Determination of Benzo[a]pyrene in Edible Oil by High Performance Liquid Chromatography-Fluorescence Detector (HPLC-FL)

In this study, an optimized high performance liquid chromatography-fluorescence detector (HPLC-FL) method for the determination of benzo[a]pyrene in edible oil was established. HPLC was performed with Thermo Fisher Scientific C18 column (250 mm×4.6 mm, 5 μm) as the chromatographic column and acetonitrile and water as the mobile phase, and the excitation wavelength and emission wavelength of fluorescence detector were 286 and 430 nm, respectively. The response was high, and the linear range was 0.5-10.0 ng/ml. The lowest limit of detection was 0.11 ng/ml, and the average recovery was 92.5%. This method is suitable for quantitative analysis of benzo[a]pyrene content in edible oil.

Determination of fluorinated quinolone antibacterials by ion chromatography with fluorescence detection

For preparing fluorinated quinolone antibiotic medicine locally used in stomatology, simultaneous determination of norfloxacin, ciprofloxacin, and enoxacin was carried out by multiphase ion chromatography with fluorescence detection. Quinolone antibiotics were separated by Dionex OmniPac PAX-500 column with an eluent of 15 mmol/L H2SO4 and 35% methanol （v/v） at a flow-rate of 1.0 ml/min and detected with fluorescence with excitation and emission wave lengths of 347 ran and 420 ran respectively. The detection limits （S/N=3） of norfloxacin, ciprofloxacin and enoxacin were 50, 105 and 80 ng/ml respectively. The relative standard deviations of retention time, peak area and peak height were less than 1.1% and good linear relationship resulted. The developed method was applied to pharmaceutical formulations and biological fluids.

Speciation Analysis of Arsenic Compounds in Seafood by Ion Chromatography-Atomic Fluorescence Spectrometry

Ion chromatography-ultra violet-hydride generation-Atomic Florescence Spectrometry was applied to detect 5 arsenic species in seafoods. The arsenic species studied include arsenobetaine(As B), arsenite(As(III)), dimethylarsinic acid(DMA), monomethylarsonic acid(MMA), and arsenate(As(V)), which were extracted from samples using 2% formic acid. Gradient elution using 33 mmol L^(-1) CH_3COONH_4 and 15 mmol L^(-1) Na_2CO_3 with 10 mL CH_3CH_2OH at pH 8.4 allowed the chromatographic separation of all the species on a Hamilton PRP-X100 anion-exchange column in less than 8 min. In this study, an ultrasound extraction method was used to extract arsenic species from seafood. The extraction efficiency was good and the recoveries from spiked samples were in the range of 72.6%–109%; the precision between sample replicates was higher than 3.6% for all determinations. The detection limits were 3.543 μg L^(-1) for As B, 0.4261 μg L^(-1) for As(III), 0.216 μg L^(-1) for DMA, 0.211 μg L^(-1) for MMA, and 0.709 μg L^(-1) for As(V), and the linear coefficients were greater than 0.999. We also developed an application of this method for the determination of arsenic species in bonito, Euphausia superba, and Enteromorpha with satisfactory results. Therefore, it was confirmed that this method was appropriate for the detection of arsenic species in seafood.

Development of Ultra Fast Liquid Chromatography (UFLC) Method for Fluorescence Detection of Domperidone in Human Serum and Application to Pharmacokinetic Study

A simple and sensitive fluorescence detection of domperidone by ultra fast liquid chromatographic method was developed and validated in human serum. For the evaluation of new drug delivery systems, conducting of pharmacokinetic studies in human volunteers is essential for approval to marketing after preclinical evaluation in animal models. The present method consists of protein precipitation, extraction of analytes from human serum into dichloromethane and separation using reversed-phase C18 column. Propranolol hydrochloride was used as an internal standard and the eluent was monitored by fluorescence detector at excitation 282 and emission 328 nm. The mobile phase used was 62:38 ratio of 10 mM phosphate buffer pH adjusted to 3.1 with OPA and methanol at a flow rate of 1 mL·min-1. The method was evaluated for assay, LLOD, LLOQ, recovery and stability studies. The retention times for domperidone and propranolol hydrochloride were found to be 6.36 and 7.94 minutes respectively. The intraday and inter-day coefficient of variation and percent error values of assay method were less than 5%;mean recovery was more than 96% for each analyte and the method was found to be precise, accurate and specific during study. The method was successfully applied for pharmacokinetic study of immediate and controlled release bioadhesive hot melt extruded buccal patches of domperidone after buccal administration to healthy human volunteers. The Cmax, Tmax, and AUC0–24 of domperidone from immediate and controlled release buccal patches were found to be 129.7 ng·mL-1, 1.5 h, 455.1 ng·h·mL-1 and 145.7 ng·mL-1, 5.25 h, 911.0 ng·h·mL-1 respectively. A simple, sensitive and reliable method for the fluorescence determination of domperidone in human serum by UFLC method was developed and validated.

Molecular Weight Distribution of Dissolved Organic Matter in Lake Hongfeng Determined by High Performance Size Exclusion Chromatography (HPSEC) With On-Line UV-Vis Absorbance and Fluorescence Detection

The molecular weight distribution (MWD) of dissolved organic matter (DOM) in lake waters from Lake Hongfeng was examined using high performance size exclusion chromatography (HPSEC) with UV-vis absorbance and fluorescence detection. The elution curves obtained by absorbance and fluorescence techniques expressed similar patterns, with the exception of diminishing of large fraction and the peaks behind several seconds in fluorescence chromatograms. According to its molecular weight (MW), DOM in water samples is divided into several fractions: large ({>3.5} kDa); medium-large ({3.5}-{2.0} kDa); medium ({2.0}-{1.0} kDa) and small ({<1.0} kDa). The average molecular weight was calculated using the elution curve detected by UV-vis absorbance and fluorescence detection techniques. The results showed that the weight-average molecular weight (Mw) and number-average molecular weight (Mn) calculated by UV-vis absorbance techniques range from 1750 to 2050 Dalton and from 1450 to 1850 Dalton, respectively. And the Mw and Mn obtained by fluorescence detection are lower by 50 to 400 Dalton. As a reference, the molecular weight of Fluka humic acid (FHA) is larger than that of water samples by about 200 Dalton. The average molecular weight of DOM for water samples collected in March and July was compared. The results revealed that the molecular weight is lower for water samples obtained in July than that obtained in March, indicating the ambient environment has an influence on the molecular weight, including photo-degradation and biological activity.

Cholesterol crystal binding of biliary immunoglobulin A: visualization by fluorescence light microscopy

AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. RESULTS: Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies.Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. CONCLUSION: Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.

Simple HPLC-Fluorescence Determination of Raspberry Ketone in Fragrance Mist after Pre-Column Derivatization with 4-Hydrazino-7-nitro-2,1,3-benzoxadiazole

Raspberry ketone {RK, 4-(4-hydroxyphenyl)butan-2-one} is a natural compound contained in raspberry, and is added to cosmetics for skin whitening. It is very important to measure the RK level in cosmetics for quality assessment, since RK structurally resembles 4-(4-hydroxyphenyl)-2-butanol, which causes leukoderma on consumers’ skin. Here, we present a simple HPLC-fluorescence method for determination of RK in a fragrance mist by pre-column derivatization with 4-hydrazino-7-nitro-2,1,3-benzoxadiazole hydrazine (NBD-H), which reacts with the carbonyl group of RK. The NBD-RK derivative was eluted from a reversed-phase ODS column, and detected with excitation at 470 nm and emission at 550 nm. The retention time of NBD-RK derivative obtained by reaction with NBD-H at 80°C for 20 min was 10.3 min. The standard curve was linear in the range of 0.2 to 10 μg/mL, with a correlation coefficient (r2) value of 0.9980. The lower limit of detection was 0.018 μg/mL (absolute amount of 1.8 pmol). The coefficients of variation were less than 8.1%. The content of RK in fragrance mist (1.00 mL) was 1.18 ± 0.07 mg (range: 1.12 to 1.28 mg, n = 5). Recovery tests were satisfactory (83.9% ± 3.9%;range: 79.6 to 88.8%, n = 5).

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

A sensitive HPLC method for the detection of amino acids with pre-column fluorescence derivatization with carbazole-9-yl-acetyl chloride has been developed. This method, in conjunction with a multi-gradient program, offers baseline resolution of the common CRAC-amino acids from a linear acetonitrile gradient. Separation of amino acid derivatives was obtained on a reversed-phase C-18 column. Derivatization and chromatographic conditions were optimized. Amino acid derivatives were eluted in 40 min with good reproducibility. The relative standard deviations (n=6) at an analytical concentration of 100 pmol are < 4%. The methods described are also suitable for the analysis of amino acids in different biological samples.

高效液相色谱-氢化物发生-原子荧光光谱法测定富硒大豆中硒代氨基酸的含量

取0.1 g脱脂后富硒大豆样品于离心管中,用5 mL pH 7.5的130 mmol·L^(-1)三(羟甲基)氨基甲烷-盐酸缓冲液超声振荡30 min,用15 mg链霉蛋白酶于37℃振荡酶解5 h,离心后取上清液,过0.22μm尼龙有机滤膜。滤液中的硒代半胱氨酸(SeCys)、硒代蛋氨酸(SeMet)、甲基硒代半胱氨酸(MeSeCys)在Agela MP-C_(18)色谱柱上分离,以含2%(体积分数)甲醇和0.5 mmol·L^(-1)四丁基溴化铵的40 mmol·L^(-1)磷酸氢二铵溶液(pH 7.0)进行等度洗脱,并采用氢化物发生-原子荧光光谱法测定3种硒代氨基酸的含量。结果表明,3种硒代氨基酸在7 min内可以实现基线分离,标准曲线的线性范围均为5~100μg·L^(-1),检出限依次为0.086,0.075,0.047 mg·kg^(-1)。按照标准加入法进行回收试验,回收率为85.5%~103%,测定值的相对标准偏差(n=7)不大于3.0%。方法用于分析富硒大豆中的硒代氨基酸,大豆中硒赋存形态多为SeMet,含量占总硒量的85.5%~94.5%。

丹磺酰肼衍生-高效液相色谱-荧光检测器法测定水中痕量3-羟基丙醛

提出了丹磺酰肼衍生-高效液相色谱-荧光检测器法测定水中痕量3-羟基丙醛(3-HPA)的方法。将1.0 g水样、2.0 mL含200 mg·L^(-1)丹磺酰肼的乙腈溶液、1.0 mL 3.0%(体积分数)乙酸溶液混合,再用乙腈定容至50 mL,于40℃衍生反应30 min。以Agilent Eclipse Plus C_(18)色谱柱为固定相,以不同体积比的乙腈-0.10%(质量分数)七氟丁酸溶液的混合溶液为流动相进行梯度洗脱,采用荧光检测器测定。结果表明:衍生试剂丹磺酰肼与3-HPA衍生物在20 min内可实现基线分离;3-HPA的质量浓度在7.6~380.0μg·L^(-1)内与衍生物的峰面积呈线性关系,检出限(3S/N)为1.1μg·L^(-1);方法用于实际水样分析,测定值的相对标准偏差(n=6)为0.71%,3-HPA的加标回收率为98.0%~102%。

薄层色谱与表面增强拉曼光谱法鉴别一次性纸杯中荧光增白剂

建立一次性食品包装用纸杯中非法添加荧光增白剂的薄层色谱-表面增强拉曼光谱(TLC-SERS)鉴别法。采用微波法与传统法分别制备了银、金、金包银和银包金4种金属溶胶,并通过紫外光谱法、电镜扫描法、电势与粒径测定法等手段进行表征,检测各种金属溶胶对吡啶的拉曼光谱增强效应以判定金属溶胶质量,并以4种双三嗪氨基二苯乙烯类荧光增白剂(DSD-FWAs)中的二磺酸型荧光增白剂71(C.I.71)、荧光增白剂85(C.I.85)、荧光增白剂90(C.I.90)、荧光增白剂113(C.I.113)为目标检测成分,建立一次性纸杯中4种荧光增白剂的TLC-SERS鉴别方法。结果显示微波法制备的银溶胶作为本项研究食品包材中增白剂鉴别方法的表面增强剂效果更佳,对市售100批样品进行TLC-SERS鉴别,6批样品中检出C.I.113,建立的方法可为食品包装材料中荧光增白剂的检测提供新的备选方法。

燃料油中硫化氢在线检测系统研制与验证

针对当前燃料油中硫化氢检测技术中存在的样品处理过程复杂、仪器损耗大、烃类干扰严重以及测试周期长等问题,提出了一种顶空气相色谱-荧光检测联用技术。利用顶空气相色谱-荧光检测联用技术实现了燃料油硫化氢的实时在线检测。测得的硫化氢浓度的相对标准偏差为3.21%,且其结果重现性较好。实际样品测试结果表明,顶空气相色谱-荧光检测联用技术与船用燃料油国家标准GB 17411—2015规定使用的英国石油学会标准IP 570/15方法测试结果相近,无显著差异,可以满足日常测试需求。同时,该检测方法针对不同理化性质的燃料油均表现出较高的适用性,有望应用于现场快速检测燃料油质量。

高效液相荧光色谱法同时测定六神曲中黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

目的建立一种用免疫亲和柱净化-柱后光化学衍生-高效液相色谱同时检测六神曲中黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A的方法。方法样品采用60%乙腈超声提取,免疫亲和柱净化,采用XBridge^(®)Phenyl苯基色谱柱(4.6 mm×250 mm,5μm),以乙腈和0.1%磷酸溶液为流动相,梯度洗脱,光化学衍生仪衍生,通过切换荧光波长检测。结果黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A标准曲线的线性范围分别为0.006~0.108、0.500~2.500和0.202~1.009 ng,6种毒素的线性关系在0.9994以上,检出限分别为1.2、100.0和40.3 ng/ml,平均加标回收率为73.2%~92.3%,相对偏差为2.1%~5.4%。结论该方法具有专属性强、操作方便等特点,能够有效用于六神曲中3种毒素的同时检测和安全质量控制。

液相色谱—原子荧光光谱联用法测定海鱼粉中甲基汞含量的不确定度评定

本文评估了用液相色谱法—原子荧光光谱法测定海鱼粉中甲基汞的不确定度。对测试过程中不确定度的来源进行分析,并对各组分的不确定度进行计算。结果表明,当海鱼粉中甲基汞的含量为0.670 mg/kg,扩展不确定度为0.034 mg/kg(k=2)。

荧光检测器高效液相色谱法测定樟脑丸中萘

建立高效液相色谱法测定樟脑丸中萘的含量。样品以甲醇为溶剂超声溶解,经0.22μm滤膜过滤后,以荧光检测器高效液相色谱法测定。荧光检测器激发、发射波长分别为290、330 nm,以甲醇为流动相,流量为1.0 mL/min,以Platisil ODS柱(250 mm×4.6 mm,5μm)色谱柱作为分析柱,柱温为25℃,进样体积为10μL,利用色谱峰面积外标法进行定量。该方法线性相关系数为0.9999,检出限为1.0×10^(-8)g/mL,样品加标回收率为98.4%~102.8%(n=6),测定结果的相对标准偏差为1.81%(n=6)。该方法可用于樟脑丸中萘的检测。

畜禽粪污异位发酵床处理残渣组成特征研究

异位发酵床是处理畜禽粪污的常用技术,目前针对异位发酵床的研究基本是在半年以内的短期跟踪,对于一年以上异位发酵床处理后残渣的组成特征及利用潜力仍不清楚,限制了发酵后残渣的处理和资源化利用。本研究选择浙江省3个典型养猪场,采集了畜禽粪污经异位发酵床处理半年(S1组)、1年(S2组)及2年(S3组)剩下的残渣,采用微生物高通量测序、色谱分离结合常规理化指标测试,对异位发酵床发酵剩余残渣中的微生物、无机物、重金属及有机物组成和含量进行测定,并据此评估了残渣的土地利用潜力。结果表明:长期运行下异位发酵床物料微生物趋于一致,S2组和S3组具有相似的微生物群落组成。发酵过程每日的翻抛有利于粪污中氮的转化,三组样品中溶解态NH_(4)^(+)含量均显著低于NO_(3)^(-)和NO_(2)^(-)含量;经长时间发酵,有机物降解后,无机物累积,残渣中无机盐含量增加,但其总电导率均低于3000μS/cm,处于植株安全生长范围;畜禽粪污的长期投加,有机物降解为可挥发的二氧化碳和水,重金属却只能被保留在发酵床中,不断富集和累积,但残渣中重金属含量仍低于《有机肥料》(NY/T525-2021)浓度限值,满足有机肥制作标准。三维荧光光谱及色谱分析显示,S1组和S2组残渣中腐殖质组分种类多,且含有多种小分子有机组分,而发酵2年的S3组残渣中腐殖质种类少、分子量大、结构更加单一稳定。本研究揭示了畜禽粪污长期异位发酵床处理后残渣组成特征及土地利用潜力,即经异位发酵床长期处理的畜禽粪污部分指标无法满足《畜禽粪污还田技术规范》(GB/T 25246-2010),因此无法直接还田施用,但能满足《有机肥料》(NY/T 525-2021),可制成有机肥料后还田,这为长期异位发酵床残渣的后续处理和资源化利用提供了参考。

高效液相色谱-荧光检测器法同时测定乳及乳制品中维生素A和维生素E

目的建立高效液相色谱-荧光检测器法同时测定乳及乳制品中维生素A和维生素E的分析方法。方法样品经水解皂化、液液萃取、蒸发浓缩后,选用PFP色谱柱分离,以甲醇-水(9:1,V/V)等度洗脱,高效液相色谱-荧光检测器检测,外标法定量,计算维生素A、维生素E的含量。结果维生素A和4种维生素E异构体在0.10~5.00μg/ml范围内呈现良好线性,方法检出限为0.0100μg/g、方法定量限为0.0400μg/g,维生素A的平均回收率在85.0%~97.0%之间,RSD≤6.41%(n=6);4种维生素E异构体的平均回收率在84.0%~101%之间,RSD≤7.04%(n=6)。结论本方法简便快速,灵敏准确,可满足乳及乳制品中维生素A和维生素E的同时测定。

顶空气相色谱-荧光技术测定燃料油中硫化氢

实际生产检测过程中,燃料油中的硫化氢(H2S)主要是通过快速液相萃取法和气相色谱法进行检测。针对两种方法中样品前处理过程复杂、受人为影响大及对仪器造成损害等问题,文章采用顶空进样气相色谱-荧光检测技术实现了对燃料油中H2S的准确测定。结果表明,顶空进样气相色谱-荧光检测技术测定H2S的相关系数达0.99以上,定量限和检出限分别达到了0.3 mg/kg和0.1 mg/kg,RSD低于4.0%。在实际油品检测中,燃料油中H2S含量的测定结果与英国石油学会标准IP 570/15的差异并不明显,相对误差<4.0%,证实了顶空进样气相色谱-荧光检测技术对于燃料油中H2S含量测定的准确性。该检测方法的建立为燃料油及石油化工行业中H2S检测方法的开发提供了借鉴,为简化油品检测前处理过程、提高检测效率提供了条件。

MTHFR C677T基因多态性方法学比较

目的评价比较实时荧光定量PCR法与PCR-金磁微粒层析法两种方法检测5,10-亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性的一致性、检测限、特异性。方法随机选取首都医科大学附属北京天坛医院采集的50例静脉抗凝全血,均通过磁珠法提取基因组DNA,依照最新版分子诊断检验程序性能验证指南(CNAS-GL039)要求对两种方法学的一致性、检测限、特异性进行性能评价。结果实时荧光定量PCR与PCR-金磁微粒层析具有高度的一致性,采用两种方法检测50例样本的一致性为100%;两种方法检测交叉反应中检测结果未受影响,基因型之间无交叉污染情况;检测限验证中前者的灵敏度更高,最低检测限可达0.078125ng/μL,后者的最低检测限为0.625ng/μL。结论实时荧光定量PCR法与PCR-金磁微粒层析法均具有操作简便、灵敏度高,特异性强的特点,可用于人类MTHFR基因多态性检测,同时能够对叶酸代谢作出有效的诊断,需根据临床实验室的需求,选择合理有效的检测方法。

综合创新实验:异三聚茚的合成,表征与应用

异三聚茚(Isotruxene)是一种新型的稠环芳香化合物,具有成为光电材料的潜力,但受到合成方法的限制,科学界对其研究十分少见。我们发展了一种通过微波反应一步合成异三聚茚的方法,通过柱层析处理得到较纯的异三聚茚,并通过核磁共振谱鉴定了主产物的结构。通过高效液相色谱(HPLC)实现了主产物和副产物的拆分,并测定了主产物的纯度。此外,我们还观测到异三聚茚与硝基化合物混合时的荧光淬灭现象,证实了异三聚茚在爆炸物检测领域的应用潜力。本实验综合了有机合成,仪器分析,物理化学等多个学科的要素,可用于模块化教学,适应不同学时的实验教学要求,既可培养学生的基本操作,又能激发学生的研究兴趣,锻炼学生的综合能力。