Speciation of iron in breast milk and infant formulas whey by size exclusion chromatography-high performance liquid chromatography and electrothermal atomic absorption spectrometry

Speciation of iron in milk was carried out by high performance liquid chromatography (HPLC) and electrothermal atomic absorption spectrometry (ETAAS). Milk whey was obtained and low molecular weight protein separation was performed by size exclusion chromatography (SEC) with a TSK Gel SW glass guard (Waters) pre-column and a TSK-Gel G2000 glass (Toso Haas) column. After studying water as a possible mobile phase, this mobile phase was carefully selected in order to avoid alterations of the sample and to make subsequent iron determination in the protein fractions easier by ETAAS. The proposed method is sensitive (limit of detection [LOD] and LOQ 1.4 and 4.7 μg l⁻¹, respectively) and precise (relative standard deviation [RSD]<10%). Iron is principally found in the proteins of 3 and 76 kDa in breast milk, and it is irregularly distributed in infant formulas.     

Speciation of zinc in low molecular weight proteins of breast milk and infant formulas by size exclusion chromatography/flame atomic absorption spectroscopy.

Size exclusion chromatography (SEC) and flame atomic absorption spectroscopy (FAAS) were used for the separation of metal-containing species in milk whey. After milk ultracentrifugation, the sample was injected into a TSK-Gel G2000 glass column and eluted with 0.2M NH4NO3-NH3, pH 6.7. Low molecular weight proteins were fractionated, and the fractions were characterized by molecular weight. Zinc distributions were obtained by FAAS using a high performance nebulizer. The method was very sensitive (limit of detection = 2.6 x 10(-3) microg/mL; limit of quantitation = 8.9 x 10(-3) microg/mL) and precise (RSDs < or =10%). This method was applied to the determination of Zn in binding compounds in breast milk whey and in commercial cow's milk-based formulas. Distribution patterns were different. The presence of Zn in most fractions in breast milk was most significant, whereas in infant formulas Zn was detected only in fractions of molecular weight <5 kDa and in the highest molecular weight peak.     

Determination of sulfonamides by packed column supercritical fluid chromatography with atmospheric pressure chemical ionisation mass spectrometric detection

Sulfonamide antibiotics are widely used to prevent bacterial infections in livestock, and residues are commonly found in milk and meat. Packed column supercritical fluid chromatography (pSFC) with detection using ultra violet (UV) and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS) provides a versatile method for the detection and quantification of six major sulfonamides. The APCI mass spectra for all the sulfonamides consisted of protonated molecules at low cone voltages. Increasing the cone voltage led to informative fragmentation patterns, which provided structural information for identification purposes. The pSFC-APCI-MS technique was shown to be linear (r2 > or = 0.999) over the concentration range 0.1-50 micrograms ml-1 using total ion current. The precision and the accuracy of the system and validation of sample preparation are acceptable, with RSD < 2% and relative error 8%. Selected ion monitoring gave detection limits as follows: sulfadiazine 41, sulfamethoxazole 45, sulfamerazine 47, sulfamethizole 59, sulfamethazine 181 and sulfadimethoxine 96 micrograms l-1, which are lower than the amounts permitted in milk products. The APCI pSFC-MS system was shown to have a high degree of reproducibility. The technique was then applied to determine the above sulfonamides in milk. The results obtained show that there are no matrix effects from the milk and that the detection limits remained as stated for the standard solutions.     

Quantification of total chromium and hexavalent chromium in UHT milk by ETAAS

Procedures for the quantification of total chromium and hexavalent chromium in UHT milk samples are presented. Total chromium was determined directly in milk with the addition of a surfactant and a mixture of Pd and Mg as a chemical modifier. For the selective separation of hexavalent chromium, the sample pre-treatment consisted in precipitation of proteins and elution of the supernatant through a Chromabond NH2 column. The metal was eluted with nitric acid. Both total chromium and hexavalent chromium were evaluated by atomic absorption spectrometry with electrothermal atomization using the same instrumental conditions. The detection limits were 0.2 and 0.15 microgram l-1 for total chromium and hexavalent chromium, respectively. The linearity ranges under the optimized conditions were 0.2-20 and 0.15-50 micrograms l-1. For total chromium the precision was 4.9 and 5.7% for the analytical and the over-all procedure, respectively, and for hexavalent chromium 4.3 and 4.9%, respectively. The validation of both procedures was performed by the standard additions method and the recoveries were higher than 93% in all cases. For total chromium, a certified reference material was also used to validate the methodology. The methods were applied to the determination of total chromium and hexavalent chromium in 60 UHT milk samples.     

Fractionation of soluble selenium compounds from fish using size-exclusion chromatography with on-line detection by inductively coupled plasma mass spectrometry

Fish accumulate significant amounts of selenium and are an important dietary source of this element. Some studies have however indicated a low bioavailability of the selenium from fish. Since little is known of the selenium forms in fish, we have studied soluble selenium compounds in fish species, and compared different techniques for fractionation of selenocompounds (size-exclusion chromatography, ultrafiltration, and precipitation with trichloroacetic acid). The size-exclusion column (Superdex 200 HR 10/30) was coupled on-line to inductively coupled plasma mass spectrometry (ICP-MS). The limit of detection was 0.20 microgram l-1 and the selenium response was linear in the investigated concentration range of 0-20 micrograms l-1 (r2 = 0.98). For plaice 47% of the selenium was extractable while the extraction efficiency for cod was 23%. The fish extracts were injected onto the column four times each and the variation in the quantitative data for different selenium-containing fractions between the runs was small (RSD < 10%). The recovery of selenium in the chromatographic step was about 70%, indicating some interaction between the fish extracts and the column material. Ultrafiltration using a membrane with a cut-off at M(r) 10,000 gave results similar to the size-exclusion fractionation, for cod about 20% of the soluble selenium had a M(r) < 10,000 and the corresponding value for plaice was 69%. Removal of high-molecular-weight compounds from the sample by trichloroacetic acid precipitation showed a similar proportion of low-molecular-weight compounds for plaice (77%), while the obtained value for cod was higher (38%) compared with the other techniques.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

高效液相色谱-串联质谱法检测奶中克拉维酸残留

采用高效液相色谱-串联质谱(HPLC-MS/MS)建立了克拉维酸在奶中的残留检测方法。2 g样品经乙醇沉淀蛋白质后,转入鸡心瓶中旋转蒸发浓缩至0.5 mL左右,用乙酸铵定容,净化后检测。流动相为乙腈和0.1%甲酸水,梯度洗脱,经Luna 5u C8色谱柱分离,采用电喷雾电离,多反应监测负离子模式对克拉维酸进行定量分析。采用基质匹配法对奶中克拉维酸的含量进行标准校正,在克拉维酸含量为10～400 μg/kg范围内呈现良好的线性关系,相关系数大于0.999;奶中加标样品的检出限(LOD,按信噪比(S/N)≥3计)为10 μg/kg,定量限(LOQ, S/N≥10)为20 μg/kg。在定量限、1/2最高残留限量、最高残留限量、2倍最高残留限量添加水平下,奶中克拉维酸的平均回收率为80.00%～91.25%,相对标准偏差为5.60%～8.77%。该方法可用于奶中克拉维酸残留的分析检测。     

Cascade ultrafiltering of 210Pb and 210Po in freshwater using a tangential flow filtering system</p> </p>

Summary A rapid method was developed using ultrafilters with a tangential flow filtering system for molecular size separation of naturally occurring ²¹⁰Pb and ²¹⁰Po in a freshwater sample. Generally, ultrafiltering of a large volume water sample for measuring the nuclides was too time consuming and not practical. The tangential flow filtering system made the filtering time short enough to adapt for in-situ ultrafiltering the large volume sample. In this method, a 20 liter water sample was at first passed through the 0.45mm pore size membrane filter immediately after sample collection to obtain suspended particle matter [>0.45mm particulate fraction (PRT)]. Two ultrafilters (Millipore Pellicon 2^ò) were used sequentially. The nuclides in the filtrate were separated into three fractions: high molecular mass (100 kDa-0.45mm; HMM), low molecular mass (10 k-100 kDa; LMM) and ionic (<10 kDa; INC) fractions. It took 80 minutes to process the sample after collection. The cut-off molecular size of each ultrafilter was confirmed by size exclusion chromatographs (SEC) of the LMN and the HMM fractions. Adsorption of the nuclides and organic compounds in the sample onto the ultrafilters was negligibly small. Good reproducibility of the nuclide concentrations in each fraction was confirmed by repeated experiments. The method was successfully applied to obtaine the molecular size distributions of ²¹⁰Pb and ²¹⁰Po in an oligotrophic lake, Lake Towada located in the northern area of Japan.</p> </p>     

Structural characterisation of Baltic amber and its solvent extracts by several mass spectrometric methods

A sample of Baltic amber ( approximately 40 million yrs old) has been extracted using pentane, toluene and 1-methyl-2-pyrrolidinone (NMP). The relationship between solubility characteristics of the extracts in relation to molecular mass and chemical makeup has been investigated. The extracts were first characterised by (13)C-NMR spectrometry, size exclusion chromatography (SEC) and UV-fluorescence spectroscopy. The fractions differed less in terms of chemical structural features than they did in terms of molecular mass. This contrasts markedly with data on fractions of coal-derived liquids, but parallels results from petroleum-derived vacuum residues. In SEC, the toluene soluble/pentane insoluble fraction gave a peak for high mass material at about 67 000 u. Material excluded from the column porosity in this fraction and in NMP solubles eluted between 8 and 11 min, corresponding to polystyrene masses between 200 000 and several million u. A column with a larger pore size distribution was calibrated using polystyrene and polymethylmethacrylate standards with detection by a light-scattering evaporative analyser. The largest polystyrene standard (15.4 million u) eluted at 13.4 min, similar to that of the earliest eluting amber-derived material in the NMP solubles fraction. Results from probe-MS and pyrolysis-GC/MS have been used to confirm the similarity of chemical structures of the three solubility fractions. Broadly, low mass ions appear to correspond to the various monomeric units of structures present in the amber, the higher mass ions to dimer units and the molecular ions to the different combinations of three or more monomeric units. The main monomer groups have been identified in detail, showing a situation very different from that of coal-derived materials, where the sizes of aromatic ring systems increase with molecular size.     

超高效液相色谱-四极杆-飞行时间质谱法快速筛查牛奶中的农药和兽药残留

利用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)技术建立了牛奶中42种农药和兽药残留的快速检测方法。目标药物包括常用的13种农药和29种兽药,采用QuEChERS（Quick, Easy, Cheap, Effective, Rugged, and Safe）方法进行样品前处理。牛奶样品经含1%甲酸的乙腈溶液提取,同时加入无水硫酸钠和氯化钾盐析,提取液经C18填料净化后直接测定。目标药物经ACQUITY UPLCTMBEH C18柱分离,以乙腈和0.1%甲酸水溶液为流动相进行梯度洗脱,采用正离子全信息串联质谱扫描模式(MSE)进行检测。结果表明,牛奶中42种农药和兽药的定量限(LOQ, S/N=10)为1～100 μg/kg; 3个加标水平的平均回收率为68.2%～129.1%,相对标准偏差为2.8%～30.8%。该方法快速简便、灵敏度较高,可用于牛奶中42种农兽药的快速筛查。     

Modified determination of dihydrostreptomycin in kidney, muscle and milk by HPLC.

A method is presented for the determination of dihydrostreptomycin in milk, muscle and kidney by reversed-phase ion-pair high-performance liquid chromatography and post-column derivatisation with beta-naphthoquinone-4-sulfonate prior to fluorescence detection. The new sample work-up procedures include acid precipitation of proteins and, in the case of muscle and kidney, removal of fats by solvent extraction followed by solid phase extraction on a cation exchanger. The fluorescence response was linear from 25 to 2000 micrograms l-1 of injected analyte. The detection limits were 10 micrograms kg-1 for milk and 15 micrograms kg-1 for muscle and kidney and the analyte recoveries were on average 93% for milk, 70% for kidney and 75% for muscle.     

高效液相色谱-串联质谱法检测牛奶中头孢洛宁残留

建立了牛奶中头孢洛宁残留检测的高效液相色谱-串联质谱方法。1 g牛奶经乙腈沉淀蛋白质后,上清液于37 ℃水浴下氮气吹干,用1 mL甲醇-0.1%甲酸水溶液（3：7,v/v）复溶,正己烷除脂净化后检测。流动相为乙腈和0.1%甲酸水溶液,梯度洗脱,经C₁₈色谱柱分离,采用多反应监测正离子模式对头孢洛宁进行定性定量分析。采用基质匹配法对牛奶中头孢洛宁的含量进行标准校正,在2~200 μg/L范围内,头孢洛宁质量浓度与其对应峰面积的线性关系良好,相关系数>0.999。牛奶中加标样品的检出限（按S/N≥3计）为0.5 μg/kg,定量限（S/N≥10计）为2 μg/kg。在定量限、1/2最高残留限量、最高残留限量、2倍最高残留限量添加水平下,牛奶中头孢洛宁的平均回收率为78.5%~86.2%,日内相对标准偏差为1.5%~6.2%,日间相对标准偏差为2.9%~5.6%。该方法可用于牛奶中头孢洛宁的残留检测。     

在线净化液相色谱-串联质谱法快速测定牛奶中的6种孕激素

建立了在线净化液相色谱-串联质谱同时测定牛奶中炔诺酮、17α-羟基孕酮、甲羟孕酮、乙酸甲地孕酮、孕酮和醋酸美伦孕酮6种孕激素的方法。本方法采用乙腈为提取溶剂提取目标化合物。提取液经在线净化柱Cyclone-P净化,经Phenyl-Hexyl色谱柱分离,流动相采用0.5%（v/v）甲酸水溶液-乙腈,在电喷雾正离子模式下以多反应监测（MRM）方式测定,内标法定量。方法在0.1~50 μg/L范围内呈线性关系,线性相关系数均大于0.999。6种分析物的测定低限为0.5 μg/kg,在牛奶中3个水平的添加回收率在90.8%~107.5%之间,相对标准偏差在6.3%~11.8%之间。该方法快速简便,灵敏度高,选择性好,可用于牛奶样品中孕激素的快速定性定量分析。     

液相色谱-串联质谱法测定牛奶中伏马菌素FB1和FB2及其水解代谢物

建立了MAX混合阴离子固相萃取柱净化-高教液相色谱-串联质谱法测定牛奶中伏马菌素FB1和FB2及其水解代谢产物HFB1和HFB2的方法.牛奶样品经水稀释后,经MAX柱直接净化,甲醇洗脱得到FB1和FB2,经液相色谱-串联质谱负离子扫描测定,1％乙酸甲醇洗脱得到HFB1和HFB2,经液相色谱-串联质谱正离子扫描测定.结果表明,添加浓度为0.1～5.0 μg/L,牛奶中FB1和FB2及其水解代谢产物的回收率为76.4％～92.3％;相对标准偏差(RSD,n=5)为5.9％～12.5％;方法检出限(LOD)均为0.03 μg/L;定量限(LOQ)均为0.1 μg/L.本方法操作简单,灵敏度、回收率和重复性均良好.     

Automated stand-alone flow injection immunoanalysis system for the determination of cephalexin in milk

A fully automated stand-alone flow injection immunoanalysis (FIIA) device for the determination of cephalexin in milk is developed with a main focus on the investigation of the influence of the sample matrix. The system is based on principles of flow-through immunoassays and on sequential addition of the assay components to an immunoreactor. Protein G is immobilised on the surface of the immunoreactor serving as affinity matrix for the polyclonal anti-cephalexin antibodies. A cephalexin-alkaline phosphatase conjugate is mixed with the analyte-containing sample and binds in a competitve manner to the corresponding antibodies in the immunoreactor. After substrate addition enzymatically generated p-aminophenol is detected at a carbon electrode at +150 mV vs. Ag/AgCl. One assay cycle takes 16 min including regeneration of the immunoreactor. The large excess of protein G allows for more than 150 regenerations without significant loss of signal height. Due to the high specificity of the anti-cephalexin antibodies, other beta-lactam antibiotics like penicillin, amoxicillin and cloxacillin do not interfere in the measurements, even when added at 10 mg l-1. To deactivate alkaline phosphatase present in milk, samples are heat-treated for 3 min prior to measurements. Cephalexin recoveries from two milk samples are 90 and 110%. The detection limit in milk is 1 microgram l-1 (mean relative standard deviation of 3%), less than the maximum residue level of 4 micrograms per kg milk fixed for some beta-lactam antibiotics in the European Union. The device is suitable for fast quantitative data generation from consecutively measured samples and thus adds to analytical screening methods.     

Size fractionation of non-volatile dissolved organic compounds and metal species in German white wines by combining on-line tangential-flow multistage ultrafiltration,a home-built carbon analyser,and inductively coupled plasma mass spectrometry

Organic metal species and their size fractions in three German white wines were characterized by combining multistage ultrafiltration (MST-UF), determination of non-volatile dissolved organic carbon (NV-DOC) by a home-built carbon analyser, and metal quantification by inductively coupled plasma mass spectrometry (ICP-MS). First, NV-DOC and metal species in selected "dry" German white wines were fractionated on-line using MST-UF in the size range of >100 kDa to <1 kDa. For this purpose a 20 mL sample of the wine under study diluted 1:10 with high-purity water was processed through a cascade system of hydrophilized polyethersulfone-based flat membranes of decreasing cut-off (100, 50, 10, 5, and 3 kDa). An aliquot of the fraction <3 kDa was additionally processed through a commercial UF tube (MidGee system, cut-off: 1 kDa) to obtain low-molecular size fractions also. A home-built carbon analyser was applied to determine NV-DOC in the wines and their size fractions. The NV-DOC found in a German reference wine and its size fractions was as follows: total NV-DOC: 8.97 mg mL(-1); F(1) (>100 kDa), 0.15%; F(2) (50-100 kDa), 0.44%; F(3) (10-50 kDa), 0.74%; F(4) (5-10 kDa), 0.76%; F(5) (5-3 kDa), 0.7%; F*(6) (3-1 kDa), 0.9%; F(7) (<1 kDa), 81.6% (related to total NV-DOC). The NV-DOC recovery was 85.2%. Accordingly, most of the NV-DOC in this wine consists of low-molecular mass organic compounds of <1 kDa, presumably carboxylic acids as typical in wine. Parallel metal determinations in these wines and their fractions were performed by ICP-MS. The measurements showed that the major part of the metals investigated, up to 25 elements, were dissolved in the size fraction of <1 kDa except Ba, Sr and Pb which appeared also in other fractions. In addition, conventional UV-VIS spectroscopy was applied to characterise the studied wines and their size fractions. According to this, the UV absorbance between 254 and 280 nm of these white wines shows a parallel trend to their NV-DOC.     

气相色谱-质谱方法快速测定馏分油累积收率和性质

采用GC/MS与偏最小二乘法结合的方法测定了汽油馏分、柴油馏分、润滑油馏分、VGO馏分及渣油馏分的累积收率以及汽油馏分、柴油馏分的相对密度,建立了采用MS数据预测这5种馏分油累积收率和汽油馏分、柴油馏分相对密度的PLS校正模型。验证集结果表明：GC／MS方法与标准方法测定的结果之间无显著性差别。用GC／MS方法可以实现馏分油累积收率及性质的快速测定,与标准方法相比,大大缩短了分析时间。     

高效液相色谱-串联质谱法测定牛奶和奶粉中的三聚氰酸

建立了牛奶和奶粉中三聚氰酸(CYA)的高效液相色谱-串联质谱(HPLC-MS/MS)测定方法。样品用乙腈提取并沉淀蛋白,经强阴离子交换柱富集和净化,AX色谱柱分离,HPLC-MS/MS法测定,内标法定量。50～2000 μg/L范围内CYA的线性关系良好(r≥0.999);在奶粉和牛奶基质中,添加200、500和1000 μg/kg 3个添加水平的回收率均在97%～121%之间,相对标准偏差(RSD)均小于4.8%;定量限(LOQ)为200 μg/kg。方法的前处理快速简便,净化效果好,准确度和精密度高,可用于牛奶和奶粉中CYA的测定。     

Separation of proteins with a molecular mass difference of 2 kDa utilizing preparative double-inverted gradient polyacrylamide gel electrophoresis under nonreducing conditions: application to the isolation of 24 kDa human growth hormone

A method for separating proteins with a molecular mass difference of 2 kDa using SDS-PAGE under nonreducing conditions is presented. A sample mixture containing several human growth hormone (hGH) isoforms was initially separated on a weak anion-exchange column. Fractions rich in 24 kDa hGH as determined by analytical SDS-PAGE were pooled and further separated by cation-exchange chromatography. The fractions pooled from the cation-exchange chromatography contained two hGH isoforms with a 2 kDa molecular mass difference according to SDS-PAGE analysis, 22 and 24 kDa hGH. The 22 and 24 kDa hGH were separated using continuous-elution preparative double-inverted gradient PAGE (PDG-PAGE) under nonreducing conditions. The preparative electrophoresis gel was composed of three stacked tubular polyacrylamide matrices, a 4% stacking gel, a 13-18% linear gradient gel, and a 15-10% linear inverted gradient gel. Fractions containing purified 24 kDa hGH were pooled and Western blot analysis displayed immunoreactivity to antihGH antibodies. PDG-PAGE provides researchers with an electrophoretic technique to preparatively purify proteins under nonreducing conditions with molecular mass differences of 2 kDa.     

Study of chromium-containing proteins in subcellular fractions of rat liver by enriched stable isotopic tracer technique and gel filtration chromatography

Ten male Wistar rats were intravenously injected with a single approximately physiological dose of enriched stable isotopic Cr-50 tracer solution (200 ng (50)Cr(3+)/100 g body wt). The fundamental distribution patterns of the chromium-containing proteins in the nucleic, mitochondrial, lysosomal, microsomal, and cytosolic subcellular fractions of the rat liver were investigated by means of Sephadex G-100 gel chromatography combined with neutron activation analysis via (50)Cr (n, gamma) (51)Cr reaction. In total, nine kinds of Cr-containing proteins were found in the five subcellular fractions, whose relative molecular masses were 96.6+/-6.2, 68.2+/-1.4, 57.9+/-4.7, 36.6+/-1.2, 24.2+/-1.8, 14.0+/-1.5, 8.8+/-0.6, 6.9+/-0.4, and 4.2+/-0.4 kDa. Approximately 64.5% of Cr proteins accumulated in the cytosolic fraction. The second enriched part was the nucleic fraction; about 12.2% Cr proteins were stored in this section. The 4.2-kDa molecular mass might contain the so-called low molecular weight chromium-containing substance; however, in this research, it was only observed in the mitochondria, lysosome, and microsome. In the mitochondrial fraction, most of the Cr proteins were present as relatively low molecular weight substances: about 56% of chromium-containing proteins had molecular masses < or =6.9 kDa. Nevertheless, more than 69% of Cr-containing proteins were observed with molecular masses > or =57.9 kDa in the liver cytosolic fraction.