Optimization of a Multiplex PCR Assay for Detecting Transgenic Soybean Components in Feed Products

A multiplex polymerase chain reaction (m-PCR) assay was developed for the simultaneous detection of multiple components of genetically modified (GM) soybean. It uses two sets of primers (I, lectin1/35S/CP4; II, lectin2/35S/CP4) specific for a soybean reference gene, the 35S promoter, and an event-specific gene. Amplified fragments of 118, 414, 195, and 320 bp were easily detected by agarose gel electrophoresis and were positively confirmed by sequencing. Primer set concentrations and annealing temperatures in the m-PCR were optimized. The optimized m-PCR conditions were obtained for primer set I at a ratio of 1:2:3 and a 59.2 °C annealing temperature and set II at the same ratio and 58.6 °C, 60.3 °C, and 61.2 °C annealing temperatures. The sensitivities of the two m-PCR primer sets (I and II) were 0.25% and 0.5%, respectively. The results showed that this m-PCR assay provides rapid, reliable, and effective identification of multiple components of GM soybean in feed.     

The Simultaneous Assay of Naproxen and Salicylic Acid in Serum Using High Pressure Liquid Chromatography

Abstract

A rapid, sensitive, reverse-phase high pressure liquid chromatographic assay was developed for the simultaneous determination of naproxen and salicylic acid. These compounds are extracted from serum and then separated on a reverse-phase column using an acidified methanol eluent. Utilization of a flow-through fluorescence detector in series with a variable wavelength micro-UV detector enhances the sensitivity of the assay. Application of the assay to therapeutic levels of the drugs in human serum is demonstrated.     

Simultaneous Determination of β-Artemether and its Metabolite Dihydroartemisinin in Human Plasma and Urine by a High-Performance Liquid Chromatography-Mass Spectrometry Assay Using Electrospray Ionisation

A sensitive and selective method is described for the determination of β-artemether (AM) and its metabolite dihydroartemisinin (DHA) in human plasma and urine using artemisinin (IS) as internal standard. The method consists of a liquid-liquid extraction using 2,2,4-trimethylpentane – ethyl acetate (7:3 v/v) with subsequent evaporation of the supernatant to dryness followed by the analysis of the reconstituted sample by liquid chromatography – mass spectrometry (LC-MS) using positive electrospray ionisation (ESI). The acquisition was performed using a mass range scan and the ions (MH+?CH3OH) m/z 267.2, (MH+?H2O) m/z 267.2 and (MH+) m/z 283.2 for AM, DHA and IS respectively were used for compound quantifications. Chromatography was performed on a C18 reversed-phase column using a gradient of acetonitrile – ammonium acetate 10 mM, glacial acetic acid 0.1% as a mobile phase. The method was validated over a concentration range of 10–1000 ng mL^?1 using 1 mL of human plasma per assay and over a concentration range of 5–500 ng mL^?1 using 2 mL of human urine per assay. The method was applied to the quantitation of β-artemether and dihydroartemisinin in human plasma and urine of volunteers participating in a drug pharmacokinetic study.     

凝胶柱净化-高效液相色谱检测食品中的苏丹红

建立了凝胶柱净化-高效液相色谱同时检测食品中苏丹红Ⅰ,Ⅱ,Ⅲ和Ⅳ的方法。样品用乙醇提取,提取液经Bio-Beads SX3凝胶柱(200 mm×10 mm i.d.)净化,用环己烷-乙酸乙酯(体积比为1∶1)洗脱。采用Symmetry Shield RP18柱(250 mm×4.6 mm i.d.,　5　μm)分离,以100%甲醇为流动相,流速1.5 mL/min;用二极管阵列检测器检测,检测波长478 nm。上述4种苏丹红组分在其质量浓度为0.1~10.0 mg/L时有良好的线性关系(r>0.999),方法的检测限为7~14 μg/kg;平均加标回收率为80.7%~96.3%(添加水平为0.25,2.5 mg/kg),相对标准偏差为2.4%~5.9%。方法灵敏可靠,能满足食品中苏丹红检测的需要。     

HPLC—ICP—MS法对动物源性食品中阿散酸、硝苯砷酸与洛克沙砷残留量的同时测定

建立了动物源性食品中阿散酸(ASA)、硝苯砷酸(NPAA)和洛克沙砷(ROX)的HPLC-ICP-MS分析方法.研究了液相色谱和ICP-MS条件并对提取剂进行了优化.在0 ～100 μg/L范围内阿散酸、硝苯砷酸和洛克沙砷的线性关系良好,线性相关系数均大于0.998; 阿散酸、硝苯砷酸、洛克沙砷检出限分别为2.75、3.85、4.20 μg/kg;保留时间的相对标准偏差(RSD)不大于0.12%.5种样品中3个添加水平的平均回收率为阿散酸86% ～99%,硝苯砷酸79% ～95%,洛克沙砷82% ～98%;相应的RSD分别为2.9% ～8.6%、2.8% ～7.9%、3.3% ～8.1%.     

固相萃取-高效液相色谱法同时测定猪肉中磺胺类和氟喹诺酮类兽药残留量

建立了一种同时测定猪肉中3种磺胺和7种氟喹诺酮药物残留量的固相萃取-高效液相色谱法。样品经2%醋酸/乙腈提取,正己烷脱脂,过ENVI-18固相萃取柱净化。使用ShimadzuVP-ODS(250mm×4.6mm,5μm)色谱柱,以甲醇-0.02mol/L磷酸盐缓冲液-三乙胺系(体积比为25.5/74.5,pH2.80)为流动相,采用二极管阵列检测器进行测定。10种药物在0.1~5.0mg/L浓度范围内线性良好,相关系数均大于0.9999。检出限为3.40~9.00μg/kg,样品的平均加标回收率在69%~104%之间,RSD<5%(n=3)。     

保健食品中9种镇静催眠类药品的UPLC-MS/MS法检测及质谱裂解特征的研究

建立了同时测定保健食品中9种镇静催眠类化合物(氯美扎酮、三唑仑、阿普唑仑、艾司唑仑、地西泮、硝西泮、奥沙西泮、劳拉西泮、氯氮卓)的超高效液相色谱-串联质谱(UPLC-MS/MS)分析方法,并研究了其质谱裂解规律。样品以甲醇为提取溶剂,经Zorbax SB-C18(3.5μm,2.1 mm×150 mm)色谱柱分离,乙腈-水(含0.1%甲酸)为流动相梯度洗脱,流速为0.3 m L/min。采用电喷雾离子源(ESI),正离子多反应监测(MRM)扫描方式检测,基质匹配标准曲线法定量。9种药物在0.5~50μg/L范围内线性关系良好,相关系数(r2)均大于0.99,定量下限为1.6~9.2μg·kg~(-1)。低、中、高3个加标水平下的平均回收率为78.1%~101.2%,日内相对标准偏差(RSD)为1.4%~9.7%,日间RSD为2.0%~11.2%。该法简便、快速、准确可靠,适合于保健食品中非法添加镇静催眠类药品的高通量筛查。而对目标化合物碎片离子及质谱裂解规律的研究,也为其定性鉴别和定量分析提供了参考。     

Simultaneous Determination of Tetracyclines and Fluoroquinolones in Poultry Eggs by UPLC Integrated with Dual-Channel-Fluorescence Detection Method

An innovative, rapid and stable method for simultaneous determination of three tetracycline (oxytetracycline, tetracycline and doxycycline) and two fluoroquinolone (ciprofloxacin and enrofloxacin) residues in poultry eggs by ultra-high performance liquid chromatography–fluorescence detection (UPLC-FLD) was established and optimized. The samples were homogenized and extracted with acetonitrile/ultrapure water (90:10, v/v) and then purified by solid-phase extraction (SPE). LC separation was achieved on an ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 mm × 100 mm), and the mobile phase was composed of acetonitrile and a 0.1 mol/L malonic acid solution containing 50 mmol/L magnesium chloride (the pH was adjusted to 5.5 with ammonia). When the five target drugs were spiked at the limit of quantification, 0.5 times the maximum residue limit (MRL), 1.0 MRL and 2.0 MRL, the recoveries were above 83.5% and the precision ranged from 1.99% to 6.24%. These figures of merit complied with the parameter validation regulations of the EU and U.S. FDA. The limits of detection and quantifications of the targets were 0.1–13.4 µg/kg and 0.3–40.1 µg/kg, respectively. The proposed method was easily extended to quantitative analyses of target drug residues in 85 egg samples, thus demonstrating its reliability and applicability.     

电感耦合等离子体质谱法同时测定食品中的铅、镉和总砷

建立了电感耦合等离子体质谱(ICP–MS)法同时测定食品中的铅、镉和总砷含量的分析方法。样品经消化后直接用ICP–MS分析,采用在线引入混合内标溶液校正基体干扰和仪器的信号漂移。铅、镉、总砷的质量浓度在0~50μg/L范围内与信号强度呈良好的线性,线性相关系数(r)均大于0.999 0。用该方法对标准物质进行测定,分别采用湿法、微波消解法对样品进行处理,两种前处理方式测定值无显著性差异,测定结果的相对标准偏差在0.45%~4.2%之间(n=6),检测结果均在参考值范围内。该方法可同时测定铅、镉和总砷含量,操作简便、重现性好、结果准确可靠,可以满足各种食品中铅、镉和总砷同时测定的要求。     

Multiplex genotyping of KRAS point mutations in tumor cell DNA by allele-specific real-time PCR on a centrifugal microfluidic disk segment

Point Mutations on the Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified as an important predictive biomarker for response to cancer therapy targeting the epidermal growth factor receptor. KRAS mutations are prevalent in up to 40 % of all colorectal carcinomas, and routinely conducted KRAS genotyping is becoming mandatory to predict therapy success and to reduce therapy costs. We report a low-cost, disposable and ready-to-use centrifugal microfluidic cartridge (termed GeneSlice) containing preloaded primers and probes. The GeneSlice cartridge enables the parallel detection of the seven most relevant KRAS point mutations by allele-specific real-time PCR. It represents a cost effective alternative to dideoxy-sequencing with a faster time-to-result (~ 2 h versus up to 20 h in case of dd-sequencing). Microfluidic processing of the GeneSlice along with allele-specific amplification and real-time detection are conducted in a slightly modified, commercially available PCR thermocycler. Intra-chip standard deviation of C_q values on the GeneSlices is negligible (GeneSlice 1: C_q,std.dev. = 0.13; GeneSlice 2: C_q,std.dev?=?0.26). In 23 of 24 experiments, the data for genotyping 6 cancer cell lines (n?=?4 per cell line) agreed with dd-sequencing. Additionally, DNA derived from microdissected formalin-fixed and paraffin embedded colorectal carcinomas of two cases was genotyped correctly and reproducibly (n?=?3 per patient; one GeneSlice excluded from evaluation). The GeneSlice therefore clearly demonstrated the potential to become a valuable tool for routine diagnostics of KRAS mutations by reducing costs and hands-on time. Figure

Photograph of a centrifugal microfluidic cartridge “GeneSlice” for multiplex genotyping of KRAS point mutations from tumor cell DNA by allele-specific real-time PCR. Information about the mutation status is required to predict success of state-of-the-art cancer therapy with antibodies     

Detection of Parvalbumin Fish Allergen in Canned Tuna by Real-Time PCR Driven by Tuna Species and Can-Filling Medium

Canned tuna is considered one of the most popular and most commonly consumed products in the seafood market, globally. However, in past decades, fish allergens have been detected as the main concern regarding food safety in these seafood products and are listed as the top eight food allergies. In the group of fish allergens, parvalbumin is the most common. As a thermally stable and calcium-binding protein, parvalbumin can be easily altered with changing the food matrices. This study investigated the effect of a can-filling medium (tomato sauce, spices, and brine solutions) on the parvalbumin levels in canned tuna. The effect of pH, calcium content, and the DNA quality of canned tuna was also investigated before the parvalbumin-specific encoded gene amplification. The presence of fish allergens was determined by melting curve analyses and confirmed by agarose gel electrophoresis. The obtained results showed that the presence of parvalbumin in commercially canned tuna was driven by can-filling mediums, thermal conductivity, calcium content, and the acidity of various ingredients in food matrices. The intra-specific differences revealed a variation in fish allergens that are caused by cryptic species. This study proved that allergens encoding gene analyses by agarose electrophoresis could be used as a reliable approach for other food-borne allergens in complex food matrices.     

高效液相色谱法同时测定食品和药品中的糖及其降解产物5—羟甲基糠醛

５－羟甲基糠醛（５－ＨＭＦ）是糖的降解产物，本文提出了用高效液相色谱法同时测定食品和药品中的蔗糖，葡萄糖，果糖和５－ＨＭＦ的方法，采用氢型阳离子交换色谱法Ａｍｉｎｅｘ－ＨＰＸ－８７Ｈ，以乙腈－０．０１ｍｏｌ／Ｌ，硫酸（４０：６０）为流动相分离糖和５－ＨＭＦ，使用串联的紫外光度检测器（２８０ｎｍ）和示差折光检测器，分别检测５－ＨＭＦ和糖。     

固相萃取/高效液相色谱法对中成药和保健品中7种降糖化学药物的测定

建立了一种固相萃取/高效液相色谱测定保健品中格列吡嗪、格列齐特、格列本脲、格列喹酮、盐酸二甲双胍、盐酸苯乙双胍和瑞格列奈7种降糖化学药物的方法.采用阳离子周相萃取柱,对提取液进行净化处理.得到的不同洗脱液分别进样,用液相色谱进行分析,采用ZORBAX SB-C18柱,以乙腈-0.02mol/L磷酸缓冲盐(pH 2.8)作为流动相进行梯度洗脱,7个色谱峰分离良好,两次进样在40min内可以完成,外标法定量.在3种不同剂型中药制剂中的添加回收率在83%～101%之间.     

固相萃取/气相色谱-质谱同时测定猪肉中4种β-激动剂类药物残留量

β-激动剂(属于苯乙胺类药物,PEAs)是一类可以引起交感神经兴奋的类似肾上腺素的药物。一些β-激动剂如克伦特罗、沙丁胺醇、特布它林、非诺特罗可使多种动物(牛、羊、猪、家禽等)体内的营养成分由脂肪组织向肌肉组织转移,从而显著提高胴体的瘦肉率和饲料转化率,因此常用做饲料     

超高效液相色谱-四极杆/线性离子阱质谱法同时测定不同产地何首乌中10种核苷类成分

建立了何首乌药材中10种核苷类成分(尿嘧啶、胞苷、鸟嘌呤、尿苷、腺嘌呤、肌苷、鸟苷、胸苷、腺苷、2'-脱氧胞苷)的超高效液相色谱-串联四极杆/线性离子阱质谱(UPLC-QTRAP-MS/MS)同时测定的分析方法。不同产地何首乌样品用超纯水在室温下超声提取,提取液经高速离心处理,取上清液,经Waters Atlantis T3色谱柱(2.1 mm×150 mm,3μm),以甲醇-5 mmol/L醋酸铵(含0.1%冰醋酸)为流动相,0.4 m L/min梯度洗脱,采用正离子多反应监测(MRM)模式测定。10种核苷在一定浓度范围内具有良好的线性关系,相关系数均大于0.99,检出限为1.05~9.68 ng/m L;平均加标回收率为97.8%~104.8%,相对标准偏差(RSD)在1.8%~5.0%之间。该方法简便、灵敏、准确,为何首乌药材内在质量的评价和控制提供了可靠的检测方法。     

Validation of a Reference Gene (BdFIM) for Quantifying Transgene Copy Numbers in Brachypodium distachyon by Real-Time PCR

Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon.     

Simultaneous Determination of 21 Sulfonamides in Poultry Eggs Using Ionic Liquid-Modified Molecularly Imprinted Polymer SPE and UPLC–MS/MS

An ionic liquid-modified molecularly imprinted polymer (IL-MIP) composite with sulfamethazine as a template molecule and methyl acrylic acid and 1-aminopropyl-3-methylimidazolium bromide as functional monomers was successfully synthesized. The achieved IL-MIP was characterized and evaluated in detail and utilized in the extraction and cleanup of sulfonamides (SAs) in poultry egg samples. The results demonstrated that the IL-MIP possessed a broad reorganization toward SAs and could selectively adsorb 21 kinds of SA compounds. Furthermore, the solid-phase extraction column based on the IL-MIP was used in the extraction and cleanup of 21 SAs in eggs, and the confirmatory detection of SAs was performed using ultraperformance liquid chromatography–tandem mass spectrometry. Under optimum conditions, the limits of detection (LODs) for all SAs ranged from 0.1 ng·g⁻¹ to 1.5 ng·g⁻¹, and the LOD of this method was better than those of the existing methods. The recoveries of SA compounds spiked in egg samples ranged from 84.3% to 105.8%, with low relative standard deviations (<15%). The developed method based on the IL-MIP extraction and cleanup was successfully used in the detection of 21 SAs in more than 100 real poultry egg samples. The results indicated that the proposed method was suitable for detecting 21 SAs in poultry eggs.     

Simultaneous Identification and Determination of Several Barbiturates in Plasma by Reverse-Phase HPLC

Abstract

A Reverse-Phase HPLC method is described for the simultaneous identification and determination of 5 barbiturates: Allobarbital, Phencbarbital, Butabarbi -tal, Butalbital and Pentobarbital. Barbital is used as internal standard. The mobile phase is Milli Q Water/ Acetonitrile/ 1.75 M Phosphoric Acid (738:200:2, v/v/v) at a flow rate of 0.8 mL/min and UV detection is carried out at 195 nm. The single extraction procedure requires only small samples and analysis is quick.     

反相高效液相色谱法同时测定食品和多维片中8种水溶性维生素

建立了以0．05mol/L KH2PO4-甲醇为流动相的梯度洗脱反相高效液相色谱同时测定水溶性维生素C,B1,B2,B6,B12,烟酸,烟酰胺和叶酸的分析方法,方法检出限为1．4－0．76ng,用该法测定了奶粉,玉米粉,饮料及复合维生素片中水溶性维生素,相对标准偏差为2．8％－8．8％,加标回收率为74．7％－112．5％。