Chemometric deconvolution of gas chromatographic unresolved conjugated linoleic acid isomers triplet in milk samples

A generally known problem of GC separation of trans-7;cis-9; cis-9,trans-11; and trans-8,cis-10 CLA (conjugated linoleic acid) isomers was studied by GC–MS on 100 m capillary column coated with cyanopropyl silicone phase at isothermal column temperatures in a range of 140–170 °C. The resolution of these CLA isomers obtained at given conditions was not high enough for direct quantitative analysis, but it was, however, sufficient for the determination of their peak areas by commercial deconvolution software. Resolution factors of overlapped CLA isomers determined by the separation of a model CLA mixture prepared by mixing of a commercial CLA mixture and CLA isomer fraction obtained by the HPLC semi-preparative separation of milk fatty acids methyl esters were used to validate the deconvolution procedure. Developed deconvolution procedure allowed the determination of the content of studied CLA isomers in ewes’ and cows’ milk samples, where dominant isomer cis-9,trans-11 is eluted between two small isomers trans-7,cis-9 and trans-8,cis-10 (in the ratio up to 1:100).     

高效毛细管电泳法测定牛奶和奶粉中残留的三聚氰胺

建立了牛奶和奶粉中三聚氰胺的高效毛细管电泳-二极管阵列检测器(HPCE-DAD)检测方法。使用长度58.5 cm、内径75 μm的毛细管柱,分离电压25 kV,进样量3.5 kPa (35 mbar)×8 s,分离温度25 ℃,缓冲溶液20 mmol/L 柠檬酸-40 mmol/L 磷酸氢二钠(pH 2.6),检测波长232 nm。分析物在1~100 mg/L 范围内线性良好,r2>0.997;牛奶和奶粉的定量限分别为0.5 mg/kg和1.0 mg/kg。在添加水平为定量限浓度至50 mg/kg时的回收率为72.2%~97.3%,相对标准偏差为2.1%~3.9%。     

Profiling and quantifying polar lipids in milk by hydrophilic interaction liquid chromatography coupled with evaporative light-scattering and mass spectrometry detection

In this work, a high-performance liquid chromatography with evaporative light scattering detection method has been developed and applied for quantification of the polar content of the lipid fraction in milk samples of different origin. From a chromatographic stand-point, a 4.6-mm I.D. hydrophilic interaction liquid chromatography column was employed to attain a baseline separation of major phospholipid classes contained in the various milk samples tested. Quantitative analysis was performed by the external calibration method using reference material solutions in the 5–100 mg/L concentration range. Analytical recoveries ranging from 57 to 100 %, and repeatability data lower than 8.04 % were obtained on a skimmed cow’s milk sample. The crude cow milk was the most abundant (0.04 %) in phospholipids and donkey milk was the poorest (0.004 %). Quantitative differences were determined in the phospholipid content of the milk samples tested. Finally, characterization of phospholipid profile and fatty acid composition of the different samples was carried out by an ion trap-time of flight mass spectrometer and gas chromatography coupled to flame ionization and mass spectrometry detection. A thorough screening of the polar lipid composition of milk samples of different origin is here outlined, for the first time.     

Improved determination of milk oligosaccharides using a single derivatization with anthranilic acid and separation by reversed-phase high-performance liquid chromatography

An improved analytical scheme for human milk neutral oligosaccharides determination was developed, in which, the oligosaccharides were pooled in two fractions (pools 1 and 2) after gel filtration, and then were quantitatively derivatized with a single fluorescent reagent, 2-anthranilic acid. Separation was by reversed-phase HPLC on an ODS-100Z column with a mobile phase of 50 mM ammonium acetate pH 4.0 and 150 mM citrate buffer pH 4.5 and monitored by a fluorescence detector at 360 nm excitation and 425 nm emission wavelengths. The method improved on the separation of neutral tetra- and hexa-saccharide isomers, namely, lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT) as well as of lacto-N-difucohexaose I (LNDFH I) and lacto-N-difucohexaose II (LNDFH II). The separation of trisacccharide isomers, 3-fucosyllactose (3-FL) and 2′-fucosyllactose (2′-FL) was also successful. Limits of detection and quantification were in the range of 1–10 ng/l and 2–30 ng/l, respectively. The methods’ accuracy was good with its precision at <20% RSD and <1% RSD, respectively, for oligosaccharide concentration and retention time. The recoveries were in the range of 80–100%. This method was successfully applied to the separation and determination of representative neutral oligosaccharide contents in Samoa women milk.     

Quantification of cow milk adulteration in goat milk using high-performance liquid chromatography with electrospray ionization mass spectrometry

A method was developed for the quantification of cow milk adulteration in goat milk, based on solvent separation of whey proteins followed by high-performance liquid chromatography with electrospray ionization mass spectrometry (HPLC/ESI-MS). The presence of cow milk was determined using beta-lactoglobulin whey protein as the molecular marker. The adulterants were identified using both retention time and molecular mass derived from multiply charged molecular ions. Standard solutions containing cow and goat milk in different volume ratios were prepared and analyzed. Good linearity covering cow milk content from 5% and above was obtained. The proposed method identifies the adulterants using accurate molecular masses for protein identification and detects the addition of cow milk to goat milk at levels as low as 5%.     

高效液相色谱-串联质谱检测牛奶中的甲状腺素

建立了高效液相色谱-串联质谱(HPLC-MS/MS)检测牛奶中甲状腺素3,3′,5,5′-四碘-L-甲腺原氨酸(T4), 3,3′,5-三碘-L-甲腺原氨酸(T3)和3,3′,5′-三碘-L-甲腺原氨酸(rT3)的方法。样品用乙腈提取,离心,上清液经氨水碱化和Cleanert PAX固相萃取小柱净化,在Zorbax Eclipse XDB-C18 (150 mm×2.1 mm, 3.5 μm)色谱柱上以0.1%(v/v)乙酸水溶液和甲醇为流动相等度洗脱分离,以电喷雾正离子(ESI+)模式电离,多反应监测(MRM)模式质谱检测,内标法定量。结果表明,甲状腺素的检出限(LOD)不大于0.03 ng/g,定量限(LOQ)不大于0.1 ng/g;在考察的浓度范围内线性关系良好(r2＞ 0.998);回收率为80.61%～101.7%,相对标准偏差(RSD)为1.48%～9.70%。室温下样品溶液中的甲状腺素保持稳定。对5个牛奶样品的测定结果显示,T3含量为0.59～1.30 ng/g, RSD为2.06%～7.70%; T4和rT3未检出。该方法具有样品处理简单,灵敏度高,重现性好,定性和定量结果可靠等特点,为牛奶中甲状腺素的测定和相关质量安全评价提供了可靠手段。     

高效液相色谱法检测液态奶中的苯酚类和水杨酸苯胺类抗蠕虫药

采用高效液相色谱法建立了硝碘酚腈、氯羟柳胺、氯氰碘柳胺、碘醚柳胺在液态奶中的多残留检测方法。取5 g样品,用含1%(v/v)三乙胺的乙腈提取,经MAX柱净化。以乙腈和0.02 mol/L乙酸铵水溶液(pH 4.0)作为流动相,经C18反相色谱柱分离后用紫外检测器检测,外标法定量。结果表明:空白加标奶样中4种药物在5~500 μg/kg范围内均呈现良好的线性关系,相关系数均大于0.99。空白加标奶样品的检出限(LOD)为3 μg/kg,定量限(LOQ)为5 μg/kg。硝碘酚腈、氯羟柳胺、氯氰碘柳胺、碘醚柳胺在1/2最高残留限量(MRL)、1倍MRL、2倍MRL添加水平下的平均回收率和相对标准偏差分别是92.20%~96.13%和5.55%~16.30%; 87.40%~94.74%和5.40%~12.21%; 86.97%~91.09%和2.67%~8.17%; 77.86%~95.36%和5.02%~13.15%。表明该检测方法简单,灵敏,适用于液态奶中水杨酸苯胺类多残留的定量分析检测。     

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。     

Direct copper determination in whole milk, non-fat milk and whey milk by electrothermal atomic absorption spectrometry

A method for the direct determination of copper in samples of whole milk, non-fat milk and whey milk by electrothermal atomic spectrometry (ETAAS) was studied. The fat separation by centrifugation at 3200 rpm and the separation of casein mycelles to obtain the whey milk by ultracentrifugation at 31 000 g were investigated. In all cases Mg(NO₃)₂ was used as chemical modifier and Triton X-100 (0.2% w/v) as emulsifying agent. The optimum pyrolysis temperature was 1500° C. The detection limit was 0.4 μg/l of copper. The precision was studied for the whole milk and the coefficients of variation (CV) were 5.7, 4.0, 2.4 and 2.8% for 0, 5, 10 and 20 μg/l of copper added. The accuracy was determined by using the Reference Material Milk A-11 (IAEA) with a certified content of 378.4 ± 24 ng Cu/g; 359 ± 16 ng/g were found. The method was applied to ten cow milk samples, the levels of copper being determined for whole milk, non-fat milk and whey milk. A statistical study was applied and it was concluded that the majority of copper is in the non-fat milk. Received: 29 February 1996 / Revised: 19 April 1996 / Accepted: 28 April 1996     

Determination of sulfonamides in milk after precolumn derivatisation by micellar liquid chromatography

A simple method to identify and determine six sulfonamides (sodium sulfacetamide, sulfamethizole, sulfaguanidine, sulfamerazine, sulfathiazole and sulfamethoxazole) in milk by micellar liquid chromatography (MLC) is reported. The assay makes use of a precolumn diazotisation-coupling derivatisation including the formation of an azo dye that can be detected at 490 nm. Furthermore, the use of MLC as an analytical tool allows the direct injection of non-purified samples. The separation was performed with an 80 mM SDS-8.5% propanol eluent at pH 7. Analysis times are below 16 min with a complete resolution. Linearities (r > 0.9999), as well as intra- and inter-day precision (below 2.7%), were studied in the validation of the method. The limits of detection and quantification ranged from approximately 0.72 to 0.94 and 2.4 to 3.1 ng mL⁻¹, respectively. The detection limit was below the maximum residue limit established by the European Community. Finally, recoveries in spiked milk samples were in the 83-103% range.     

亲水作用色谱-串联质谱法测定乳及乳制品中左旋肉碱

建立了亲水作用色谱-串联四极杆质谱测定乳及乳制品中左旋肉碱的分析方法。样品经2%(v/v)乙酸水溶液提取、乙腈沉淀蛋白质净化,以乙酸铵和乙腈为流动相,经Acquity UPLC BEH HILIC色谱柱分离后采用电喷雾质谱多反应监测(MRM)方式扫描,外标法定量。结果表明,左旋肉碱的质量浓度在1~100 μg/L范围内线性关系良好(r²> 0.99),定量限为0.01 mg/kg。标准加入法测定左旋肉碱在高、中、低3个加标水平的回收率为96.0%~103.4%,相对标准偏差(RSD)为1.2%~4.3%。该方法前处理简单、快速,检测结果准确、灵敏,可为各类乳及乳制品中左旋肉碱的含量水平测定、研究和控制提供技术支持。     

气相色谱/三重四极杆串联质谱法测定牛奶及奶粉中213种农药多残留

在系统优化固相萃取吸附剂填料类型、洗脱溶剂种类及体积的基础上,建立了牛奶和奶粉中213种农药残留的气相色谱-三重四极杆串联质谱(GC-MS/MS)方法。试样用乙腈均质提取,采用石墨化炭黑/氨基柱(ENVI-Carb/NH₂)净化后,用GC-MS/MS多反应离子监测(MRM)模式进行检测,外标法定量。结果表明,197种农药在10~1000 μ g/L,16种农药在50~1000 μ g/L范围内线性关系良好,相关系数均大于0.99,方法的检出限(S/N=3)为0.03~7.59 μ g/kg,定量限(S/N=10)为0.10~21.94 μ g/kg,平均添加回收率为66.9%~120.1%,相对标准偏差(RSD)为1.23%~17.6%。该方法样品处理简单快速,相比其他多残留分析方法净化效果好,灵敏度和选择性高,适用于日常检测工作。     

Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context

The ability to quantify the casein content by an exact and cost-effective approach represents an issue of crucial importance in the dairy industry as the natural variations in milk protein concentration can markedly affect the yield of the cheesemaking processes, thus causing a direct and significant economic impact on the producers. In this work, the separation and quantification of α_s1-, α_s2-, κ- and β-casein was carried out by direct RP-HPLC analysis of milk. The identification of each casein was established by electrospray ionization mass spectrometry. The data show that this method is able to effectively separate the bovine casein fractions, it provides simplified analytical conditions (with special regard to mobile phase composition and gradient profile) and faster separation while ensuring adequate precision to achieve reliable quantifications in milk samples from dairy production.     

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。     

Determination of ammonium in milk and dairy products by ion chromatography

To control the quality and the biochemical evolution of milk and dairy products during their technological transformations, it is interesting to determine their ammonium concentrations. A chromatographic method for the determination of this compound is proposed. The method is based on the separation of ammonium by cation-exchange chromatography and its detection by suppressed conductivity. With an appropriate sample preparation, this method enabled identification and quantification of ammonium with good repeatability (relative standard deviation of about 5%). Moreover, good sensitivity (less than 0.5 mg/kg) and no interference between ammonium and other matrix components were determined. It was also shown that this method offers a very promising alternative for studying changes in ammonium concentration of milk or caseinate after their heat treatments and in different dairy products such as yoghurt and cheeses (hard cooked and mould ripened cheeses).     

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。     

Analytical Method for Quantification β-Cyclodextrin in Milk, Cream and Butter by LC

A simple, rapid and sensitive liquid chromatographic method using a refractive index detector has been developed and validated for identification and quantification of β-cyclodextrin in milk, cream and butter. The chromatographic system consists of a YMC ODS-AQ column packed with C18 reversed phase silica packing material as stationary phase using a mixture of methanol and water 7:93% (v/v) as the mobile phase. Linearity was established for the β-cyclodextrin concentration in the range 0.01–4 mg mL^?1, with a coefficient (r) of 0.9998. Repeatability of the method was assessed; the coefficient of variation for β-cyclodextrin range were 1.24, 3.01, and 5.36% for milk, cream and butter. Recoveries ranged between 99.91 and 94.53%. This method was highly reproducible and reliable for quantification of treated milk with β-cyclodextrin for removal cholesterol from the milk fat.     

MALDI-MS and multivariate analysis for the detection and quantification of different milk species

The extensive consumption of milk and dairy products makes these foodstuffs targets for potential adulteration with financial gains for unscrupulous producers. Such practices must be detected as these can impact negatively on product quality, labelling and even health. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) is a potentially useful technique, with proven abilities in protein identification and more recently through the use of internal standards for quantification purposes of specific proteins or peptides. In the current work, we therefore aim to explore the accuracy and attributes of MALDI-ToF-MS with chemometrics for the detection and quantification of milk adulteration. Three binary mixtures containing cows' and goats', cows' and sheep's, and goats' and sheep's milk and a fourth tertiary mixture containing all types of milk were prepared and analysed directly using MALDI-ToF-MS. In these mixtures, the milk concentrations of each milk varied from 0% to 100% in 5% steps. Multivariate statistical methods including partial least squares (PLS) regression and non-linear Kernel PLS regression were employed for multivariate calibration and final interpretation of the results. The results for PLS and KPLS were encouraging with between 2% and 13% root mean squared error of prediction on independent data; KPLS slightly outperformed PLS. We believe that these results show that MALDI-ToF-MS has excellent potential for future use in the dairy industry as a rapid method of detection and enumeration in milk adulteration.     

Quantitative separation of oxytocin,norfloxacin and diclofenac sodium in milk samples using capillary electrophoresis

A simple, sensitive and rapid method has been developed for simultaneous separation and quantification of three different drugs: oxytocin (OT), norfloxacin (NOR) and diclofenac (DIC) sodium in milk samples using capillary electrophoresis (CE) with UV detection at 220 nm. Factors affecting the separation were pH, concentration of buffer and applied voltage. Separation was obtained in less than 9 min with sodium tetraborate buffer of pH 10.0 and applied voltage 30 kV. The separation was carried out from uncoated fused silica capillary with effective length of 50 cm with 75 µm i.d. The carrier electrolyte gave reproducible separation with calibration plots linear over 0.15–4.0 µg/mL for OT, 5–1000 µg/mL for NOR and 3–125 µg/mL for DIC. The lower limits of detection (LOD) were found to be 50 ng/mL for OT, and 1 µg/mL for NOR and DIC. The method was validated for the analysis of drugs in milk samples and pharmaceutical preparations with recovery of drugs within the range 96–100% with RSD 0.9–2.8%. Copyright © 2009 John Wiley & Sons, Ltd.     

Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants

A new RP-HPLC method for the separation and quantification of the most common genetic variants of bovine milk proteins is described. A reversed-phase analytical column C8 (Zorbax 300SB-C8 RP, 3.5 microm, 300A, 150 x 4.6 I.D.) was used. All the most common casein (CN) and whey protein genetic variants, including beta-CN(I) were detected and separated simultaneously in less then 40 min, with the exception of alpha(S1)-CN(B) and CN(C) variants. Purified protein genetic variants were employed in calibration and showed different absorbances at 214 nm. The procedure was developed using 40 raw individual milk samples of cows belonging to four different breeds and certified skim milk powder BCR-063R. Method validation consisted in testing linearity, repeatability, reproducibility and accuracy. A linear relationship (R(2)>0.99) between the concentrations of proteins and peak areas was observed over the concentration range, with low detection limits. Repeatability and reproducibility were satisfactory for both retention times and peak areas. The RSD of peak areas ranged from 0.92 to 4.32% within analytical day and from 0.85 to 9.52% across analytical days. The recoveries, calculated using mixtures of samples previously quantified, ranged from 98.1 to 103.7%.