Detection of cow's milk in ewe's or goat's milk by HPLC

Summary High-pressure liquid chromatography (HPLC) was used to detect added cows' milk in goat and ewe's milk. Analysis of the whey proteins enabled detection of mixtures in proportions of less than 1%. The method does not distinguish between goat and ewe's milk.     

Silicon determination in milk by electrothermal atomic absorption spectrometry using palladium as chemical modifier

A direct method for silicon determination in milk samples by Electrothermal Atomic Absorption Spectrometry was developed. Palladium was used as chemical modifier at a concentration of 610 mg L(-1); with this modifier, silicon was stable up to 1800 degrees C. The precision and accuracy of the method were investigated. The detection limit was 16.2, 2.7 and 7.2 micro g L(-1) for cows' milk, human milk and infant formula, respectively. The method was applied to silicon determination in 17 infant formula samples, 13 human milk samples and 12 cows' milk samples.     

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.     

Detection of streptomycin and dihydrostreptomycin residues in milk,honey and meat samples using an optical biosensor

Immuno-biosensor inhibition assays for the detection of streptomycin and dihydrostreptomycin residues in whole cows' milk, honey, pig kidney and pig muscle are reported. The antibody showed high cross-reactivity with dihydrostreptomycin in various foodstuffs (buffer 103%, milk 96%, honey 84%, kidney extract 129% and muscle extract 98%). There was no significant cross-reaction with other aminoglycosides or commonly used antibiotics. A streptomycin derivative was used to prepare a stable, reusable sensor chip surface. The assay allowed the direct analysis of bovine whole milk (fat content approximately 3.5%). Honey samples required dilution with buffer, while kidney and muscle samples from pigs were homogenized in an aqueous extraction buffer and clarified by centrifugation. The limit of detection for each assay was determined from known streptomycin-free samples (n = 20; mean - (3 x standard deviation)) and the results were as follows: milk 30 microg kg(-1), honey 15 microg kg(-1), kidney 50 microg kg(-1) and muscle 70 microg kg(-1). Repeatability (or relative standard deviation) between runs were calculated (n = 3) at the respective Community maximum residue limits (MRL) and 0.5 x MRL with the exception of honey since no European MRL exists at present. Results were determined as 4.3% (200 microg kg(-1)) and 2.8% (100 microg kg(-1)) in milk, 13.3% (40 microg kg(-1)) and 9.5% (20 microg kg(-1)) in honey, 7.1% (1000 microg kg(-1)) and 7.6% (500 microg kg(-1)) in kidney and 7.1% (500 microg kg(-1)) and 11% (250 microg kg(-1)) in muscle.     

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.     

Validation of the Charm MRL-3 for fast screening of beta-lactam antibiotics in raw milk

The biochemicals utilized in the Charm MRL beta-Lactam test (8 min test) were applied to faster flowing lateral components to create a new 3 min, one-step beta-lactam test called Charm MRL-3 (Charm Sciences Inc., Lawrence, MA). This new test was validated at T&V-ILVO according to Commission Decision 2002/657/EC. The following analytical parameters were checked: test specificity, detection capability, and test robustness (impact of deviation of the test protocol, and impact of the milk composition, batch differences of reagents). Further, the suitability of the Charm MRL-3 to screen heat-treated milk or milk from animal species other than the cow was also tested. Finally, the test was integrated in the monitoring of dairy samples to check the occurrence of false-negative or false-positive results, and the test was also included in a national ring trial and an international proficiency study. The results proved that the Charm MRL-3 is a fast, simple, and reliable cows' milk test that can be used at the farm level in order to prevent tanker milk contamination, or at the entrance of the dairy plant to screen tanker milk for the presence of beta-lactam antibiotics.     

Determination of oxolinic acid in cow's milk and human urine by means of a single-use phosphorimetric sensor

A single-use phosphorimetric drop plane sensor for the determination of oxolinic acid (OXA) is proposed. The sensor was formed by a 30x16 mm(2) rectangular strip of Mylar type polyester as solid support that contained a circular sensing zone, 6 mm in diameter and 20 mum in thickness, formed by PVC plasticized with tributylphosphate adhered to its surface. When the strip was introduced for 1 hour into a sample solution, the analyte was retained in the sensing zone, making it possible to directly measure the phosphorescence intensity emitted by the OXA in the solid phase, at lambda(exc)=330 nm and lambda(em)=449 nm. The variables that affect the construction of the sensor have been studied, along with the experimental variables that influence the fixation of the analyte in the sensor. The method's detection limit was 0.01 mg l(-1) with an applicable concentration range from 0.04 to 1.50 mg l(-1) and a repeatability of 2.6% at the concentration range of 0.8 mg l(-1). The method was applied to samples of human urine and cows' milk, with recovery percentages ranging between 97.6 and 108.7%.     

Preconcentration of milk proteins using octadecylated monolithic silica microchip

Sample preparation is a bottleneck in systems for chemical analysis and it is a required step in order to remove interference and preconcentrate the target analytes. Much research in recent years has focused on porous monolithic materials since they are highly permeable to liquid flow and show high mass transfer compared with common packed beds. This study has focused on the use of a glass microchip containing an inorganic silica-based monolithic material modified with octadecyl groups for preconcentration of milk proteins from skimmed cows’ milk that vary in molecular weight, hydrophobicity, and abundance. Comparison between the fabricated device and a commercial cartridge for the preconcentration of proteins in skimmed cows’ milk showed the ability of the device to successfully enrich protein mixtures from the sample. The three replicate experiments showed that the RSD of the mass to charge ratio of milk proteins ranged from 0.01 to 0.46%. In addition, it was found that there were no significant differences between the observed and reported masses of the milk proteins and the relative percentage error of the molecular masses ranged between 0.03 and 0.90%. The fact that the small amounts of sample required and short sample preparation time suggest that this new microfluidic device may be a viable alternative to existing procedures for protein extraction from real samples.     

Distribution of radionuclides in the environment in northern Italy after the Chernobyl accident.

Soon after the Chernobyl nuclear accident, the air-pumping stations in Pavia (northern Italy) were alerted. In a few days, a rapid increase in radionuclide concentration in air particulates was observed. Consequently, an environmental radioactivity monitoring programme was started in which several matrices such as soil, grass, vegetables and cows' milk were subjected to direct gamma-ray spectrometry. The radioactivity distribution and its variation with time is presented, discussed and compared with other available data. Detection limits, precision and accuracy are also reported, and depth profiles in soils for 137Cs are presented and correlated with soil quality parameters. A survey of environmental radioactivity in soil, in a search for residual Chernobyl fallout, was carried out and a map of the 137Cs distribution over a large area in northern Italy is presented and discussed.     

Determination of oxytetracycline in bovine kidney and medicated milk replacer by liquid chromatography

Improvements and optimization of AOAC INTERNATIONAL Official Method 995.09 for the detection of oxytetracycline in bovine kidney at the new U.S. tolerance of 12 ppm are reported. Recoveries from kidney fortified at 4 concentrations over the range of 3-40 ppm averaged 84-98%. Results from the kidney of a calf fed medicated milk replacer containing oxytetracycline are also reported. Additionally, adaptation of this method to the detection of oxytetracycline in medicated milk replacer is discussed.     

Determination of ultrafiltrable zinc in human milk by electrothermal atomic absorption spectrometry.

Percentages of non-protein-bound zinc in human milk have been reported by different workers, but ultrafiltration and zinc determination in human milk have not been comprehensively examined. However, zinc contamination and zinc membrane binding have been described for the determination of non-protein-bound zinc in serum. In this work, ultrafiltration was studied in terms of zinc contamination and zinc membrane binding. An MPS-1 micropartition system fitted with a YMT membrane was used. Zinc contamination was found to be less than 276 nmol dm-3 and the zinc recovery was 85 +/- 4%. The conditions for electrothermal atomic absorption spectrometry were also studied. The detection limit was found to be 26.4 nmol dm-3 and the upper linear range was 4 mumol dm-3. The precision varied from 3% (within-run) to 17% (between-run). The recovery of standard additions was 95 +/- 7% (n = 30, different human milk ultrafiltrate samples). Physiological values varied from 0.46 to 84 mumol dm-3 (4-56% of zinc in whole human milk). Expressed in mumol dm-3, zinc in human milk ultrafiltrate decreased slightly through the lactation period, whereas expressed as a percentage of the total zinc in milk, zinc in human milk ultrafiltrate remained constant from day 2 to day 69 post partum.     

Distribution of strontium in milk component

The distribution of strontium between the milk components, i.e., serum, casein micelles, whey and hydroxyapatite was determined. The sorption on hydroxyapatite was investigated using batch method and radiotracer technique. The aqueous phase comprised of either milk or whey. The sorption of strontium on hydroxyapatite depended on the method of its preparation and on the composition of the aqueous phase. The sorption of strontium was increased with an increase of pH. The presence of citrate species resulted in decrease of the sorption of strontium on hydroxyapatite. The sorption of ⁸⁵Sr on hydroxyapatite decreased with the increasing concentration of Ca²⁺ ions. Addition of Ca²⁺ ions to milk resulted in milk pH decrease. The decrease in pH value after calcium addition to milk is related to exchanges between added calcium and micellar H⁺. The average value of strontium sorption on casein micelles in milk with presence of hydroxyapatite was (47.3 ± 5.6) %. The average value of sorption of ⁸⁵Sr on casein micelles in milk without the addition of hydroxyapatite was (68.9 ± 2.2) %.     

Gas-liquid chromatographic determination of milk fat and cocoa butter equivalents in milk chocolate: interlaboratory study

A collaborative trial was conducted to validate an analytical approach comprising method procedures for determination of milk fat and the detection and quantification of cocoa butter equivalents (CBEs) in milk chocolate. The whole approach is based on (1) comprehensive databases covering the triacylglycerol composition of a wide range of authentic milk fat, cocoa butter, and CBE samples and 947 gravimetrically prepared mixtures thereof; (2) the availability of a certified cocoa butter reference material for calibration; (3) an evaluation algorithm, which allows reliable quantitation of the milk fat content in chocolate; (4) a subsequent correction to take account of the triacylglycerols derived from milk fat; (5) mathematical expressions to detect the presence of CBEs in milk chocolate; and (6) a multivariate statistical formula to quantitate the amount of CBEs in milk chocolate. Twelve laboratories participated in the validation study. CBE admixtures were detected down to a level of 0.5 g CBE/100 g milk chocolate, without false-positive or -negative results. The applied quantitation model performed well at the statutory limit of 5% CBE addition to milk chocolate, with a prediction error of 0.7%, and HorRat values ranging from 0.8 to 1.5. The relative standard deviation for reproducibility (RSDR) values for quantitation of CBEs in analyses of chocolate fat solutions ranged from 2.2 to 3.8% and for analyses of real chocolate samples, from 4.1 to 4.7%, demonstrating that the whole approach, based solely on chocolate fat blends, is applicable to real milk chocolate samples.     

Spreeta-based biosensor immunoassays to detect fraudulent adulteration in milk and milk powder

Biacore biosensors (Biacore AB, Uppsala, Sweden) have proven to be robust analytical tools for the automated immunochemical detection of different adulterants and contaminants in milk and milk powder. However, the significant cost of the instruments is a disincentive for their wide application in food control laboratories. Therefore, a low-cost alternative optical biosensor (Spreeta, Texas Instruments, Attleboro, MA) was built into an affordable liquid handling system. Using this prototype biosensor, an inhibition immunoassay for bovine K-casein was evaluated for the detection of cow's milk in ewe's and goat's milk and for the detection of bovine rennet whey powder in milk powder. Comparable sensitivities were obtained for both adulterants in the Spreeta-based prototype biosensor and a Biacore 3000 instrument. The limit of detection for cow's milk was 0.17% (v/v) and bovine rennet whey powder could be detected in milk powder above 1% (w/w). The Spreeta sensor was also useful in the control of fraudulent water additions to milk, simply by measuring differences in the bulk response.     

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     

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%.     

Raman spectroscopic quantification of milk powder constituents

Raman spectroscopy has significant potential for the quantification of food products. Milk powder is an important foodstuff and ingredient that is produced on large scale (over 20 million tonnes per annum). Raman spectroscopy, unlike near- and mid-infrared spectroscopies, has not been used extensively to quantify milk powder constituents. The effect of sample presentation on spectroscopic calibrations of protein and fat for 136 New Zealand milk powders was assessed using Raman spectroscopy. Prediction models were produced to quantify a protein concentration range of 32.19-37.65% w/w for skim milk powder, and a protein concentration range of 23.34-25.02% w/w and a fat concentration range of 26.26-29.68% w/w for whole milk powder (where ratios of prediction to deviation exceeded 2.6 with one exception). The resultant calibrations were not influenced by sample orientation; the sample temperature during data collection did affect the calibrations. Calcium fortification in the form of calcium carbonate was identified within a sub-set of samples, reinforcing the efficacy of Raman spectroscopy for identifying both crystalline and non-crystalline constituents within milk powder.     

The routine analysis of some specific isomers of polychlorinated biphenyl congeners in human milk

A mixture of 14 polychlorinated biphenyl (PCB) isomers of various congeners, representing approximately 70% of all human milk PCBs reported in the literature, was made up to identify and approximate their residues in human milk. Individual isomer levels varied from 5 to 103 nanograms per gram of milkfat with the 2,2',4,4',5,5'-hexachlorobiphenyl isomer as the major PCB contaminant of human milk. PCB isomer numbers 74, 118, 153, 138 and 180 made up approximately 75% of all PCBs as measured by this mixture. There was close agreement of total PCB isomer content in breast milk between electron capture gas chromatography and gas chromatographic mass spectrometry determinations. A major interference was encountered however for PCB isomer no. 52, whose residue level in the breast milk was approximately 3 X higher by gas liquid chromatography than by mass spectrometry.     

超高效液相色谱-串联质谱法测定牛奶和奶粉中残留的左旋咪唑

建立了一种专属、灵敏的超高效液相色谱-串联质谱(UPLC-MS/MS)测定牛奶和奶粉中左旋咪唑残留量的方法。在碱性环境下用乙酸乙酯超声波提取试样中的左旋咪唑,再经稀盐酸反提取、强阳离子交换(SCX)固相萃取柱净化,采用BEHC18超高效液相色谱柱、乙腈-0.1%甲酸(体积比为15∶85)流动相分离,以电喷雾离子源正离子检测方式进行质谱分析。实验结果表明,在2.0～100.0 μg/L浓度范围内左旋咪唑呈良好的线性关系,其相关系数r=0.997。在低、中、高3个浓度添加水平下,左旋咪唑的回收率为64.5%～102.0%,相对标准偏差小于13.1%。牛奶中左旋咪唑检出限为0.4 μg/kg,奶粉中左旋咪唑检出限为2.0 μg/kg。     

Determination of selected persistent organochlorine pollutants in human milk using solid phase disk extraction and narrow bore capillary GC-MS

Summary A simple and rapid procedure based on solid phase disk extraction (SPDE), adsorption chromatography on acidified silica gel and GC-MS analysis was developed for the determination of 8 organochlorine pesticides and 19 PCB congeners in human milk. By using the SPDE procedure, a high throughput and parallel sample processing could be achieved. Method variables were optimized on whole cow's milk (3.5% fat) fortified at levels close to concentrations found in human milk. Recoveries of target analytes were acceptable and ranged from 69 to 102% and 86 to 120% for whole and skimmed milk, respectively. By the use of two stage clean-up and narrow bore capillary columns, detection limits as low as 20 pg mL⁻¹ could be obtained. The method was used for the determination of organochlorine pollutants in human milk from 19 individuals from Romania. The concentrations of PCBs were low, whereas those of organochlorine pesticides were higher than the values reported from other European countries.