To support labeling, claims, and authenticity of food products, industry needs reliable methods for the analysis of fatty acids, including trans fatty acids (TFA). In finished products, precise ...quantification of TFA can be problematic due to the occurrence of various positional and geometrical isomers originating from different sources, such as animal fats or processed vegetable oils and fats. The risk of underestimating TFA amounts is particularly high when inappropriate GC conditions are used. Complex sample preparation procedures involving purification of TFA isomers by silver ion chromatography have been well-documented and used for research purposes. However, in the food industry, time and cost constraints do not permit multiple analytical steps; therefore, streamlined methods are necessary. Direct methods include preparation of fatty acid methyl esters directly from food samples without prior extraction. The appropriate resolution is obtained using high-resolution GC with a highly polar 100 m capillary column, and quantification is achieved using experimentally determined response. We found that it is possible to quantify TFA in the range of 0.01 to 5.00 g/100 g of lipids in a wide range of food products. In addition, the use of direct transmethylation, response factors, and high-resolution GC allow accurate quantification of other fatty acids, including polyunsaturated and long-chain polyunsaturated fatty acids.
The increasing availability of different monoclonal antibodies (mAbs) opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast ...and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL) respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells.
The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to longer mouse survival, as compared to single mAb injection, up to complete inhibition of tumour growth in 100% mice treated with both anti-HLA-DR and anti-CD5.
Altogether these data suggest that the combined use of two mAbs, such as anti-HLA-DR and anti-CD5, may significantly enhance their therapeutic potential.
Long-chain polyunsaturated fatty acids (LC-PUFA) have various positive biological effects. Fish oil represents a major source of LC-PUFA; therefore it is extensively used to enrich food products as, ...for example, infant formulae, dairy products and fruit juices. However, in the presence of oxygen and metals, LC-PUFA readily degrade, producing off-flavors and decreasing the nutritional value of the product. The deterioration of sensory properties (taste and odor) can be easily perceived by the consumer, due to the formation of volatile compounds that are formed by decomposition of lipid hydroperoxides, also known as primary oxidation products. In this study, we used the headspace solid-phase microextraction-gas chromatography/mass spectrometry technique (HS-SPME-GC/MS) to characterize and quantify volatile compounds in a food matrix supplemented with fish oil. We demonstrated that the HS-SPME-GC/MS method is a valuable tool to monitor lipid oxidation at early stages. We identified t-2-hexenal and c-4-heptenal as possible oxidation markers during the storage of milk enriched with 5% of cod oil.
Long-chain polyunsaturated fatty acids (LC-PUFAs) of the
n-3 series and especially eicosapentaenoic and docosahexaenoic acids (EPA and DHA, respectively) have important biological properties. The ...main dietary sources of LC-PUFAs are fish and fish oil. Geometrical isomerization is one of the main reactions happening during the thermal treatment of polyunsaturated fatty acids. Refined fish oils are used to supplement food products in LC-PUFAs and the quality of these nutritional ingredients have to be controlled. In the present study, a suitable method for the quantification of EPA and DHA geometrical isomers in fish oils by gas–liquid chromatography (GC) is presented. A highly polar capillary column (CP-Sil 88, 100
m) operating under optimal conditions was used. Method selectivity was studied by GC–mass spectrometry. The performance characteristics of the quantification method were studied using samples of fish oil deodorized at 220
°C for 3
h. The linearity of the method was assessed by analyzing composite samples obtained by mixing fish oil deodorized at 220
°C with semi-refined fish oil (control). Precision was evaluated by analyzing the same samples in triplicate. Results showed that the validated method is suitable to quantify low amounts of geometrical (
trans) isomers of EPA and DHA in refined fish oils. The limits of quantification of the EPA and DHA geometrical isomers are 0.16 and 0.56
g/100
g of fish oil, for EPA and DHA, respectively. Commercially available LC-PUFA oil samples were evaluated by using the validated method. The results show that the oils analyzed contain low amounts (<1% of total fatty acids) of geometrical isomers of EPA and DHA.
Lipid oxidation has great impact on the quality of food products through flavor and taste deterioration, reduction in nutritive value, and potential toxicity of the oxidized food components. Flavor ...and taste deterioration can be easily perceived and it represents one of the major causes of consumer complaints in the food industry. The deterioration of sensory properties is due to the decomposition products of hydroperoxides that easily isomerize and degrade into volatile compounds. Volatile products are responsible for flavor and taste deterioration. In this study, we present the development of the solid‐phase microextraction gas chromatography‐mass spectrometry (SPME‐GC‐MS) technique to quantify low amounts (μg/g range) of secondary oxidation products, i.e. hexanal. The optimization of SPME parameters is a difficult task because of the possibility of further formation of volatile products during analysis. Different parameters such as type of fiber, exposure time of the fiber to the sample headspace and the optimal temperature of absorption have also been investigated. The complete validation of the method was achieved by the determination of linearity, limits of detection and quantification and repeatability. We demonstrated that the SPME method is a valuable tool for the quantification of low amounts of secondary oxidation products such as hexanal. Therefore, this technique can be used to detect early formation of volatiles.
Detection of foreign fat in milk fat can be performed by analyzing triacylglycerols (TAGs) by gas-liquid chromatography (GLC) using the standardized methodology. The standard methodology recommends ...the use of a packed column, which allows the separation of milk TAGs according to their chain length (total carbon number). This procedure is not widely applied because these columns are not commercially available. This study describes a fast methodology by using a short apolar open-tubular capillary column. The developed experimental conditions can be used to obtain the chromatographic resolution required in the standardized procedure, and the separation of milk fat TAGs (C24 to C54) is achieved in less than 4 min. As indicated by the standardized method, the quantification was performed by calibration using the certified reference material CRM-519 butterfat as standard substance. The methodology was fully validated and relative repeatability values were compared with the values provided in the standardized procedure. The developed method was applied to detect adulteration of milk fat with partially hydrogenated vegetable oils (PHVOs). PHVOs contain variable amount of trans-18:1 acids and two different PHVOs having different trans-18:1 acid levels (13 and 38%) were added to milk fat at levels ranging from 5 to 30%. The obtained mixtures were analyzed by GLC and formulas established by the European Union were applied. Calculated S values indicated that PHVOs in milk fat could be analyzed at these levels. Approximate amounts of PHVOs added to the composite samples could be calculated using the standardized formula. The impact of adulteration of milk fat with PHVOs, which contains an important amount of trans-9 and trans-10 18:1 acid isomers, was investigated as a complementary analytical criteria. We showed in composite samples, that the trans-18:1 acid isomeric distributions are distinct when referenced to the original milk fat profile and that trans-9 18:1 acid isomer is a good indicator of the occurrence of PHVOs in milk fat. Our results showed clearly that a short apolar capillary column can be used instead of a packed-column and that the mathematical model developed for the detection of foreign fat was suitable to detect adulteration of milk fat with PHVOs.
Heat flux differential scanning calorimetry (DSC) and electron spin resonance spectroscopy (ESR) were used to assess the activity and the thermal stability of antioxidants in four vegetable oils. ...Sunflower oil (SO) and high oleic sunflower oil (HOSO), both rich in diunsaturated fatty acids (FA), low trans oil (LT) and partially hydrogenated palm oil (PHPO), both containing monounsaturated FA, were analyzed by isothermal heat flux DSC, with or without 300
mg/kg of antioxidant: ascorbyl palmitate (AP), α-tocopherol (αT), δ-tocopherol (δT) and propyl gallate (PG). DSC experiments showed that δT is the most effective antioxidant for SO and PG for the less unsaturated oils. SO and PHPO were also analyzed by ESR at 120 and 145
°C, respectively. ESR results confirm the strongest antioxidant activity of δT and PG for SO and PHPO, respectively. Therefore, the present study demonstrates that DSC and ESR are valuable technologies to study activity and stability of antioxidants at high temperature. Moreover, experiments performed in the presence of the spin-trap
N-
tert-butyl-α-phenylnitrone (PBN), suggest that δT delay lipid oxidation through a different reaction mechanism when compared to αT. A different mechanism between tocopherols isomers in delaying lipid oxidation has been hypotized.
The determination of the occurrence and level of cocoa shells in cocoa products and chocolate is an important analytical issue. The recent European Union directive on cocoa and chocolate products ...(2000/36/EC) has not retained the former limit of a maximum amount of 5% of cocoa shells in cocoa nibs (based on fat-free dry matter), previously authorized for the elaboration of cocoa products such as cocoa mass. In the present study, we report a reliable gas−liquid chromatography procedure suitable for the determination of the occurrence of cocoa shells in cocoa products by detection of fatty acid tryptamides (FATs). The precision of the method was evaluated by analyzing nine different samples (cocoa liquors with different ranges of shells) six times (replicate repeatability). The variations of the robust coefficient of variation of the repeatability demonstrated that FATC22, FATC24, and total FATs are good markers for the detection of shells in cocoa products. The trueness of the method was evaluated by determining the FAT content in two spiked matrices (cocoa liquors and cocoa shells) at different levels (from 1 to 50 mg/100 g). A good relation was found between the results obtained and the spiking (recovery varied between 90 and 130%), and the linearity range was established between 1 and 50 mg/100 g in cocoa products. For total FAT contents of cocoa liquor containing 5% shells, the measurement uncertainty allows us to conclude that FAT is equal to 4.01 ± 0.8 mg/100 g. This validated method is perfectly suitable to determine shell contents in cocoa products using FATC22, FATC24, and total FATs as markers. The results also confirmed that cocoa shells contain FATC24 and FATC22 in a constant ratio of nearly 2:1. Keywords: Cocoa product; cocoa shell; fatty acid tryptamide; gas−liquid chromatography
Seven methods commonly used for fatty acid analysis of microgrganisms and foods were compared to establish the best for the analysis of lyophilized lactic acid bacteria. One of these methods involves ...fat extraction followed by methylation of fatty acids, while the other methods use a direct methylation of the samples, under different operating conditions (e.g., reaction temperature and time, reagents, and pH). Fatty acid methyl esters were identified by gas chromatography‐mass spectrometry and quantified by on‐column capillary gas chromatography. Two reliable methods for the analysis of fatty acids in bacteria were selected and further improved. They guarantee high recovery of classes of fragile fatty acids, such as cyclopropane and conjugated acids, and a high degree of methylation for all types of fatty acid esters. These two direct methylation methods have already been successfully applied to the analysis of fatty acids in foods. They represent a rapid and highly reliable alternative to classical time‐and solvent‐consuming methods and they give the fatty acid profile and the amount of each fatty acid. Using these methods, conjugated linoleic acids were identified and quantified in lactic acid bacteria.
A new European legislation (2000/36/CE) has allowed the use of vegetable fats other than cocoa butter (CB) in chocolate up to a maximum value of 5% in the product. The vegetable fats used in ...chocolate are designated as cocoa butter replacements and are called cocoa butter equivalents (CBE). The feasibility of CBE quantification in chocolate using triacylglycerol (TAG) profiles was conducted by analyzing 55 samples of CBs and 31 samples of CBEs using a liquid chromatograph equipped with an evaporative light scattering detector (HPLC-ELSD). Statistical evaluation of the data obtained has been performed, and a simulation study has been carried out to assess the viability to use this method for quantifying the amount of CBE in real mixtures and in chocolates. The TAGs POP, POS, PLS, and the ratios POP/PLS, POS/PLP (P, palmityl; O, oleyl; S, stearyl; L, linoleyl) are particularly significant to discriminate between CB and CBE. Analysis of 50 mixtures between 5 different CBEs and 10 different CBs at 2 different concentration levels is presented. The data are visualized and interpreted. A mathematical model has been developed to assess the amount of CBE in real mixtures. This predictive model has been successfully applied and validated on dark chocolates including authorized CBE. The results are affected by ±2.1% absolute average error. In particular, estimations between 10 and 20% of CBE show a very good match. On the other hand, values equal to or smaller than 5% show a larger prediction error (detection limit of the method). For the main purpose of this method (i.e., quantification of CBE at 5% max in chocolate, which represents about 15% of the total fat) this model shows very good results. For milk chocolate, the mathematical model can also be used if TAG are integrated from partition number (PN) 46 to 54. Consequently, the model proposed provides sufficient information to verify the real application of the European legislation. Keywords: Adulteration; cocoa butter; cocoa butter equivalent; chocolate; triacylglycerol; data visualization; high-performance liquid chromatography; vegetable fat
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