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%.     

Ratios between content of fatty acids as markers for mixture of cow and goat milk

The fat of mixture of goat and cow milk, extracted by di-ethylic ether, was gas chromatographically analysed in order to propose an analytical procedure suitable to reveal small additions of cow milk to goat milk. The obtained results show evident differences in the percentages of some fatty acids. An attempt to apply the peak area ratios, corresponding to the methyl esters of fatty acids, suggested by the literature for cow milk did not yield good results. Other ratios suitable to reveal even the presence of low percentages (up to 5 %) of cow milk in goat milk were found and proposed in this paper. Such ratios are sensitive and can be defined markers towards cow or goat milk and also towards extraneous fat like lard or colza seeds oil.     

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.     

Characterization of maleuric acid derivatives on transgenic human monoclonal antibody due to post-secretional modifications in goat milk

A fully human antibody to tumor necrosis factor-alpha was expressed in the mammary glands of transgenic goats. The goat expressed antibody (gAb) is heterogeneous and has several isoforms due to typical cellular post-translational modifications. In addition, one post-secretional modification on gAb was discovered by high-resolution cation exchange chromatography (CIEX). The presence of these variants in the final product was shown to be dependent upon the initial milk storage and traditional purification methodologies used. These observations allow for the development of new sample recovery and purification processes to eliminate these variants. Various enzymatic treatments were used to characterize different gAb heavy chain C-terminal lysine and sialic acid variants. In addition, an unknown derivative with the additional mass of 140 Da was found in transgenic gAb using mass spectrometry (MS). The modification sites were identified as the N-termini of gAb light chains and heavy chains using Q-TOF MS. Characterization of transgenic gAb isoforms was facilitated by utilizing different enzymes, CIEX and MS techniques. A maleuric acid modification on the N-terminal portion of gAb was shown to be consistent with the available data characterizing this new derivative of transgenic gAb isoforms in goat milk.     

Determination of enrofloxacin and its metabolite ciprofloxacin in goat milk by high-performance liquid chromatography with diode-array detection. Optimization and validation

A high-performance liquid chromatography-diode array detection method (HPLC-DAD) combined with liquid chromatography-mass spectrometry was developed for the determination of enrofloxacin and its metabolite ciprofloxacin in goat milk. The HPLC-DAD method validation was compliant with the "DG SANCO 1805/2000" European regulation. The residues were extracted from milk with phosphate buffer, purified on a C18 Speedisk cartridge SPE (Baker) and then analysed using HPLC-DAD set at 277 nm. The decision limit (CCa) calculated by spiking samples at 100 microg/kg with both analytes, taking into account the maximum residue limit (MRL) of 100 microg/kg established by the European Union for the sum of enrofloxacin and its metabolite ciprofloxacin in milk, was 105.3 microg/kg for enrofloxacin and 105.5 microg/kg for ciprofloxacin. The detection capability (CCbeta) was 110.7 and 110.9 microg/kg for enrofloxacin and ciprofloxacin, respectively. The mean recoveries of the method, calculated by spiking samples at 50, 100 and 150 microg/kg were 84% for enrofloxacin and 88% for ciprofloxacin. The limit of quantification was 20 microg/kg for both analytes. The HPLC-DAD validated method was successfully applied for the first time in goats milk, and proved to be suitable for the sensitive and accurate quantification and confirmation analysis of enrofloxacin and ciprofloxacin for regulatory purposes.     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱快速测定羊乳中11种磷酸二酯酶-5抑制剂

建立了超高效液相-四极杆/静电场轨道阱系统(UPLC-Q-Exctive-MS/MS)快速检测羊乳中磷酸二酯酶-5抑制剂(豪莫西地那非、伐地那非、氨基他达拉非、那红地那非、红地那非、伪伐地那非、那莫西地那非、硫代艾地那非、他达拉非、西地那非及羟基豪莫西地那非)的方法。样品经乙腈提取,PRiME HLB净化后,以0.1%甲酸水-乙腈为流动相,采用ACQUITY UPLC HSS T3(2.1×100 mm,1.8 mm)色谱柱分离。在电喷雾正离子模式下(ESI+)下优化影响全扫分辨率,结合二级质谱提高准确度。结果表明,在0~100 mg/L浓度范围,目标化合物线性关系良好,方法检出限为2.5~5.0 mg/kg,本方法在3个水平下的加标回收率为75.4%~97.8%,RSD为1.6%~6.2%。本方法成功应用于羊乳中11种磷酸二酯酶-5抑制剂的快速检测。     

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk

In the dairy industry one of the most common frauds is mixing high-value milk (sheep’s and goats’) with milk of lower value (cows’). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows’ milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows’ and goats’ milk and cows’ and sheep’s milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows’ milk in sheep’s and goats’ milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

Death kinetics of <Emphasis Type="Italic">Escherichia coli</Emphasis> in goat milk and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> in cloudberry jam treated by ohmic heating

The influence of ohmic heating on the death kinetic parameters of Escherichia coli ATCC® 25922 in goat milk and spores of Bacillus licheniformis ATCC® 14580 in cloudberry jam was investigated and compared with that of conventional heating. Ohmic treatment of goat milk shortened the decimal reduction time D in comparison with the D values obtained at conventional treatment. Similarly, the z value, increase of temperature required for a ten-fold reduction of D, was also lower at ohmic treatment. The death kinetics of Bacillus licheniformis ATCC® 14580 spores in cloudberry jam was also studied employing both types of heat treatment. Similar conclusions were obtained for the D values as in the case of goat milk. However, the differences between the z values obtained for ohmic and conventional heating were not significant.     

Determination of cow's milk and ripening time in nonbovine cheese by capillary electrophoresis of the ethanol-water protein fraction

A novel method is reported for analyzing adulteration of goat and ewe cheeses with cow's milk: capillary zone electrophoresis (CZE) in isoelectric, acidic buffers (50 mM imino diacetic acid, IDA, pH = pI 2.3). The cheese samples were extracted with a 20:80 v/v ethanol-water mixture in presence of 3 M urea and 1% beta-mercaptoethanol for 1 h. After centrifugation and lipid extraction, the samples were dissolved in 50 mM IDA, 6 M urea and 0.5% hydroxyethyl cellulose and analyzed by CZE at 700 V/cm. A total of 18 characteristic peaks were resolved among the three types of cheeses and 18 variables were defined as their respective areas. There was excellent similarity among the electrophoretic patterns obtained with cheeses of a given type of milk, while cheeses made with different types of milk were easily distinguishable. Most peaks were common to all cheeses, but the profile differed depending on the type of milk used. Principal component analysis, linear discriminant analysis, and partial least squares regression (PLS) were used for statistical analysis of the data obtained by CZE. In particular, by using PLS multivariate regression, the contents of cow's milk in presumably pure goat and ewe cheeses, as well as in binary and ternary mixtures, could be predicted with relative standard deviations of ca. 6-7%. In addition, the ripening time in goat and ewe cheeses could also be predicted.     

Front face synchronous fluorescence as a tool for the quality assurance of Greek milk

This research focuses on implementing the low cost and rapid front face synchronous fluorescence (SyFS) in order to ensure the quality assurance of Greek milk. Specifically, samples originated from the Greek domestic production of goat, sheep, cow, as well as foreign cow milk samples and adulterated cow milk samples. SyFS spectra were acquired in the excitation area of 250–500 nm with (Δλ)= 100 nm. Greek and foreign cow milk samples were differentiated based on intensity variations at wavelengths 350–515 nm, 540–579 nm, and 580–600 nm. The emissions at these wavelength positions correspond to tryptophan, vitamin A, and riboflavin. The supervised model with 94 samples exhibited p-value = 7,98E-11, RMSEE= 0,29171, RMSEcv= 0,29284 and RMSEP= 0,98013, AUROC for Greek samples= 0,61 and AUROC for foreign= 0,85. We differentiated milk samples according to the animal type with PCA and OPLS-DA models of 107 samples exhibiting RMSEE= 0,225842, RMSEcv= 0,228054 and RMSEP= 0,518635, AUROC for sheep samples= 0,99, AUROC for goat samples= 0,98 and AUROC for cow samples= 0,96. In fact, the emission band 350–591 nm characterized sheep milk and corresponds to aminoacids and fatty acids, cow milk was related to the 350–600 nm emission band related to the b-carotene and to the goat milk the emission bands 350–505 nm and 520–600 nm were attributed to tryptophan, NADH and Rivoflabin. Finally, we investigated whether SyFS coupled with chemometrics may provide preliminary evidence on adulterated cow milk samples. All models were validated with permutation testing, p-values and ROC curves.     

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.     

Determination of immunoglobulin G in bovine colostrum and milk by direct biosensor SPR-immunoassay

An automated biosensor surface-plasmon resonance-based assay was developed for the determination of immunoglobulin G (IgG) in bovine milk and colostrum with either goat or rabbit antibovine IgG or protein G used as detecting molecule. The method is configured as a direct and nonlabeled immunoassay, with quantitation against an authentic IgG calibrant. Whole colostrum or milk is prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flowrate, contact time, and regeneration, were optimized, and nonspecific binding was evaluated. Performance parameters included working range of 15-10 000 ng/mL, method detection limit of 0.08 mg/mL, overall instrument response reproducibility relative standard deviation (RSD(R)) of 0.47%, mean between-run RSD(R) of 10.5% for colostrum, and surface stability over 200 analyses. The proposed method was compared with independent alternative methods. The technique was applied to the measurement of IgG content during early lactation transition from colostrum to milk, as well as in consumer milk, colostrum, and hyperimmune milk powders.     

Raman spectroscopy based analysis of milk using random forest classification

The development of a classification system based on the Raman spectra of milk samples is proposed in present study. Such development could be useful for nutritionists in suggesting healthy food to infants for their proper growth. Previously, molecular structures in milk samples have been exploited by Raman spectroscopy. In the current study, Raman spectral data of milk samples of different species is utilized for multi-class classification using a dimensionality reduction technique in combination with random forest (RF) classifier. Quantitative and experimental analysis is based on locally collected milk samples of different species including cow, buffalo, goat and human. This classification is based on the variations (different concentrations of the components present in milk such as proteins, milk fats, lactose etc.) in the intensities of Raman peaks of milk samples. Principal component analysis (PCA) is used as a dimensionality reduction technique in combination with RF to highlight the variations which can differentiate the Raman spectra of milk samples from different species. The proposed technique has demonstrated sufficient potential to be used for differentiation between milk samples of different species as the average accuracy of about 93.7%, precision of about 94%, specificity of about 97% and sensitivity of about 93% has been achieved.     

Detection and characterization by high-performance liquid chromatography and mass spectrometry of two truncated goat alphas2-caseins

The identification and characterization of truncated forms of goat alphas2-Cn variants A and E are reported. The two proteins, which have experimental Mr values of 24 183 and 24 227 Da, were detected as minor components in a goat milk sample from an autochthonous breed of southern Italy, 'Rossa Mediterranea', by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). Characterization of the amino acid sequences, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the polypeptide chains correspond to the 1-204 sequence of mature alphas2-Cn variant A (component with Mr of 24 183 Da) and E (component with Mr of 24 227 Da), respectively. These components seem to be the product of a differential splicing of pre-messenger RNA during the translation process of the alphas2-Cn variants A and E.     

Quantitative determination of volatile organic compounds (VOC) in milk by multiple dynamic headspace extraction and GC-MS

A method for the accurate determination of volatile organic compounds (VOC) in milk samples has been developed and tested. It combines multiple dynamic headspace extraction with GC-MS. Absolute amounts of VOC in the liquid phase are obtained by determining the first order kinetic dependence of the stepwise extraction of the analytes and internal standards from the liquid matrix. Compounds released from milk were collected on a train of traps filled with different solid sorbents to cover all components having a number of carbon atoms ranging from 4 to 15. They were analysed by GC-MS after thermal desorption of VOC from the collecting traps. Quantification of VOC in milk was performed using deuterated compounds as internal standards. The method was used to follow seasonal variations of monoterpenes in goat milk and to detect the impact of air pollution on the quality of milk.     

Direct determination of recombinant bovine somatotropin in plasma from a treated goat by liquid chromatography/high-resolution mass spectrometry

Recombinant bovine somatotropin (rbST) is used in dairy cattle to enhance milk production. Despite the ban on this hormone in some countries, especially in Europe, there is so far no method available for the direct detection of rbST either in milk or in plasma. An analytical strategy has been developed to analyze rbST in plasma, including a purification procedure based on a precipitation with ammonium sulphate, followed by a solid-phase extraction (SPE)-based clean-up on C4 sorbent and precipitation with cold methanol. The hormone was then digested with trypsin and analyzed by liquid chromatography/high-resolution mass spectrometry (LC/HRMSn) on a linear ion trap coupled with an Orbitrap. The tryptic N-terminal peptide, specific to the difference between the endogenous and recombinant form of the somatotropin, was fragmented and product ions were analyzed at high mass resolution. Applying this approach to goat plasma allowed the direct detection of 10 ng mL(-1) of rbST in fortified samples. It also showed the presence of rbST in plasma collected from a goat treated with the hormone, even 2 days after administration. These results are of a great interest in the field of somatotropin control and undoubtedly constitute a first step in the development of a method for the detection of rbST not only in bovine plasma, but also in other biological matrices such as milk.     

柱前衍生-超高效液相色谱法测定3种奶粉中18种氨基酸

近年来羊奶粉和骆驼奶粉备受消费者青睐,它们具有潜在的低致敏性,因此成为牛乳不耐受人群尤其是婴幼儿的母乳替代品,其营养价值备受关注。牛奶粉、羊奶粉和骆驼奶粉中氨基酸含量的比较研究鲜有报道。利用酸水解得到游离氨基酸,选择6-氨基喹啉-N-羟基琥珀酰亚胺氨基甲酸酯(AQC)进行柱前衍生,超高效液相色谱分离并检测,外标法定量。18种氨基酸在各自线性范围内线性关系良好,相关系数(r ²)大于0.999;以3倍和10倍信噪比(S/N)确定方法的检出限(LOD)和定量限(LOQ),分别为1.3~2.5 (mg/100 g)和3.9~7.5 (mg/100 g)。方法验证采用奶粉标准参考物质SRM 1849a,测定值符合其含量范围,6次测定值的相对标准偏差(RSD)为2.04%~3.65%。采用建立的方法分别对市售和网购的牛奶粉、羊奶粉和骆驼奶粉进行18种氨基酸成分和含量分析,旨在从氨基酸角度对这3种不同来源乳品进行对比。该方法快速,灵敏度高,准确可靠,适用于不同基质乳粉中18种氨基酸成分和含量的确定。     

Determination of pesticides and some metabolites in different kinds of milk by solid-phase microextraction and low-pressure gas chromatography-tandem mass spectrometry

A new analytical method is proposed to determine more than 40 multiclass pesticides in different kinds of processed (whole, skimmed and powdered) and unprocessed (goat and human) milk samples using solid-phase microextraction (SPME). A comparative study between headspace (HS) and direct immersion (DI) was carried out. The effect of milk dilution and the use of acid to reduce the influence of the matrix in DI-SPME mode were also evaluated. DI of the SPME fiber into previously diluted and acidified milk samples achieved the best sensitivity results. Pesticides were determined using low-pressure gas chromatography-tandem mass spectrometry (LP-GC-MS/MS). Both of the selected techniques have been shown to be effective at reduce fat interference and can determine analytes present at very low concentrations (limits of quantification between 0.02 and 1.00 g L^–1). Performance characteristics such as linearity, recovery, precision, and lower limits, together with an estimation of the measurement uncertainty using validation data, are presented for each pesticide. All of the pesticides presented recovery rates of between 81 and 110% and precision values lower than 12% (expressed as the relative standard deviation). The overall uncertainty of the method was estimated at three different concentrations (10, 25 and 50 g L^–1) and was lower than 25.5% in all cases. The proposed analytical methodology was applied to the analysis of target pesticides in 35 samples: 15 commercial, 3 human and 17 goat milk samples. The metabolite p,p-DDE was the compound most frequently found in both the breast and goat milk samples, at concentration levels <20 g L^–1. However, pesticide residues were not found in any of the other 15 commercial milk samples (skimmed, powdered and whole milk) analyzed.     

MALDI‐TOF mass spectrometry for the monitoring of she‐donkey's milk contamination or adulteration

Donkey's milk (DM), representing a safe and alternative food in both IgE‐mediated and non‐IgE‐mediated cow's milk protein allergy, can be categorized as precious pharma‐food. Moreover, an economically relevant interest for the use of DM in cosmetology is also developing. The detection of adulterations and contaminations of DM is a matter of fundamental importance from both an economic and allergenic standpoint, and, to this aim, fast and efficient analytical approaches to assess the authenticity of this precious nutrient are desirable. Here, a rapid matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF MS)‐based method aimed to the detection of bovine or caprine milk in raw DM is reported. The presence of the extraneous milks was revealed by monitoring the protein profiles of the most abundant whey proteins, α‐lactalbumin (α‐LA) and β‐lactoglobulin, used as molecular markers. The possibility of obtaining a quantitative analysis of the level of cow or goat milk in DM based on the MALDI‐TOF peak areas of α‐LAs was also explored. The results showed that the experimental quantitative values were in good agreement with the real composition of each mixture. As pretreatment of the milk samples is not required, and owing to the speed and the high sensitivity of MALDI‐MS, the protocol here reported could represent a reliable method for routine analyses aimed to assess the absence of contamination in raw fresh DM samples. Copyright © 2013 John Wiley & Sons, Ltd.