反相高效液相色谱法测定牛奶中的主要蛋白质

建立了测定牛奶中的主要蛋白质的反相高效液相色谱法,对前处理方法进行了优化,采用Agilent Zorbax 300SB-C8(250 mm×4.6 mm,5 μm)色谱柱,三氟乙酸-水-乙腈流动相梯度洗脱,214 nm检测,柱温45 ℃,外标法定量.测定κ-CN, αs2-CN, αs1-CN, β-CN, Whey和Igg的线性关系良好,相关系数均大于0 999,加标回收率在74.8%～132.5%之间.大豆蛋白质不影响分离与检测.采用本方法分析了9种牛奶样品中上述蛋白质的含量与总量,结果表明,本方法测定结果准确可靠.不同样品中4种酪蛋白与乳清蛋白的比例基本接近,但是不同品牌之间存在一定差异.     

毛细管区带电泳测定液态奶及奶粉中的A2 β-酪蛋白及总β-酪蛋白

β-酪蛋白（β-CN）是牛乳中的主要酪蛋白。它包含13种变异体,其中A2是牛β-CN的主要变异体之一。定量测定A2 β-CN对A2乳制品的评价十分重要。该文建立了对液态奶和奶粉中A2 β-CN及总β-CN分离并定量的毛细管区带电泳（CZE）分析方法。使用熔融石英毛细管（50 μm×30/40 cm有效/总长度）,以50 mmol/L Na₂HPO₄+140 mmol/L柠檬酸+0.2%（质量分数）HPMC+4 mol/L尿素溶液（pH为2.7）作为分离缓冲溶液,在214 nm下检测。总β-CN和A2 β-CN的校准峰面积与其质量浓度间呈良好的线性关系,相关系数在0.9968~0.9997之间,加标回收率为85.5%~106.4%。对两款巴氏奶和两款奶粉中A2 β-CN和总β-CN进行分析,A2 β-CN和总β-CN的日内精密度分别为2.4%~4.7%和2.6%~4.8%,日间精密度分别为4.0%~6.3%和3.9%~6.7%。所建立的CZE方法可分离液态奶及奶粉中的A2 β-CN,并可对A2 β-CN及总β-CN进行定量。通过计算A2 β-CN在总β-CN中的占比来评价A2乳制品的质量,为保护消费者的利益提供了方法依据。     

Determination of vegetal proteins in milk powder by sodium dodecyl sulfate-capillary gel electrophoresis: interlaboratory study

An interlaboratory study, with the participation of 8 laboratories, was conducted to evaluate a sodium dodecyl sulfate-capillary gel electrophoresis method for determination of adulteration of milk powder with soy and pea proteins. Calibration standards (0-8%, w/w, soy and pea protein in total protein) and adulterated skim milk powders (0-5%, w/w, soy and pea proteins in total protein) were produced. Vegetal proteins were determined after removal of milk proteins by pretreatment of the samples with tetraborate-EDTA buffer, pH 8.3. Repeatability standard deviations ranged from 9 to 15% and reproducibility standard deviations ranged from 25 to 30% in the samples containing 5% vegetal protein in total protein.     

Toward milk speciation through the monitoring of casein proteotypic peptides

The possibility of detecting extraneous milk in singles species cheese‐milk has been explored. A mass spectrometry (MS)‐based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the α_s1‐casein (CN) f8‐22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4‐22 peptide was selected as a marker for the two caprine α_s1‐CN A and B variants, which differ by a Pro¹⁶ (B)‐>Leu¹⁶ (A) substitution. MALDI analysis of the digest allowed the detection of α_s1‐CN f8‐22 and caprine α_s1‐CN f4‐22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)‐MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the α_s1‐CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI‐MS method. The isotopic‐label‐free quantification of isoform‐ or variant‐specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures. Copyright © 2010 John Wiley & Sons, Ltd.     

Multiresidue determination of organochlorine pesticides and polychlorinated biphenyls in milk by gas chromatography with electron-capture detection after extraction by matrix solid-phase dispersion

A multiresidue analytical method based on matrix solid-phase dispersion was developed to analyze liquid milk for 22 organochlorine pesticides (OCPs) and 6 polychlorinated biphenyls (PCBs). Initial extraction is performed by loading 3 mL milk onto a 2.0 g octadecyl (C18)-bonded silica cartridge with n-hexane as the eluant. Neutral alumina column chromatography with sodium sulfate as the drying agent is used for further cleanup. The eluate is concentrated to 0.5 mL, and target analytes are determined by capillary gas chromatography with electron-capture detection. The optimized method was validated by determining accuracy (recovery percentages), precision (repeatability and reproducibility), and sensitivity (detection and quantitation limits) from analyses of milk samples fortified at 10 and 1 microg/L levels. Average recoveries were between 74 and 106% for all residues except beta-HCH, beta-endosulfan, and endosulfan sulfate. Both repeatability and reproducibility relative standard deviation values were < 22% for all residues. Detection limits ranged from 0.02 to 0.12 microg/L and quantitation limits were between 0.02 and 0.62 microg/L. The proposed analytical method may be used as a fast and simple procedure in routine determinations of OCPs and PCBs in milk.     

Electrokinetic injection across supported liquid membranes: New sample pretreatment technique for online coupling to capillary electrophoresis. Direct analysis of perchlorate in biological samples

A simple and sensitive method for quantifying perchlorate in biological samples using CE and capacitively coupled contactless conductivity detection was developed. An online combination of a supported liquid membrane, an inert polypropylene membrane impregnated with 1-hexanol, and electrokinetic injection of perchlorate across the supported liquid membrane directly into the separation capillary reduced the need for laborious sample pretreatment procedures, resulting in a cheap and rapid method with low LODs capability. Baseline separation of perchlorate and other anions in biological samples was achieved in background electrolyte solution consisting of 15 mM nicotinic acid and 1 mM 3-(N,N-dimethylmyristylammonio)propanesulfonate at pH 3.3. The analytical method showed excellent parameters in terms of reproducibility; RSD values for peak areas and corrected migration times at a spiked concentration of 100 μg/L of perchlorate were below 10 and 0.4%, respectively. Linear calibration curves were obtained for perchlorate in the concentration range 10-1000 μg/L (r(2) >0.999) with LODs between 2 and 5 μg/L for human urine, breast milk, serum, cow's milk, and red wine. Recoveries at 25 μg/L of perchlorate were between 97 and 106% for all biological samples. The low LODs rivaling those of presently used analytical methods support the use of this method for quantification of perchlorate in biological samples in the future.     

Separation and determination of denatured caseins by hydrophobic interaction chromatography Part II. Method validation and applications

A method recently described for the separation of denatured alpha-, beta- and kappa-caseins by hydrophobic interaction chromatography was validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method is based on fast and easy solubilization of commercial and real samples by 4.0 M guanidine thiocyanate and elution on a TSK-Gel Phenyl-5PW column (TosoHaas) in the presence of 8.0 M urea in the mobile phase. No preliminary precipitation or separation of the casein fraction is required. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) was obtained over the concentration range 0.5-60 microM. The detection limits for alpha-, beta- and kappa-caseins ranged between 0.30 and 0.65 microM. The precision of the method was evaluated; the relative standard deviation for alpha-, beta- and kappa-casein determination ranged between 2.2 and 2.7% for standard solutions and between 3.5 and 6.2% for real sample solutions. The mean casein content found in 10 aliquots of BCR-063R calculated with respect to the total protein content (estimated on the basis of certified total nitrogen content) was 79.1+/-2.7%. Results of linear fitting of standard additions data for alpha-, beta- and kappa-caseins to BCR-063R were compared with linear fitting of alpha-, beta- and kappa-casein calibration data. The method was applied to commercial caseins and to 31 real, raw samples [processed cow's milk (pasteurised, UHT-treated), follow-up milk powders, cream, cheeses, casein-free infant formulae, cookies for babies containing milk proteins] with the aim of showing the wide applicability of the method in order to determine alpha-, beta- and kappa-caseins.     

Analytical performance of commercially available and unavailable phenolic compounds using real samples by high-performance liquid chromatography-diode-array detection

This work examines a novel approach of coupling high-performance liquid chromatography with diode-array detection as applied to the study of the analytical performance of the main phenolic classes contained in real samples. The chromatographic behavior of the main phenolic classes (benzoic and cinnamic acids and flavan-3-ols, flavonols and flavones as subclass of flavonoids) is carefully examined using real legume samples. Extracts from lentils proved to be excellent “real sample” because they contained the most important classes of phenolics that were subjected to current analysis. Benzoic and cinnamic acids, flavan-3-ols, flavonol and flavones were definitely or provisionally identified. Robustness, extraction efficiencies and accuracy were thoroughly scrutinized to reach quality results. Extracts showed an excellent stability according to their retention times in relative standard deviation (R.S.D.≤3%) and high reproducibility in peak area (R.S.D.≤5%). High reproducibility was also observed in calibration slopes (R.S.D.≤7%). Detection limits ranged between 0.09 and 0.64 μg/ml and, thus, showed full capacity for the determination of these compounds. Accuracy was carefully evaluated by studying extraction efficiencies and recoveries from well-known phenolic standards. The high recoveries (≥95%) obtained from the well-known phenolic structures of all representative phenolic classes allowed us to evaluate the accuracy of poorly known and commercially unavailable phenolic structures. The satisfactory analytical performance of currently employed methods had justified their recruitment for the quantification of phenolics in the study samples.     

Isotachophoretic determination of herbicides prometryne, desmetryne, terbutryne and hydroxy-derivatives of atrazine and simazine in extracts of milk

A method is described for the determination of the triazine herbicides prometryne, desmetryne, terbutryne, OH-atrazine and OH-simazine in purified extracts of milk using analytical capillary isotachophoresis. The reproducibility of isotachophoretic analyses was 3.5% and the detection sensitivity reached 2 ng. Recovery of triazines from fortified samples of homogenized full milk (0.05 mg/L) was about 65%.     

Determination of baicalein, baicalin and quercetin in Scutellariae Radix and its preparations by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection (CE-ED) was developed for the determination of baicalein, baicalin and quercetin in Scutellariae Radix and its pharmaceutical preparations. The effects of some important factors such as the acidity and concentration of running buffer, separation voltage, injection time, and the potential of working electrode were investigated to acquire the optimum condition. The working electrode was a 300-mum diameter carbon disc electrode positioned opposite the outlet of capillary. The three analytes could be well separated within 12 min in a 40 cm length capillary at the separation voltage of 12 kV in a 100 mmol l(-1) borate buffer (pH 9.0). The response was linear over three orders of magnitude with detection limits (S/N=3) ranged from 0.224 to 0.548 mumol l(-1) for all three analytes. This proposed method demonstrated good long-term stability and reproducibility with R.S.D. of less than 5% for both migration time and peak current (n=7). It has been successfully applied to analyze the actual samples with satisfactory assay results.     

Liquid chromatographic determination of maleic hydrazide in technical and formulated products: collaborative study

Fourteen collaborating laboratories assayed maleic hydrazide (MH), 6-hydroxypyridazin-3(2H)-one, in technical and formulated products by reversed-phase liquid chromatography (LC) with sulfanilic acid as an internal standard. The active MH in the samples (6 lots) ranged from 16% (expressed as the potassium salt) to 98% (MH in the technical). A small amount of 1 M KOH was added to the technical MH and analytical standards to create the potassium salt of the analyte which is soluble in water. Test samples and standards were extracted with water containing the internal standard before analysis by LC on a C8 column with an ion-pairing eluting solution and UV detection at 254 nm. The concentration of MH was calculated by comparing the peak area response ratios of the analyte and the internal standard with those in the analytical standard solution. Eleven laboratories weighed each test sample twice with single analysis. Three laboratories weighed each sample once and made duplicate injections on the LC system. The data were analyzed using the 11 laboratories' results. A second data analysis was done including all laboratory results using a Youden pair approach, selecting one of 2 duplicate assay values randomly for each laboratory and sample. In the first data analysis, the repeatability standard deviation ranged from 0.07 to 1.39%; reproducibility standard deviation ranged from 0.22 to 1.39%. In the second data analysis (using all laboratory data), repeatability standard deviation ranged from 0.09 to 0.86%; reproducibility standard deviation ranged from 0.22 to 1.31%.     

原料奶在HPLC-MS负离子模式下的指纹图谱研究

采用高效液相色谱质谱联用技术,在负离子扫描模式下建立了原料奶的乙腈提取成分的指纹图谱。采用乙腈和0.1％的乙酸为流动相进行二元梯度洗脱,柱温30℃,分析时间为85 min。确定了11个共有峰,以7号峰为参照物,通过相对峰面积和相对保留时间进行了方法学考察。结果表明,本方法具有良好的重现性,各指纹峰相对保留时间的RSD<0.79％,相对峰面积的RSD<2.84％。在原料奶指纹图谱基础上,选取有代表性有害物如防腐剂苯甲酸等进行了标准添加,建立了添加图谱,对沈阳地区超市的50个纯牛奶样品进行了筛查,取得初步应用结果。     

Development and validation of an HPLC method for the determination of ten sulfonamide residues in milk according to 2002/657/EC

A HPLC method with diode-array detection, at 265 nm, was developed and validated for the determination of ten sulfonamides (SAs): sulfadiazine (SDZ), sulfathiazine (STZ), sulfamethoxine (SMTH), sulfamethizole (SMZ), sulfamethoxypyridazine (SMPZ), sulfamonomethoxine (SMMX), sulfamethoxazole (SMXZ), sulfisoxazole (SIX), sulfadimethoxine (SDMX), and sulfaquinoxaline (SQX) in milk. A mixture of ethyl acetate, n-hexane, and isopropanol was used for the extraction of target analytes from milk. The mobile phase, a mixture of 0.1% v/v formic acid, CH(3) CN, and CH(3) OH was delivered to the analytical column under a gradient program. The procedure was validated according to the European Union regulation 2002/657/EC in terms of selectivity, stability, decision limit, detection capability, accuracy, and precision. Mean recoveries of sulfonamides from milk samples spiked at three concentration levels (0.5×MRL, 1×MRL, and 1.5×MRL) (MRL, maximum residue level) were 93.9-115.9% for SDZ, 97.8-102.9% for STZ, 94.6-107.0% for SMTH, 98.3-111.5% for SMZ, 95.3-108.4% for SMPZ, 97.9-106.0% for SMMX, 97.6-111.3% for SMXZ, 94.3-104.6% for SIX, 96.4-109.1% for SDMX, and 98.2-111.2% for SQX. All RSD values were lower than 8.8%. The decision limits CCa calculated by spiking 20 blank milk samples at MRL (100 μg/kg) ranged from 101.61 to 106.84 μg/kg, whereas the detection capability CCb ranged from 105.64 to 119.01 μg/kg.     

Determination of furosine in food products by capillary zone electrophoresis-tandem mass spectrometry

A new method for qualitative and quantitative analysis of furosine in food products by capillary electrophoresis coupled to tandem mass spectrometry (CE-MS(2) ) has been optimized and validated. In analytical conditions, the pH value of the run buffer was similar to that of the hydrolyzed samples, which helps in avoiding interactions of the analyte with silanol groups of the inner wall of the fused silica capillary. In all previous CE methods proposed in literature, no SPE treatment has been used. The method has been applied to the analysis of food products, such as flour, having low amounts of furosine. Flour samples of different origin (wheat, chestnut, lupin, einkorn, chickpeas) have been investigated. Different food products such as pasta, milk, and tigelle bread (a typical Italian unleavened bread) were also analyzed. Some of the samples have also been analyzed by reversed-phase HPLC, and when compared to CE-MS, data obtained showed good agreement. CE-MS, in comparison to HPLC, showed advantages in terms of time of analysis and cost. The validation procedure provided good values for LOD and LOQ (respectively 0.07 and 0.25 mg L(-1) ), recovery (77 and 97%), and precision investigated in terms of intraday repeatability (RSD%: 4-6% for peak areas; 1-2% for migration times) and intermediate precision (below 16% for peak areas, and below 7% for migration times). Therefore, the technique reported can be proposed as a powerful analytical method and a valid alternative to common traditional analytical techniques.     

Separation and determination of denatured alpha(s1)-, alpha(s2)-, beta- and kappa-caseins by hydrophobic interaction chromatography in cows', ewes' and goats' milk,milk mixtures and cheeses

Caseins alpha(s1)-, alpha(s2)-, beta- and kappa- from raw cows', ewes' and goats' milk were separated and determined by hydrophobic interaction chromatography (HIC) by using a Propyl column (Eichrom) in the presence of 8.0 M urea in the mobile phase. The method is based on fast and easy solubilization of real raw samples by 4.0 M guanidine thiocyanate followed by the HIC analysis, without any preliminary precipitation or separation of the casein fraction. Elution conditions have been optimized by analyzing commercial single bovine standard caseins and their mixture. In the optimized chromatographic conditions the four casein fractions were separated in less than 45 min. A linear relationship between the concentration of casein and peak area (UV absorbance detector at 280 nm) has been obtained over the concentration range of 0.5 to 40 microM. The detection limit for alpha-, beta- and kappa-caseins ranged between 0.35 and 0.70 microM. The precision of the method was evaluated, the coefficient of variation for alpha-, beta- and kappa-casein determination ranging between 3.0 and 6.0%. The method has been validated by the analysis of reference skim milk powder (BCR-063R) certificated for total nitrogen content. The method was applied to commercial casein mixture and to the qualitative and quantitative analysis of casein fractions in unprocessed, raw cows', goats' and ewes' milk (10 samples analyzed for each species), in one sample of unprocessed buffalos' milk and in commercial cheeses (mozzarella, robiola, ricotta and stracchino). Binary mixtures of milk (cow/goat and cow/ewe) were also analyzed and the ratio between casein peak areas (alpha(s1)/kappa, alpha(s2)/beta, beta/kappa and alpha(s2)/alpha(s1)) of the HIC chromatograms was proposed and discussed in order to evaluate a possible application of this method to detect milk adulteration.     

应用搅拌棒吸附萃取-热脱附-气相色谱-质谱测定烟叶和茶叶中拟除虫菊酯类农药残留

应用搅拌棒吸附萃取(SBSE)技术分别萃取烟叶和茶叶中的5种拟除虫菊酯,并利用热脱附系统将萃取到的物质进行热脱附,然后通过气相色谱-质谱联用仪(GC-MS)进行分析测定。实验过程中对影响SBSE的因素及影响热脱附的条件进行了优化。在优化条件下,采用外标法分别对烟叶和茶叶中的5种拟除虫菊酯类农药残留进行了定量分析。结果表明,烟叶中5种拟除虫菊酯的检出限范围为3.3~11.4 ng,加标回收率为94.8%~103.4%,6次测定的相对标准偏差（RSD）为5.3%~8.6%;茶叶中5种拟除虫菊酯的检出限范围为4.2~10.5 ng,加标回收率为98.2%~110.1%,6次测定的RSD为5.0%~9.6%。实验证明该法具有较高的准确度、灵敏度和较好的重现性,可用于烟叶和茶叶中拟除虫菊酯类农药残留的快速分析测定。     

Evaluation of Sample Preparation Strategies for Human Milk and Plasma Proteomics

Sample preparation is the most critical step in proteomics as it directly affects the subset of proteins and peptides that can be reliably identified and quantified. Although a variety of efficient and reproducible sample preparation strategies have been developed, their applicability and efficacy depends much on the biological sample. Here, three approaches were evaluated for the human milk and plasma proteomes. Protein extracts were digested either in an ultrafiltration unit (filter-aided sample preparation, FASP) or in-solution (ISD). ISD samples were desalted by solid-phase extraction prior to nRPC-ESI-MS/MS. Additionally, milk and plasma samples were directly digested by FASP without prior protein precipitation. Each strategy provided inherent advantages and disadvantages for milk and plasma. FASP appeared to be the most time efficient procedure with a low miscleavage rate when used for a biological sample aliquot, but quantitation was less reproducible. A prior protein precipitation step improved the quantitation by FASP due to significantly higher peak areas for plasma and a much better reproducibility for milk. Moreover, the miscleavage rate for milk, the identification rate for plasma, and the carbamidomethylation efficiency were improved. In contrast, ISD of both milk and plasma resulted in higher miscleavage rates and is therefore less suitable for targeted proteomics.     

Composition and genetic variability of proteins from nuclear fractions of mouse (DBA/2J and C57BL/6J) liver and brain

Proteins from nuclear plasma of mouse liver and brain and from the nuclear membranes of mouse liver were separated by two-dimensional electrophoresis. For the purpose of comparison, liver cytosol proteins were also investigated. The protein samples were prepared from two inbred strains of the mouse (DBA/2J, C57BL/6J) and their hybrids. The patterns obtained were compared with regard to the composition and genetic variability (qualitative and quantitative variants) of proteins from different nuclear fractions and organs. The percentage (greater than 30%) of spots common to different organs (liver, brain), but from the same nuclear fraction (plasma) was greater than the percentage (less than 20%) of spots common to different cell and nuclear fractions (cytosol, nuclear plasma and nuclear membranes) of the same organ (liver). Quantitative genetic variants occurred much more frequently than qualitative genetic variants (5.1% vs. 0.2%; liver nuclear plasma). The incidence of genetic variants was much higher in liver (5.3%) than in brains (0.0%), and higher in solubilized nuclear proteins (5.3%) than in structure-bound nuclear proteins (2.1%).     

Development and validation of an HPLC confirmatory method for the determination of seven tetracycline antibiotics residues in milk according to the European Union Decision 2002/657/EC

An HPLC method with diode-array detection, at 355 nm, was developed and validated for the determination of seven tetracyclines (TCs) in milk: minocycline (MNC), TC, oxytetracycline (OTC), methacycline (MTC), demeclocycline (DMC), chlortetracycline (CTC), and doxycycline (DC). Oxalate buffer (pH 4) was used with 20% TCA as a deproteinization agent for the extraction of analytes from milk followed by SPE. The separation was achieved on an Inertsil ODS-3, 5 microm, 250 x 4 mm(2 )analytical column at ambient temperature. The mobile phase, a mixture of A: 0.01 M oxalic acid and B: CH(3)CN, was delivered using a gradient program. The procedure was validated according to the European Union decision 2002/657/EC determining selectivity, stability, decision limit, detection capability, accuracy, and precision. Mean recoveries of TCs from spiked milk samples (50, 100, and 200 ng/g) were 93.8-100.9% for MNC, 96.8-103.7% for OTC, 96.3-101.8% for TC, 99.4-107.2% for DMC, 99.4-102.9% for CTC, 96.3-102.7% for MTC, and 94.6-102.1% for DC. All RSD values were lower than 8.5%. The decision limits CC(a) calculated by spiking 20 blank milk samples at MRL (100 microg/kg) ranged from 101.25 to 105.84 microg/kg, while detection capability CC(b )from 103.94 to 108.88 microg/kg.     

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin M1 in liquid milk: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic method for determination of aflatoxin M1 in milk at proposed European regulatory limits. The test portion of liquid milk was centrifuged, filtered, and applied to an immunoaffinity column. The column was washed with water, and aflatoxin was eluted with pure acetonitrile. Aflatoxin M1 was separated by reversed-phase liquid chromatography (LC) with fluorescence detection. Frozen liquid milk samples both naturally contaminated with aflatoxin M1 and blank samples for spiking, were sent to 12 collaborators in 12 different European countries. Test portions of samples were spiked at 0.05 ng aflatoxin M1 per mL. After removal of 2 noncompliant sets of results, the mean recovery of aflatoxin M1 was 74%. Based on results for spiked samples (blind pairs at 1 level) and naturally contaminated samples (blind pairs at 3 levels) the relative standard deviation for repeatability (RSDr) ranged from 8 to 18%. The relative standard deviation for reproducibility (RSDR) ranged from 21 to 31%. The method showed acceptable within- and between-laboratory precision data for liquid milk, as evidenced by HORRAT values at the low level of aflatoxin M1 contamination.