Significance of minor components of milk fat

In order to establish criteria for the evaluation of the genuineness of milk fat and its derivatives, research has been carried out on some minor components (mono- and diglycerides and cholestrol esters). Most determinations of milk fat diglycerides showed significant qualitative and quantitative differences compared with other fat sources, and especially with tallow. In particular, the milk fat diglycerides C30–C32–C34–C36 showed characteristic fingerprints. Moreover, the sterol fractions (free and esterified) have also been evaluated. The cholesterol ester profile in milk lipids includs fatty acides from 10 to 18 carbon atoms, while tallow basically showed only C14–C16–C18. In comparison, phytosteral esters with C16 and C18 fatty acids have been found in vegetable fats. These results lead to the hypothesis of possible use of these methods in addition to those most commonly applied (TG-FAME). Furthermore, the natural variability of milk fat components makes a final evaluation of these methodologies difficult unless experimental work is extended to more samples and performed by several research groups.     

Programmable Temperature Vaporizer (PTV) Applied to the Triglyceride Analysis of Milk Fat

The PTV (Programmable Temperature Vaporizer) is a split/splitless injector which allows the sample to be introduced at a relatively low temperature, thus affording accurate and reproducible sampling. After injection the PTV is rapidly heated to transfer the vaporized components into the capillary column. This type of injector has proved to be an efficient tool for the evaluation of fatty acids, essential oils, and pesticides in food analysis. In this work the suitability of PTV for the analysis of milk fat purity by the Official EU method was evaluated. This method is based on the gas chromatographic determination of triglycerides only according to their total number of carbon atoms followed by the application of formulae deriving from multiple linear regressions. The analysis is currently carried out with a packed column or a short capillary column and an on-column injection system. Several samples of pure milk fat and mixtures of milk fat with foreign fat were analyzed with the same capillary column and by using both PTV and on-column injection systems. The results show that the gas chromatographic profile obtained by PTV is comparable with that obtained by the on-column injector, while repeatability and reproducibility data meet the requirements indicated in the Official Method. Therefore, this study demonstrates that it is possible to use the PTV instead of the on-column injector to determine the purity of milk fat with this method.     

A chemometric approach to the detection of milk adulteration based on protein profiles determined by capillary electrophoresis

The general objective of this study was to utilize chemometrics in the interpretation of capillary electrophoresis milk protein profiles, for the detection of pasteurized milk adulteration with rehydrated milk powder or a rehydrated dairy-based milk substitute. The specific objectives were 1) to collect quantitative data on major casein and whey proteins in authentic and adulterated milks in a single CE analysis; and 2) to apply a pattern recognition procedure, Soft Independent Modeling of Class Analogies (SIMCA), on collected CE protein data, for the development of a statistical model useful in the detection of pasteurized milk adulteration. Authentic samples were fresh milk collected from various farms over a period of six months. Adulterated samples were authentic fresh milk partially or totally substituted with rehydrated milk powder or a rehydrated commercial milk substitute at different levels. Quantitative protein data obtained by capillary free zone electrophoresis for beta-lactoglobulin, alpha-lactalbumin, beta-casein, and alpha-casein of 86 samples, authentic and adulterated samples, were used as a training set to build a SIMCA multivariate statistical model. The detection of sample outliers was useful for the elimination of unusual samples and optimization of the multivariate model. From the 35 commercial pasteurized milks tested, which were treated as unknowns, a total of 14 samples (40%) were not assigned to the authentic or fresh milk group, meaning that these samples had some type of adulteration at the levels included in the training set (> 15%). Decision-making on detecting adulteration of unknown commercial pasteurized milk samples was eased since predictions were based on statistical probabilities.     

Development and validation of a solid-phase extraction and gas chromatography-tandem mass spectrometry method for the determination of isopropyl-9H-thioxanthen-9-one in carton packaged milk

A simple and efficient method for the determination of isopropyl-9H-thioxanten-9-one (ITX) in different fat content milk samples and baby milk samples stored in packaged cartons was developed and validated. Samples were extracted using solid-phase extraction (SPE) and analysed by gas chromatography-tandem mass spectrometry operated in selected reaction monitoring mode (SRM). Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low microg/L were achieved, whereas linearity was established within 0.5-500 microg/L range. Good precision was obtained both in terms of intra-day repeatability and inter-day precision on two concentration levels (RSD% lower than 2%). Good percentage recoveries were obtained (92.0-102.0%) even in the presence of high amount of fat. Finally, the developed method was successfully applied to analyse a number of commercial milk samples with different fat content and baby milk samples.     

High-performance liquid chromatographic assay of erythromycin from biological matrix using electrochemical or ultraviolet detection

Two chromatographic methods were developed for the determination of erythromycin A (EA) residues in animal tissues (muscle, liver, kidney and fat of cattle, pigs and poultry) and cow's milk. In addition to a more traditional method using electrochemical detection, we developed an original alternative method based on UV detection at 236 nm, by pretreating to create a chromophore in the molecule. An internal standard was used with both methods to check the variability of the analytical system. Analysis times and performance were compared. The recovery of EA from various matrices was greater than 95%. For both methods the quantification limit for EA was 0.25 microgram ml-1 for plasma, 0.025 microgram g-1 for milk and 0.125 microgram g-1 for the other biological matrices. The methods can be used to check for EA residues in these matrices; in fact, the statutory maximum residue limits (MRLs) of EA are 0.4 microgram g-1 in muscle, kidney, liver and fat of beef cattle, sheep, pigs and poultry, and 0.04 microgram g-1 in cow's and sheep's milk.     

An integrated digital polymerase chain reaction chip for multiplexed meat adulteration detection

Meat adulteration detection is a common concern of consumers. Here, we proposed a multiplex digital polymerase chain reaction method and a low-cost device for meat adulteration detection. Using a polydimethylsiloxane microfluidic device, polymerase chain reaction reagents could be pump-free loaded into microchambers (40 × 40 chambers) automatically. Due to the independence of multiplex fluorescence channels, deoxyribonucleic acid templates extracted from different animal species could be distinguished by one test. In this paper, we designed primers and probes for four types of meat (beef, chicken, pork, and duck) and labeled each of the four fluorescent markers (hexachlorocyclohexane [HEX], 6-carboxyfluorescein [FAM], X-rhodamine [ROX], and cyanine dyes 5 [CY5]) on the probes. Specific detection and mixed detection experiments were performed on four types of meat, realizing a limit of detection of 3 copies/µL. A mixture of four different species can be detected by four independent fluorescence channels. The quantitative capability of this method is found to meet the requirements of meat adulteration detections. This method has great potential for point-of-care testing together with portable microscopy equipment.     

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.     

Interlaboratory study of a multiresidue gas chromatographic method for determination of organochlorine and pyrethroid pesticides and polychlorobiphenyls in milk,fish, eggs,and beef fat

An interlaboratory study was conducted to validate a gas chromatographic (GC) method for determination of 21 organochlorine pesticides, 6 pyrethroid pesticides, and 7 polychlorobiphenyl (PCB) congeners in milk, beef fat, fish, and eggs. The method was performed at low contamination levels, which represent relevant contents in food, and is an extension of the European standard (method NF-EN-1528, Parts 1-4). It enlarges the applicable scope of the reference EN method to pyrethroid pesticides and proposes the use of solid-phase extraction (SPE) as a cleanup procedure. Cryogenic extraction was made, and SPE cleanup was performed with 2 successive SPE cartridges: C18 and Florisil. After injection of the purified extract onto a GC column, residues were measured by electron capture detection. Food samples (liquid milk, beef fat, mixed fish, and mixed eggs) were prepared, tested for homogeneity, and sent to 17 laboratories in France. Test portions were spiked with 27 pesticides and 7 PCBs at levels from 26 to 45, 4 to 27, 31 to 67, and 19 to 127 ng/g into milk, eggs, fish, and fat, respectively. Based on results for spiked samples, the relative standard deviation for repeatability ranged from 1.5 to 6.8% in milk, 3 to 39% in eggs, 4.5 to 12.2% in fish, and 7 to 13% in fat. The relative standard deviation for reproducibility ranged from 33 to 50% in milk, 29 to 59% in eggs, 31 to 57% in fish, and 30 to 62% in fat. This method showed acceptable intra- and interlaboratory precision data, as corroborated by HORRAT values at low levels of pesticide and PCB contamination. The statistical evaluation of the results was performed according to the International Organization for Standardization (ISO; ISO 3534 standard) and 5725-2 Guideline.     

Discriminating animal fats and their origins: assessing the potentials of Fourier transform infrared spectroscopy,gas chromatography,immunoassay and polymerase chain reaction techniques

The objective of the reported study was to assess the abilities of various methods to differentiate the sources of fats used in feedstuff formulations. The main target was the identification of tallow (ruminant fat) and its differentiation from non-ruminant fats. Four different techniques were compared in terms of their suitability for enforcing existing and upcoming legislation on animal by-products: (1) Fourier transform infrared spectroscopy (FT-IR) applied to fat samples, (2) gas chromatography coupled with mass spectrometry (GC–MS) to determine fatty acid profiles, (3) immunoassays focusing on the protein fraction included in the fat, and (4) polymerase chain reaction (PCR) for the detection of bovine-specific DNA. Samples of the different fats and oils as well as mixtures of these fats were probed using these analytical methods. FT-IR and GC–MS differentiated pure fat samples quite well but showed limited ability to identify the animal species or even the animal class the fat(s) belonged to; no single compound or spectral signal that could permit species identification could be found. However, immunoassays and PCR were both able to identify the species or groups of species that the fats originated from, and they were the only techniques able to identify low concentrations of tallow in a mixture of fats prepared by the rendering industry, even when the samples had been sterilised at temperatures >133 °C. Fats used in animal nutrition come mainly from the rendering industry, thereby confirming the suitability of PCR and immunoassays for their identification. However, neither of these latter techniques was able to detect premier jus tallow, representing the highest quality standard of fat with extremely low protein concentration.     

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     

应用Milkoscan FT120对牛奶掺假的研究

采用红外扫描乳品测定仪对原料乳中脂肪、蛋白、干物质、乳糖、柠檬酸密度、冰点、碳水化合物、全糖、游离脂肪酸等成分参数的检测建立原料乳光谱数据库,在具有代表性原料乳中加入植脂末模拟牛奶掺假,通过应用掺假模块鉴别生鲜牛奶是否掺假.     

Crude fat, diethyl ether extraction, in feed, cereal grain, and forage (Randall/Soxtec/submersion method): collaborative study

A method for determining crude fat in animal feed, cereal grain, and forage (plant tissue) was collaboratively studied. Crude fat was extracted from the animal feed, cereal grain, or forage material with diethyl ether by the Randall method, also called the Soxtec method or the submersion method. The proposed submersion method considerably decreases the extraction time required to complete a batch of samples. The increase in throughput is very desirable in the quest for faster turnaround times and the greater efficiency in the use of labor. In addition, this method provides for reclamation of the solvent as a step of the method. The submersion method for fat extraction was previously studied for meat and meat products and was accepted as AOAC Official Method 991.36. Fourteen blind samples were sent to 12 collaborators in the United States, Sweden, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.09 to 9.26% for crude fat. Among-laboratory (including within) relative standard deviation (reproducibility) ranged from 1.0 to 21.0%. The method is recommended for Official First Action.     

Crude fat, hexanes extraction, in feed, cereal grain, and forage (Randall/Soxtec/submersion method): collaborative study

A method for determining crude fat in animal feed, cereal grain, and forage (plant tissue) was collaboratively studied. Crude fat was extracted from the animal feed, cereal grain, or forage material with hexanes by the Randall method, also called the Soxtec method or the submersion method. The use of hexanes provides for an alternative to diethyl ether for fat extractions. The proposed submersion method considerably decreases the extraction time required to complete a batch of samples compared to Soxhlet. The increase in throughput is very desirable in the quest for faster turnaround times and the greater efficiency in the use of labor. In addition, this method provides for reclamation of the solvent as a step of the method. The submersion method for fat extraction was previously studied for meat and meat products and was accepted as AOAC Official Method 991.36. Fourteen blind samples were sent to 14 collaborators in the United States, Sweden, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.23 to 5.80% for crude fat. Among-laboratory (including within) relative standard deviation (reproducibility) ranged from 1.88 to 14.1%. The method is recommended for Official First Action.     

Determination of narasin and monensin in bovine, swine, and chicken tissues by liquid chromatography with tandem mass spectrometry: first action 2011.24

The single-laboratory validation (SLV) of an LC-MS/MS method for determination and confirmation of two ionophores, narasin and monensin, in animal tissues is described. The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/x) regression and selectivity of the method for narasin and monensin in the presence of lasalocid, salinomycin, maduramycin, nicarbazin, and sulfadiazine. Recoveries varied from 86.2 to 103.5% for narasin and 89.1 to 105.1% for monensin. Intertrial repeatability precision [relative standard deviation of repeatability (RSDr)] varied from 3.9 to 13.8% for narasin and 3.3 to 16.3% for monensin in fortified tissue. Precision of the method was verified in incurred tissues. The LOQ of the method was validated and ranged from 0.45 ng/g in milk, to 4.0 ng/g in chicken fat, but was 0.75 ng/g for most tissues. Two confirmatory ions for each analyte were examined across all matrixes, resulting in estimated false-negative rates of 0.00% (95% confidence interval of 0.00-0.68%) for monensin ions (540 samples) compared to the U.S. and European Union (EU) acceptance criteria. The confirmatory ions for narasin demonstrated 0.00% false-negative rates (95% confidence interval of 0.00-0.58%) when compared to either the U.S. or EU criteria in 630 samples. The method was robust when small changes in method parameters were made and stability of fortified tissues, extracts, and calibration solutions were estimated. The data satisfy the requirements of the AOAC Stakeholder Panel on Veterinary Drug Residue for SLV studies, and the method was adopted Official Methods of Analysis First Action 2011.24 by the AOAC Expert Review Panel on Veterinary Drug Residues.     

Detection of incurred dihydrostreptomycin residues in milk by liquid chromatography and preliminary confirmation methods.

An LC method for the determination of the aminoglycosides streptomycin (STR) and dihydrostreptomycin (DHS) in milk was developed/modified on the basis of published papers. Mean recoveries were 87 and 95% for STR and DHS, respectively. Recoveries are dependent on the concentration level and batch of solid-phase extraction columns used, and independent of fat content and homogenization. The relative standard deviations are 15.6 and 9.6% for STR and DHS, respectively, at a level of 100 micrograms kg-1. Limits of detection (8 and 12 micrograms kg-1, respectively) and quantification (12 and 18 micrograms kg-1, respectively) are far below the EU maximum residue limit of 200 micrograms kg-1. Lyophilized DHS samples can be used for internal control of the analysis as the DHS concentration is not influenced by the lyophilization process and subsequent storage at 6 degrees C. In incurred milk samples no false negative results of preliminary confirmation tests (Charm II Aminoglycoside test, Ridascreen Streptomycin ELISA) with respect to DHS concentrations > or = 20 micrograms kg-1 as determined by the LC method are observed. DHS concentrations of incurred samples determined by ELISA are higher than those obtained by the LC method. These differences were more pronounced with incurred than with spiked milk samples, thus leading to the conclusion that in incurred samples substances are present which co-react in the ELISA and which are not detected by the LC method.     

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.     

快速气相色谱法测定蔬菜中菊酯类农药残留量

采用一种较快速，简单的方法测定蔬菜中菊酯类农药残留物。样本中农残经乙酸酯萃取，凝胶渗透色谱及固相提取净化后，用气相色谱－电子捕获检测器测定，色谱柱为ＨＰ－５小口长石英毛细管柱。本法对６种常用菊酯类农药的回收率范围为８９．６％－９９．７％，检测限为０．０４ｍｇ／ｋｇ。对同一批普施用菊酯类农药的白菜样本进行化验，本法跟美国食物及药品管理局农药残留标准测定方法所得结果非常吻合。     

Direct determination of Fe and Zn in different components of cow milk by FAAS with a high performance nebulizer

Methods for the direct determination of iron and zinc in different components of cow milk (whole milk, non fat milk and whey milk) by flame atomic absorption spectrometry (FAAS) were performed using two nebulizers (nebulizer of platinum with a glass impact bead and a high performance nebulizer). The non fat milk and the whey milk were obtained by physical procedures (centrifugation and ultracentrifugation) in absence of chemical treatment. A limit of detection of 0.024 and 0.007 mug ml(-1) for iron and zinc, respectively, were obtained by using a high performance nebulizer. The precision obtained varied between 1.4-4.0% and 0.4-1.9% for iron and zinc, respectively. The accuracy of the methods was studied with the analysis of SRM-1549 and A-11 non fat milk reference materials. The methods were applied to ten cow milk samples. Dates about the distribution of iron and zinc into the different components of cow milk were present.     

Rapid method for the determination of organochlorine pesticides in milk

This method involved one step solvent extraction of milk with ethyl acetate-acetone-methanol by ultrasonication. The supernatants were further cleaned-up and enriched by solid-phase extraction using octadecyl (C18)-bonded silica cartridges, then assayed by capillary gas-liquid chromatography with electron capture detection. The recoveries of eleven organochlorine pesticides (OCPs) from raw milks were quantitative, ranging from 90-110% at 10 times the limit of detection (LOD). The LOD ranged from 0.5 micrograms/l whole milk for alpha-hexachlorocyclohexane to 2.5 micrograms/l whole milk for 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane. The day-to-day variation of the method was evaluated over 7 days using 3 different pools of spiked cow milks (at the LOD, 5 and 10 times the LOD). The coefficient of variations (C.V.s) were 16 +/- 6, 10 +/- 2 and 9 +/- 3% (mean S.D.), respectively. The method showed no emulsion problems common with conventional non-polar solvent extraction, and the use of solid-phase extraction considerably reduced the sample clean-up process compared with the existing methods. The method also showed that OCPs in milk could be extracted quantitatively without extraction of total fat, and that OCPs spiked into cows milk could be used to construct calibration curves for human milk determinations.     

Petrifilm rapid S. aureus Count Plate method for rapid enumeration of Staphylococcus aureus in selected foods: collaborative study

A rehydratable dry-film plating method for Staphylococcus aureus in foods, the 3M Petrifilm Rapid S. aureus Count Plate method, was compared with AOAC Official Method 975.55 (Staphylococcus aureus in Foods). Nine foods-instant nonfat dried milk, dry seasoned vegetable coating, frozen hash browns, frozen cooked chicken patty, frozen ground raw pork, shredded cheddar cheese, fresh green beans, pasta filled with beef and cheese, and egg custard-were analyzed for S. aureus by 13 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample and 3 levels of inoculated test sample, each in duplicate. The mean log counts for the methods were comparable for pasta filled with beef and cheese; frozen hash browns; cooked chicken patty; egg custard; frozen ground raw pork; and instant nonfat dried milk. The repeatability and reproducibility variances of the Petrifilm Rapid S. aureus Count Plate method were similar to those of the standard method.