Separation of casein micelles from whey proteins by high shear microfiltration of skim milk using rotating ceramic membranes and organic membranes in a rotating disk module

This paper investigates the microfiltration of skim milk in order to separate caseins micelles from two whey proteins, α-lactalbumin (α-La) and β-lactoglobulin (β-Lg), using a modified dynamic filtration pilot (MSD) consisting in 6 ceramic 9-cm diameter membrane disks of 0.2 μm pores, rotating around a shaft inside cylindrical housing. A comparison was made with another dynamic filtration module consisting in a disk rotating near a fixed PVDF 15.5 cm diameter membrane with 0.15 μm pores. Maximum permeate fluxes were 120 L h⁻¹ m⁻² with the MSD module at 1930 rpm and at 40 °C, and 210 L h⁻¹ m⁻² at 2500 rpm and 45 °C, with the rotating disk module. Casein rejection was around 99% at high speed for both membranes. α-La transmission decreased with increasing transmembrane pressure (TMP) from 75% to 60% for ceramic membranes and from 25% to 10% for the PVDF one. β-Lg transmissions were lower, ranging from 23% to 15% for ceramic membranes and from 20% to 5% for the PVDF one. In a concentration test with the PVDF membrane at 2000 rpm, the flux decayed from 200 L h⁻¹ m⁻² at initial concentration to 80 L h⁻¹ m⁻² at VRR = 3.2 and 22.1% of the initial α-La mass was recovered in the permeate, against 8.1% for β-Lg. Permeate fluxes in the mass transfer limited regime (J_lim) of the MSD and rotating disk module operated at various speeds were well correlated by the equation J_lim = 17.13 V_av where V_av denoted the disk azimuthal velocity averaged over the membrane area. Measurements of J_lim, taken from Ref. [G. Samuelsson, P. Dejlmek, G. Tragardh, M. Paulsson, Minimizing whey protein retention in crossflow microfiltration of skim milk. Int. Dairy J. 7 (1997) 237–242] during MF of skim milk using tubular ceramic membranes at velocities from 1.5 to 8 m s⁻¹ with permeate co-current recirculation were found to obey the same correlation.     

Cation-exchange chromatographic isolation of the methionine-bound sulfur in casein hydrolysates for stable-isotope-ratio analysis

Summary A chromatographic method is described for the isolation of the methionine-bound sulfur in casein for stable-isotope-ratio analysis. Casein is cleaved by acid hydrolysis and the high-molecular-weight secondary products formed are removed by adsorption on octadecyl-silica. Methionine is separated from the bulk of the polar amino acids by cation-exchange chromatography and subsequently oxidized to methionine sulfone by performic and peracetic acid. In a further step the methionine is isolated from the accompanying nonpolar amino acids by cation-exchange chromatography with a mobile phase of very low eluent strength.     

Validation of a high-performance liquid chromatographic method with fluorescence detection for the simultaneous determination of tetracyclines residues in bovine milk

A high-performance liquid chromatography-fluorescence detection method was optimized and validated to determine tetracyclines residues in bovine milk. Post-column derivatization using metal complexation in non-aqueous reagent increased the fluorescence of chelates by a factor up to 2.54 compared to water (signal-to-noise ratio enhancement). Overall recoveries ranged from 61 to 115%, with RSD_r from 5 to 15% (n = 54). Detection limits ranged from 5 to 35 μg kg⁻¹. Limits of quantification were established at 50 μg kg⁻¹. Decision limits (CCα) were 109, 108 and 124 μg kg⁻¹ and detection capabilities (CCβ) 119, 117 and 161 μg kg⁻¹ for oxytetracycline, tetracycline and chlortetracycline, respectively. The method was applied successfully in a national monitoring program.     

Fractionation analysis of iodine in bovine milk by preconcentration neutron activation analysis

Iodine is an essential trace element for human beings. The main source of iodine is generally food items such as fish and milk. Either the lack or the excess of iodine can cause health problems. There exists an increasing interest in the determination of total iodine as well as various species of iodine in milk. We have developed an epithermal neutron activation analysis method with a Compton suppression (ENAA-CS) counting system for the determination of ng mL⁻¹ levels of iodine. We have also employed chemical separation methods prior to ENAA-CS to measure the fraction-specific concentrations of iodine in bovine milk. We have measured the following iodine concentrations in homogenized milk (3.25%milk fat): 0.48 ± 0.02 μg mL⁻¹ of total iodine, 0.020 ± 0.003 μg mL⁻¹ of lipid-bound iodine, 0.039 ± 0.002, 0.019 ± 0.002 and 0.021 ± 0.004 μg mL⁻¹ of protein-bound iodine depending on the protein separation method and 0.45 ± 0.02 μg mL⁻¹ of inorganic species.     

Gas chromatography analysis of major free mono‐ and disaccharides in milk: Method assessment,validation, and application to real samples

Saccharides are functional constituents of milk. Although d ‐lactose represents almost the totality of the saccharides in the milk, minor species, like d ‐glucose, d ‐galactose, myo‐inositol and, as a result of severe thermal treatments, monosaccharides like d ‐tagatose, are also detectable. Although chromatography has been the main analytical approach used for accomplishing this task, quite surprisingly a validated gas chromatographic method aimed at the simultaneous determination of these compounds is still needed. Hence, our contribution is devoted to fill this gap. After the optimization of clean‐up and derivatization (conversion of saccharides in their trimethyl silyl ethers) steps, the adoption of a highly cross‐linked silphenylene stationary phase permitted to obtain high resolution and a fast chromatographic run. Validation was accomplished in terms of limit of detection, limit of quantification, linearity, precision, and trueness. The accuracy of the method was successfully tested on a number of partially skimmed milk samples. Excellent limits of detection for all analytes make this method eligible, also with respect to a gas chromatographic/mass spectrometry approach, for routine analysis and quality control in the dairy industries.     

Rapid determination of melamine and cyanuric acid in milk powder using direct analysis in real time-time-of-flight mass spectrometry

The use of fast semi-automated method employing direct analysis in real time (DART) ion source coupled to time-of-flight mass spectrometry (TOFMS) for analysis of melamine (MEL) and cyanuric acid (CYA) in milk powder and milk based products has been demonstrated in this study. Simple sample extraction procedure employing methanol–5% aqueous formic acid mixture, which enabled disruption of melamine–cyanurate complex, was followed by direct, high-throughput (30 s per run) examination of sample extracts spread on a glass rod by mass spectrometry under ambient conditions, without any prior chromatographic separation. After optimization of instrument parameter settings, limits of detection (LODs) 170 and 450 μg kg⁻¹ were achieved for MEL and CYA, respectively. In the final phase of study, the possibility of minimizing spectral interference, thus improving method performance characteristics through the use of ultrahigh resolving power offered by Orbitrap based mass analyzer is demonstrated.     

Improvements in the selection of real components forming a substitute mixture for petroleum fractions

Complex mixtures, particularly petroleum fractions, usually need to be suitably modeled before providing the simulation and other types of chemical engineering calculations. The most convenient way is to describe the original mixture by a substitute mixture. The formerly published approach based on the employment of substitute mixtures of real components can be improved in order to get a closer match between the behavior of the original and substitute mixtures. In the first phase of the algorithm, a new concept of a band around the characterization curves brings wider possibilities for the selection of real components into the substitute mixture. The second phase, which is used to determine the composition of the substitute mixture, can be also improved by considering the global or bulk properties of the original mixture if available. Typically, some of the properties e.g. liquid density, molecular mass and PNA (Paraffinic/Naphthenic/Aromatic carbon) analysis can be measured and used to improve the adjustment of the composition. The improved algorithm is illustrated by an example.     

Confirmatory analysis of non-steroidal anti-inflammatory drugs in bovine milk by high-performance liquid chromatography with fluorescence detection

HPLC with fluorescence detection is considered for confirmatory analysis of group B veterinary drugs by the European Union legislation. A procedure for confirming the presence of anti-inflammatory non-steroidal drug (NSAID) residues in bovine milk by reversed phase high-performance liquid chromatography with fluorescence detection is herein described. The native fluorescence of nine drugs belonging to different NSAID sub-classes, namely flurbiprofen, carprofen, naproxen, vedaprofen, 5-hydroxy-flunixin, niflumic acid, mefenamic acid, meclofenamic acid and tolfenamic acid, allowed for detection in bovine milk down to 0.25–20.0 μg/kg. Confirmation of the nine NSAIDs is attained by fluorescence detection at characteristic excitation and emission wavelengths. The procedure described is simple and selective. Limits of quantification (LOQs) ranging between 0.25 and 20 μg/kg were measured; satisfactory trueness and within-laboratory reproducibility data were calculated at LOQ spiking levels, apart from 5-hydroxy-flunixin. The procedure developed is used in our laboratory for confirmation of each one of the above mentioned NSAIDs in bovine milk, to support results after HPLC quantitative analysis with UV–vis detection.     

Monitoring of melamine contamination in fat watery milk by the photoluminescence analysis

A photoluminescence method to detect the toxic melamine contamination in fat watery milk has been proposed. Despite the quite different luminescence origins of milk and melamine patterns, their wide emission spectra under UV excitation are similar and in the range of 2.2-3.5 eV. The complex milk photoluminescence spectrum composed of riboflavin, furosine, lactulose, Vitamin E and tryptophan emitting species can be modified if milk pattern is undergone by acid treatment (for example, in vinegar). At the same time the melamine emission is not subjected to any modification in vinegar. It allows quantitatively discriminating the melamine contamination in milk in linear range, at least, 0.05-7 g/l from different photoluminescence spectra of milk (water) with and without melamine. Limit of melamine detection achieves 0.01 g/l.     

Development and application of a capillary electrophoresis-electrochemiluminescent method for the analysis of enrofloxacin and its metabolite ciprofloxacin in milk

Enrofloxacin (ENR) is a fluoroquinolone developed exclusively for the use in veterinary practice for the treatment of respiratory and gastrointestinal infections, and ciprofloxacin (CIP) is its main active metabolite. Their contents are regulated by the EU Council Regulation no. 2377/90 in animal edible tissues. We developed a sensitive and rapid method for the determination of ENR and CIP by capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection. The method is based on the detection of aliphatic tertiary or secondary amino moieties in ENR and CIP with end-column tris(2,2-bipyridyl)ruthenium(II) electrochemiluminescence. Parameters that affect separation and detection were optimized. Under the optimized conditions, the calibration functions were linear in the range of 0.03–1 μg ml⁻¹ for ENR and 0.05–1.2 μg ml⁻¹ for CIP. The detection limits of ENR and CIR were 10 ng ml⁻¹ and 15 ng ml⁻¹, respectively, based on the signal-to-noise ratio of 3. The relative standard derivations of the peak height and the migration time for ENR and CIP were less than 4.13%. The developed method was successfully applied to determine ENR and CIP in milk with a solid-phase extraction procedure.     

Easy sample treatment for the determination of enrofloxacin and ciprofloxacin residues in raw bovine milk by capillary electrophoresis

An easy, selective, and sensitive method has been developed for the determination of enrofloxacin (ENR) and its main active metabolite, ciprofloxacin (CIP), in raw bovine milk using CE with UV detection at 268 nm. Milk samples were prepared by a clean‐up/extraction procedure based on protein precipitation with hydrochloride acid followed by being defatted by centrifugation and SPE using a hydrophilic‐lipophilic balance cartridge. Optimum separation was obtained using a 50 mM phosphoric acid at pH 8.4 and the total electrophoretic run time was 6 min. Sample preparation by this method yielded clean extracts with quantitative and consistent mean recoveries from 89 to 97% for CIP and from 93 to 98% for ENR. LODs obtained were lower to the maximum residue limits for these fluoroquinolones. The precision of the ensuing method is acceptable; thus, the RSD for peak area and migration time was less than 8.5 and 0.5% for CIP and 9.9 and 0.9% for ENR, respectively. The results showed that the proposed method was efficient showing good recoveries, sensitivity, and precision for the studied compounds and could be satisfactorily applied in routine analysis for the monitoring of ENR and CIP residues in milk, due to its ruggedness and feasibility demonstrated.     

Application of microbial receptor assay to quantitation of residues of spectinomycin in bovine milk and comparison with liquid chromatographic analysis.

Spectinomycin-contaminated bovine milk samples were assayed by liquid chromatographic (LC) and microbial receptor methods. LC involved a newly developed analytical method to quantitate the concentration of spectinomycin in the contaminated milk samples. The receptor assay used reagents and the reaction system used for the Charm II spectinomycin assay. Three standard curves (selected range, full range, and second-order polynomial) were plotted for the receptor assay and used to quantitate spectinomycin in contaminated milk samples. The levels of spectinomycin obtained by the receptor assay, using only the standard curve in the selected range, were comparable to the results obtained by LC analysis.     

Separation and quantification of the major casein fractions by reverse-phase high-performance liquid chromatography and urea-polyacrylamide gel electrophoresis. Detection of milk adulterations

A urea-formaldehyde resin (UF) continuous bed has been prepared through in-situ condensation polymerization in a confined tube. The monolith is an agglomerate of 2-microm irregular particles. Nitrogen adsorption shows that the monolith has a bimodal pore size distribution. It has low resistance to flow. A dyed monolith is obtained through modification of the UF monolith with Cibacron blue F3GA. Although its dye concentration and dynamic capacity are low compared to Sepharose type affinity media, the dyed monolith can separate some proteins in the affinity mode of liquid chromatography.     

Selective sample pretreatment by molecularly imprinted polymer monolith for the analysis of fluoroquinolones from milk samples

Water-compatible pefloxacin-imprinted monoliths synthesized in a water-containing system were used for the selective extraction of fluoroquinolones (FQs). The MIP monolith was synthesized by using methacrylic acid as the functional monomer, di(ethylene glycol) dimethacrylate as a cross-linker and methanol–water (10:3, v/v) as the porogenic solvent. The ability of the derivated MIP for selective recognition of FQs (ciprofloxacin, difloxacin, danofloxacin and enrofloxacin) and quinolones (flumequine, and oxolinic acid) was evaluated. The derivated monolith showed high selectivity and was able to distinguish between FQs and quinolones. A simple rapid and sensitive method using polymer monolith microextraction (PMME) based on the MIP monolith combined with HPLC with fluorescence detection was developed for the determination of four FQs from milk samples. Owing to the unique porous structure and flow-through channels in the network skeleton of the MIP monolith, phosphate buffer diluted milk samples were directly supplied to PMME; allowing non-specific bound proteins and other biological matrix to be washed out, and FQs to be selectively enriched. The limit of detection of the method was 0.4–1.6 ng/mL and recovery was 92.4–98.2% with relative standard deviations less than 5.9%.     

Computational optimization of the configuration of a spatially resolved spectroscopy sensor for milk analysis

A global optimizer has been developed, capable of computing the optimal configuration in a probe for spatially resolved reflectance spectroscopy (SRS). The main objective is to minimize the number of detection fibers, while maintaining an accurate estimation of both absorption and scattering profiles. Multiple fibers are necessary to robustify the estimation of optical properties against noise, which is typically present in the measured signals and influences the accuracy of the inverse estimation. The optimizer is based on a robust metamodel-based inverse estimation of the absorption coefficient and a reduced scattering coefficient from the acquired SRS signals. A genetic algorithm is used to evaluate the effect of the fiber placement on the performance of the inverse estimator to find the bulk optical properties of raw milk. The algorithm to find the optimal fiber placement was repeatedly executed for cases with a different number of detection fibers, ranging from 3 to 30. Afterwards, the optimal designs for each considered number of fibers were compared based on their performance in separating the absorption and scattering properties, and the significance of the differences was tested. A sensor configuration with 13 detection fibers was found to be the combination with the lowest number of fibers which provided an estimation performance which was not significantly worse than the one obtained with the best design (30 detection fibers). This design resulted in the root mean squared error of prediction (RMSEP) of 1.411 cm⁻¹ (R² = 0.965) for the estimation of the bulk absorption coefficient values, and 0.382 cm⁻¹ (R² = 0.996) for the reduced scattering coefficient values.     

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL⁻¹) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL⁻¹ its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).     

Residue analysis of 15 penicillins and cephalosporins in bovine muscle, kidney and milk by liquid chromatography–tandem mass spectrometry

A sensitive and specific liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method for most of those penicillins and cephalosporins for which EU maximum residue limits (MRL) were set in Council regulation (EEC) 2377/90 was developed and validated in bovine muscle, kidney and milk. The analytes were extracted with acetonitrile/water and cleaned-up by a single reversed-phase solid-phase extraction step. Highest sensitivity for the analytes was obtained when amoxicillin, ampicillin, cephalexin, cephapirin, desacetylcephapirin, cephalonium, cefquinome and cefazolin were measured in the positive electrospray ionisation mode (ESI (+)) and cefoperazone, benzylpenicillin, phenoxymethylpenicillin, oxacillin, cloxacillin, dicloxacillin and nafcillin in the negative electrospray ionisation mode (ESI (−)). Chromatography was performed with a formic acid/methanol gradient. Collision-induced dissociation (CID) with argon was used for fragmentation of the pseudomolecular ions to achieve the required specificity. Possible adverse matrix effects on the electrospray ionisation process caused by co-eluting matrix components were investigated. The method was validated closely to the new EU guidelines and applied to positively screened samples from official food control allowing the identification and quantification of the residual β-lactams.     

Development and application of a capillary electrophoresis based method for the simultaneous screening of six antibiotics in spiked milk samples

An easy to use and low time consuming capillary electrophoresis (CE) method was developed and applied to the simultaneous determination of six antibiotics (ampicillin, amoxicillin, cloxacillin, penicillin, tetracycline and chloramphenicol) in spiked milk samples. Samples of milk were cleaned up by solid-phase extraction (with a C₁₈ cartridge) after protein precipitation. Analysis was performed by CE and results compared with the obtained via HPLC, both coupled to a UV-vis detector (210 nm). CE employed a 58.5 cm long fused-silica capillary (50 cm to detector), 75 μm i.d., a 2.7 × 10⁻² M KH₂PO₄, 4.3 × 10⁻² M Na₂B₄O₇ separation buffer, pH 8; an applied voltage of 18 kV; a hydrostatic injection of 0.5 psi during 3 s; and a run temperature of 25 °C. Under the described conditions, amoxicillin was not separated by HPLC, while CE was able to separate, and, therefore, allow detection. Regardless of amoxicillin, comparable results were obtained by HPLC and CE. The average recoveries of antibiotics, from milk fortified at 2.5 and 5 μg/mL, was over 72% with R.S.D.s within 5%. Recovery levels were essentially dictated by the used SPE cartridge.     

Food contaminant analysis at high resolution mass spectrometry: application for the determination of veterinary drugs in milk

Veterinary drugs (VDs) can remain in milk as a consequence of their use in livestock. In order to control the levels of VD residues in milk, screening methodologies can be applied for a rapid discrimination among negative and non-negative samples. In a second stage, non-negative samples are classified as negative or positive samples by using a confirmation method. Pre-target screening methods in low resolution MS (LRMS) are normally applied, but the number of analytes is limited, whereas the information obtained by full scan acquisition in high resolution mass spectrometry (HRMS) is improved. Here, three screening methods (running time<4 min) based on Orbitrap, quadrupole-time of flight (QqTOF) and triple quadrupole (QqQ) have been compared, using in all cases ultra-high performance liquid chromatography (UHPLC). For HRMS, the identification of the VDs was based on retention time (RT) and accurate mass measurements. Confirmation was based on the monitoring of fragments generated without precursor selection. The performance characteristics of the screening method provided reliable information regarding the presence or absence of the compounds below an established value, including uncertainty region and cut-off values. Better results in terms of cut-off values (≤ 5.0 μg kg(-1), except for spiramycin with a cut-off of 13.4 μg kg(-1) for milk samples and 43.1 μg kg(-1) for powdered milk based, emamectin with a cut-off of 42.2 μg kg(-1) for milk samples and doxycycline, with a cut-off value of 15.8 μg kg(-1) in powdered milk-based infant formulae) and uncertainty region were obtained using the Orbitrap-based screening method, which was submitted to further validation and used to analyze different real milk samples. The proposed method can be used in routine analysis, providing reliable results.