanol for elution. Good clean-up and high recovery >90% were observed for all analytes. The use of water/ACN/THF/AcOH (54:36:5:5, v/v) was found to serve as the optimum mobile phase composition and allowed for the separation of analytes from endogenous compounds present in honey. SPE parameters, such as maximum loading capacity and breakthrough volume, were also determined for each analyte. Limit of detection, linear range, recovery, repeatability of retention times, and peak heights were 3.11 × 10⁻⁸–4.44 × 10⁻⁸ g g⁻¹, 0.50–50.0 μg mL⁻¹ (R ² > 0.99), 90.7–96.9%, 3.0–3.6%, and 1.0–2.6%, respectively. Precision of the overall analytical procedure, estimated by five replicate measurements for quercetin, hesperetin and chrysin in citrus honey, as well as the relative standard deviations were 4.3%, 3.8%, and 5.5%, respectively.

     

Sensitive Determination of Thiols Using SPE Coupled to LC with Fluorescence Detection

The purpose of the present work is to develop a simple, rapid, sensitive and accurate method for the derivatization and subsequently preconcentration of Hg(II) and the determination of its derivative, diphenylmercury, in natural water samples using gas chromatography-flame ionization detection. The method is based on the diphenylation using phenyl boronic acid, subsequent extraction of phenylmercury into a single drop of an organic solvent (toluene), followed by gas chromatography-flame ionization detection GC-FID analysis of the extract. The pH of the feed solution was kept in pH 5 with acetate buffer solution. Thus, the optimized conditions are: organic solvent, toluene; derivatization time, 10 min; extraction time, 15 min; microdrop volume, 1.6 μL; stirring rate, 600 rpm; sample volume, 5 mL. The limit of detection (LOD), calculated on the basis of five replicates was 0.02 μg mL^?1. The relative standard deviation of the method (RSD%, n = 5) was 3.0. Linear range was between 0.05 and 5 μg mL^?1 and preconcentration factor obtained for phenyl-mercury was 105.     

Determination of Non-Steroidal Anti-Inflammatory Drugs in Natural Waters Using Off-Line and On-Line SPE Followed by LC Coupled with DAD-MS

Two different procedures for simultaneous determination of six NSAIDs (diflunisal, diclofenac, fenoprofen, ibuprofen, naproxen and tolmetin) in environmental waters are described. Final analysis of target compounds is performed by reversed-phase liquid chromatography – diode array detection and mass spectrometry (HPLC-DAD and LC-MS), whereas sample preparation is based on solid-phase extraction (SPE). A variety of sorbents and their respective advantages and disadvantages are discussed. For the off-line SPE of NSAIDs from water samples, a LiChrolut RP-18 was selected out of all investigated sorbents. In case of on-line coupling of SPE with chromatographic system LiChrosphere RP-18 was selected as the best one in terms of recovery of NSAIDs evaluated, RSD and availability. The applicability of the method was also evaluated. Method detection limits were in the range of 0.7−94 ng L⁻¹. Recoveries ranged from 96 to 109% and relative standard deviations were lower than 5%. The procedures were shown to be linear over a wide range of concentration, exhibited satisfactory repeatability and accuracy, and reached limits of detection in the low ng L⁻¹ range. No breakthrough volume was observed neither for off-line SPE (in the studied range of 100, 200, 300, 500, 700, 1000 and 2000 mL of tap water sample) nor for on-line SPE (in the wide range of 10 mL, 20 mL, 30 mL, 50 mL, 70 mL, 100 mL and 200 mL of tap water sample).     

Determination of Testosterone Metabolites in Rat Hepatocytes with and without Cryopreservation by On-Line SPE Column-Switching LC and MS Detection

In a recently published paper development of a sensitive automated “on-line” solid-phase extraction (SPE)/RP-HPLC assay for 6β-hydroxytestosterone (6β-OHT) with corticosterone as the internal standard (IS) was reported and its potential for quantification of various testosterone metabolites in culture media reflecting metabolic activity of cultured human and animal hepatocytes demonstrated [1]. In this following contribution the technique has been extended to determination of another five testosterone metabolites in cultured rat hepatocytes using an identical “on-line” SPE/RP-HPLC procedure and detection by tandem MS-MS with an atmospheric pressure chemical ionization (APCI) source in the selected reaction monitoring (SRM) mode as that described in [1]. All six testosterone metabolites, namely 2α-OHT, 2β-OHT, 6α-OHT, 6β-OHT, 7α-OHT and 16α-OHT, could be sufficiently separated from each other and thus an unequivocal assignment to the individual structures was achieved. Validation data are presented specifying the limits of quantitation as well as the mean values of the coefficients of variation (CV) for the target analytes and the accuracy obtained at five different days. Regio- and stereoselective testosterone hydroxylation by rat hepatocytes was measured in a long-term culture system with and without exposure to rifampicin as an inducer of liver CYP 3A4 activity. In addition, testosterone hydroxylation was analyzed in cultures of cryopreserved hepatocytes that had been stored at −196 °C. The rat hepatocytes were cultured after thawing for up to 11 days and induction of testosterone hydroxylase activity could be demonstrated in cultures which underwent a new cryopreservation protocol. This paper is dedicated to Professor Hinrich Cramer on the occasion of his 75th birthday.     

Analysis of Amisulpride in Human Plasma by SPE and LC with Fluorescence Detection

A rapid, precise, accurate, and selective high-performance liquid chromatographic method with fluorescence detection has been validated and used for analysis of amisulpride in human plasma after a simple solid-phase extraction procedure. Compounds were separated on a CN column with 0.03?M potassium dihydrogen phosphate (pH 6.5)-acetonitrile 65:35 (v/v) as mobile phase. Fluorescence detection was performed at excitation and emission wavelengths of 274 and 370?nm, respectively. Calibration plots were linear over the concentration range 10-1,000?ng?mL(-1) in human plasma, and the lower limit of quantification was 10?ng?mL(-1). Accuracy was between 0.4 and 6.4% and precision was between 3.1 and 7.5%. Amisulpride was sufficiently stable through three freeze-thaw cycles, during storage for 6?h at room temperature, and for 2?months at -22?°C. The method is suitable for the analysis of clinical samples from pharmacokinetic studies.     

Predictions of Reversed-Phase Gradient Elution LC Separations Supported by QSRR

Previous studies demonstrated that quantitative structure-retention relationships (QSRR) combined with the linear solvent strength (LSS) model allow for prediction of gradient reversed-phase liquid chromatography retention time for any analyte of a known molecular structure under defined LC conditions. A QSRR model derived at the selected gradient time and at the same gradient time was tested. The aim the present study was to evaluate the accuracy of QSRR predictions used during the predictions of LC gradient retention times with variable gradient times. For this purpose, predictions of retention times at two gradient times were used to find the optimal, different gradient times. In the first step, experimental retention data for the model set of analytes were used to derive appropriate QSRR models at two gradient times. These QSRR models were further used to predict gradient retention times for another set of testing analytes at the two selected above gradient times. Then, applying linear solvent-strength (LSS) theory, the predicted retention times for test analytes were used to find other optimal gradient times for those analytes. Satisfactory predictions of gradient retention times for test analytes were obtained at gradient times different from those applied for model analytes.     

LC and SPE Determination of Chlorophacinone in Corn Grains, Soil and Rat Plasma

A liquid chromatographic method was used for the analysis of chlorophacinone in corn, soil and rat plasma. Sample preparation differed from corn, soil and serum. Simple liquid extraction procedures were applied to soil and corn; solid phase extraction using Oasis HLB cartridges and easy protocol was used for plasma. Liquid chromatography was on X-terra C18 column. The mobile phase used did not contain ion-pairing reagent, it was constituted with methanol/ammonium bicarbonate (0.01 M, pH = 7), 70/30, v/v at 1 mL min⁻¹. UV detection was by using photodiode detector operating at 282 nm. Complete validation of the proposed procedure for corn, soil and serum was made.     

固相萃取-高效液相色谱法测定水中呋喃丹

取200mL水样,流经C_(18)固相萃取(SPE)小柱富集呋喃丹。用5.0mL四氢呋喃从SPE柱上洗脱呋喃丹,收集洗脱液,在35℃吹氩蒸干。用1.0mL甲醇溶解残渣,所得溶液供高效液相色谱分析。用Eclipse XDB C_(18)色谱柱及由甲醇-水(1+1)混合溶液作流动相进行分离,并以285nm作激发波长,320nm作发射波长对呋喃丹作荧光检测。测得呋喃丹的质量浓度在5.0×10~(-4)～5.0mg·L~(-1)范围内与相应的峰面积值呈线性关系,检出限(3S/N)为0.02μg·L~(-1)。以水样为基体加入3个浓度水平(1.0,50.0,200.0μg·L~(-1))标准溶液对方法作回收及精密度试验,测得回收率在94.0%～99.6%之间,相对标准偏差(n=7)在1.2%～4.2%之间。     

Separation and Quantitative Analysis of Capsaicinoids in Chili Peppers by Reversed-Phase Argentation LC

A reversed-phase argentation liquid chromatographic method has been achieved for the separation and determination of capsaicinoids (capsaicin, dihydrocapsaicin and nordihydrocapsaicin) in chili peppers. Capsaicinoids are determined on a C18 column with methanol–water (60:40, v/v) containing 0.03 mol L^?1 silver nitrate as eluent and with ultraviolet detection at 280 nm. Three capsaicinoids are successfully separated from each other and from other interfering components by the high selectivity of reversed-phase argentation LC. The average recovery of the method is around 97%. Good reproducibility is obtained with relative standard deviations varying from 1.68 to 4.52%. The method was successfully applied to characterize and determine the capsaicin and dihydrocapsaicin in chili peppers.     

Simultaneous Determination of Cyanazine, Chlorotoluron and Chlorbenzuron in Environmental Water Samples with SPE Multiwalled Carbon Nanotubes and LC

In this paper, a new method based on solid-phase extraction with multiwalled carbon nanotubes as the packed materials for sensitive determination of cyanazine, chlorotoluron and chlorbenzuron in environmental water samples was demonstrated. Related parameters that may influence the enrichment efficiency of multiwalled carbon nanotubes such as the kind and volume of elute, sample flow rate, sample pH, and volume of the water samples were investigated. Under the optimum conditions, the detection limits of cyanazine, chlorbenzuron and chlorotoluron were 0.015, 0.012, 0.034 ng mL⁻¹, respectively. The experimental results indicated a good linearity (R ² > 0.9947) over the concentration range of 0.4–40 ng mL⁻¹ for cyanazine and chlorotoluron, and 0.8–80 ng mL⁻¹ for chlorbenzuron. The relative standard deviations of the three analytes were 3.54, 1.55 and 1.38%, respectively. The established method also was applied to the analysis of the real-world water samples and excellent achievements were obtained with average spiked recoveries from 87.8 to 110.1%. All the results indicated that this procedure could allow the simultaneous determination of these three compounds in environmental water samples at trace levels.     

高效液相色谱法测定饲料中的喹乙醇

报道饲料中喹乙醇的高效液相色谱分析方法。以乙酰苯胺为内标,样品用二甲替甲酰胺（ＤＭＦ）提取,在Ｃ８柱上进行色谱分析,紫外检测波长２６０ｎｍ,流动相为甲醇－水（２０８０,Ｖ／Ｖ）,喹乙醇的回收率为９８．５８％～１０１．６３％,相对标准偏差为２．６７％～４．２５％。     

Optimization and Validation of a Confirmatory Method for Determination of Ten Sulfonamides in Feeds by LC and UV-Diode Array Detection

A simple, rapid, and reliable multiresidue method, based on liquid chromatography (LC) and ultraviolet (UV)-diode array detection (DAD), is described for assaying ten sulfonamides (sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfamonomethoxine, sulfachloropyridazine, sulfamethoxazole, sulfaquinoxaline, and sulfadimethoxine) in feeds. The chromatographic separation is accomplished using a C18 column, eluted with a mobile phase consisting of acetate buffer, acetonitrile, and methanol. The sample preparation requires a simple extraction with chloroform/acetone and a purification step by solid-phase extraction. The analytical parameters of precision, detection and quantification limits, recovery, and ruggedness have been evaluated by a validation procedure following the European guidelines of Regulation 882/2004/EC and Decision 657/2002/EC.     

LC Determination of Thiols Derivatized with 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole after SPE

Solid-phase extraction (SPE) coupled with high-performance liquid chromatography?Cfluorescence detection (LC?CFL) was developed for the determination of three thiol compounds including glutathione, cysteine and acetylcysteine. 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole was used for derivatization of thiols. Factors affecting derivatization and extraction efficiency were optimized. Sample solution (2?mL) was extracted on a SPE column for 2?min and then eluted with 400???L methanol. The analytes were injected onto the LC system for separation on a C₁₈ column, and eluted with methanol?Cacetate buffer. The analytes were detected by fluorescence at an emission wavelength of 515?nm with excitation at 385?nm. The linearity of the method was in the range of 0.1?C60???M, with correlation coefficients ranging from 0.9979 to 0.9990. The detection limits of the method were in the range of 5?C20?nM. The proposed method was applied to the analysis of human plasma samples with recoveries of 86?C112.9%.     

Determination of Apigenin by LC with Electrochemical Detection

A rapid, sensitive, and specific method for quantification of olmesartan, the prodrug of olmesartan medoxomil, in human plasma, using zidovudine as internal standard, is described. Sample preparation involved a simple solid-phase extraction procedure. The extract was analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (LC–MS–MS). Chromatography was performed isocratically on a 5 μm C₁₈ analytical column (50 mm × 4.6 mm i.d.) with water–acetonitrile–formic acid 20:80:0.1 (v/v) as mobile phase. The response to olmesartan was a linear function of concentration over the range 4.82–1,928 ng mL⁻¹. The lower limit of quantification in plasma was 4.82 ng mL⁻¹. The method was successfully applied in a bioequivalence study of an olmesartan formulation after administration as a single oral dose.     

Determination of the Lipophilicity of Xanthines by Reversed-Phase Liquid Chromatography

JPC – Journal of Planar Chromatography – Modern TLC -