Nathalie De Clercq

High resolution Orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CCαs reached from 0.04-0.88μgkg(-1) for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07-2.50μgkg(-1) was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.

A validated analytical method to study the long-term stability of natural and synthetic glucocorticoids in livestock urine using ultra-high performance liquid chromatography coupled to Orbitrap-high resolution mass spectrometry.

Due to their growth-promoting effects, the use of synthetic glucocorticoids is strictly regulated in the European Union (Council Directive 2003/74/EC). In the frame of the national control plans, which should ensure the absence of residues in food products of animal origin, in recent years, a higher frequency of prednisolone positive bovine urines has been observed. This has raised questions with respect to the stability of natural corticoids in the respective urine samples and their potential to be transformed into synthetic analogs. In this study, a ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) methodology was developed to examine the stability of glucocorticoids in bovine urine under various storage conditions (up to 20 weeks) and to define suitable conditions for sample handling and storage, using an Orbitrap Exactive™. To this end, an extraction procedure was optimized using a Plackett-Burman experimental design to determine the key conditions for optimal extraction of glucocorticoids from urine. Next, the analytical method was successfully validated according to the guidelines of CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone and methylprednisolone ranged, respectively, from 0.1 to 0.5μgL(-1) and from 0.3 to 0.8μgL(-1). For the natural glucocorticoids limits of detection and limits of quantification for dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.1 to 0.2μgL(-1) and from 0.3 to 0.8μgL(-1). The stability study demonstrated that filter-sterilization of urine, storage at -80°C, and acidic conditions (pH 3) were optimal for preservation of glucocorticoids in urine and able to significantly limit degradation up to 20 weeks.

Optimisation of enzymatic synthesis of cocoa butter equivalent from high oleic sunflower oil

BACKGROUND High oleic sunflower oil (HOSO) and a fatty acid (FA) mixture were inter-esterified in a solvent-free system catalysed by Lipozyme RM IM to produce a cocoa butter equivalent (CBE). The effects of reaction conditions on the percentage of saturate-oleoyl-saturate (SOS) and saturate-saturate-oleoyl (SSO) triacylglycerols (TAGs) were studied. The process was further optimised by response surface methodology. A five-factor response surface design was used to investigate the influences of the five major factors and their mutual relationships. The five factors were substrate ratio (A, FA/HOSO, mol mol⁻¹), enzyme load (B, wt% based on substrates), water content (C, wt% based on substrates), reaction temperature (D,°C) and reaction time (E, in hours) varying at three levels together with two star point levels. RESULTS The highest yield (59.1% SOS) and lowest acyl migration (2.9% SSO) was obtained at 10% enzyme load, 1% water content, 1:7 substrate mole ratio, 65°C reaction temperature and 6 h reaction time. All the investigated factors except substrate ratio had significant effect on acyl migration. CONCLUSION The quadratic response models sufficiently described the acidolysis reaction. All parameters had significant effect on the percentage of SOS TAGs. Based on the models, the reaction was optimised to obtain a maximum yield of SOS TAGs.

A novel approach to the quantitative detection of anabolic steroids in bovine muscle tissue by means of a hybrid quadrupole time-of-flight-mass spectrometry instrument☆

In recent years, the analysis of veterinary drugs and growth-promoting agents has shifted from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as Time-of-Flight (ToF) and Fourier Transform (FT)-MS). In this study, the performance of a hybrid analysis instrument (i.e. UHPLC-QuadrupoleTime-of-Flight-MS (QqToF-MS)), able to exploit both full scan HR and MS/MS capabilities within a single analytical platform, was evaluated for confirmatory analysis of anabolic steroids (gestagens, estrogens including stilbenes and androgens) in meat. The validation data was compared to previously obtained results (CD 2002/657/EC) for QqQ-MS and single stage Orbitrap-MS. Additionally, a fractional factorial design was used to shorten and optimize the sample extraction. Validation according to CD 2002/657/EC demonstrated that steroid analysis using QqToF has a higher competing value towards QqQ-MS in terms of selectivity/specificity, compared to single stage Orbitrap-MS. While providing excellent linearity, based on lack-of-fit calculations (F-test, α=0.05 for all steroids except 17β-ethinylestradiol: α=0.01), the sensitivity of QqToF-MS proved for 61.8% and 85.3% of the compounds more sensitive compared to QqQ-MS and Orbitrap-MS, respectively. Indeed, the CCα values, obtained upon ToF-MS/MS detection, ranged from 0.02 to 1.74μgkg(-1) for the 34 anabolic steroids, while for QqQ-MS and Orbitrap-MS values ranged from 0.04 to 0.88μgkg(-1) and from 0.07 to 2.50μgkg(-1), respectively. Using QqToF-MS and QqQ-MS, adequate precision was obtained as relative standard deviations for repeatability and within-laboratory reproducibility, were below 20%. In case of Orbitrap-MS, some compounds (i.e. some estrogens) displayed poor precision, which was possibly caused by some lack of sensitivity at lower concentrations and the absence of MRM-like experiments. Overall, it can be concluded that QqToF-MS offers good quantitative and confirmatory performance using the ToF-MS/MS mode whereas the full scan HR-ToF-MS allows screening for potential new designer drugs.

Development and validation of a high-resolution mass-spectrometry–based method to study the long-term stability of natural and synthetic glucocorticoids in faeces

Faecal glucocorticoid analysis is a powerful non-invasive tool for the study of the animal endocrine status and stress physiology, which is mainly carried out by immunoassays, characterised by some limitations. In this study, an ultra high-performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry (U-HPLC-HRMS) method was developed to confirm the presence of glucocorticoids in bovine faeces during a long-term stability study. Because of the complex nature of faeces, an appropriate extraction and purification procedure was developed. To this extent, a Plackett-Burman experimental design was successfully applied to determine the key conditions for optimal extraction of glucocorticoids from faeces. The targeted analysis, including natural and synthetic glucocorticoids, was successfully validated according to CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone, methylprednisolone and the metabolites 20α-dihydroprednisolone and 20β-dihydroprednisolone ranged, respectively, from 0.15 to 2.95 μg kg(-1) and from 0.40 to 5.20 μg kg(-1). Limits of detection and limits of quantification for the natural glucocorticoids dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.55 to 2.10 μg kg(-1) and from 0.70 to 5.00 μg kg(-1). The stability study of glucocorticoids in faecal matrix demonstrated that lyophilising the faeces, storage at -80°C, and aerobic conditions were optimal for preservation and able to significantly (p < 0.05) limit degradation up to 10 weeks.

Enzymatic and Other Modification Techniques to Produce Cocoa Butter Alternatives

Publisher Summary Cocoa butter (CB) is an essential ingredient in chocolate as it forms the continuous phase of chocolate. It is responsible for the gloss, texture, and typical melting behavior of chocolate. Although cocoa butter is the ideal ingredient, the varying supply and increasing price depending on fluctuating cocoa bean prices forced manufacturers to seek alternatives. Cocoa butter can be replaced by other vegetable fats, collected under the name Cocoa Butter Alternatives (CBAs). The CBAs are divided into three main categories according to their functionality and similarity to cocoa butter: the Cocoa Butter Equivalents (CBEs), the Cocoa Butter Replacers (CBRs), and the Cocoa Butter Substitutes (CBSs). A CBE should allow processing of chocolate products in an identical manner to that of cocoa butter-based products. The specific fatty acid profile and their distribution on the glycerol backbone in natural oils and fats affect their functional properties like crystallization and melting behavior. Therefore, the use of only one technique is not sufficient and several techniques need to be combined together to produce a cocoa butter alternative fat. Interesterification is an important modification technique resulting in the redistribution of the fatty acids along the glycerol backbone, which results in a change of the physicochemical properties of the fat.

Quality attributes of dark chocolates formulated with palm sap-based sugar as nutritious and natural alternative sweetener

Consumer demand for healthier alternative sweeteners and attempts to replace the most common sweetener used in chocolate, namely sucrose, continue to increase in recent times. One sucrose alternative that has not been fully explored in chocolate is palm sap-based sugar. This work investigated the impact of sucrose replacement by coconut sugar (CCS1 and CCS2) and palm sugar (CPS1, CPS2 and CPS3) on the quality attributes of dark chocolate, more particularly colour, hardness, flow behaviour and aroma profile. The results showed that chocolates formulated with palm sap-based sugar were lighter in colour and harder than the reference chocolate made with sucrose, which could be attributed to a lower particle density and a higher moisture of palm sap-based sugar than that of sucrose. Analysis of the major volatile compounds recorded the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) and high concentration of pyrazine-based compounds in the palm sap-based sugar-sweetened chocolates. The former compound (DDMP) was, however, absent in the sucrose-sweetened dark chocolate. The physicochemical properties of the sugars also had a significant effect on the rheological behaviour of the final chocolates with chocolates formulated with coconut sugar recording the highest Casson viscosity. With regard to fat melting, chocolates sweetened with palm sap-based sugar and sucrose exhibited similar melting range temperature. Palm sap-based sugar nevertheless seems to have great potential for dark chocolate applications with additional health benefits.

A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit

Carotenoids are important secondary metabolites, which have been recognized as an essential component of the human diet because of their valuable beneficial health effects. With this rationale, there is a continuous aim to define the distribution of these compounds in plants, to better understand their metabolism and to increase their concentration levels in fruits and vegetables. This study aimed at deepening the knowledge on the regulatory role of phytohormones in carotenoid metabolism. More specifically, it was envisaged to reveal the phytohormones involved in the metabolism of α-carotene, β-carotene, lycopene, lutein and zeaxanthin. To this purpose, the phytohormone profiles of 50 tomato fruits were determined by high-resolution Orbitrap mass spectrometry and evaluated towards the associated carotenoid levels. Data mining was performed by differential expression and orthogonal partial least squares analyses. This metabolomics approach revealed 5 phytohormonal metabolites, which significantly influenced (Variable Importance in Projection scores ≥0.80) carotenoid metabolism. These metabolites were identified as cis-12-oxo-phytodienoic acid, cucurbic acid, 2-oxindole-3-acetic acid, 1-acetylindole-3-carboxaldehyde, and cis-zeatin-O-glucoside. The involvement of the individual phytohormones towards carotenoid metabolism was investigated by regression analysis (P values ≤0.05, R2 varying between 0.280 and 0.760) and statistical correlation (P values ≤0.01, correlation varying between 0.403 and 0.846). It was concluded that these phytohormones all have significant contributing value in the regulation of carotenoid metabolism, thereby exhibiting down- and up-regulating influences. As a result, this knowledge encloses the potential for improving tomato fruit nutritional quality by targeted control of agronomic conditions, exogenous use of plant bioregulators, or genetic engineering.

The impact of stress on the prevalence of prednisolone in bovine urine: A metabolic fingerprinting approach

Recent studies support the hypothesis that the glucocorticoid prednisolone can be formed from cortisol under influence of stress. To evaluate this hypothesis, urine samples of supposedly non-stressed bovines (at the farm) and bovines subjected to two different forms of stress, i.e. upon slaughter (natural stress) or following administration of a synthetic analog of the adrenocorticotropic hormone (pharmacologically-induced stress) were analysed, and their urinary cortisol and prednisolone levels evaluated. At the farm, none of the examined samples exhibited urinary prednisolone levels higher than the CCα (0.09 μg L(-1)). Upon slaughter or following synthetically induced stress, significantly positive correlations between cortisol and prednisolone could be demonstrated, 0.52 and 0.69, respectively. Of all prednisolone-positive urine samples (n=84), only one showed a prednisolone levels (i.e. 6.45 μg L(-1)) above the threshold level of 5 μg L(-1) suggested by the European Reference Laboratories. Subsequently, an untargeted analysis was performed (metabolic fingerprinting) to characterize the urinary metabolite patterns related to the three different cattle groups. In this context, multivariate statistics assigned a total of 169 differentiating metabolites as playing a key role in the urinary pattern in response to stress. Three of these ions were defined as steroids using an in-house created database. As a result, the metabolic fingerprinting approach proved to be a powerful tool to classify unknown bovine urine samples that tested positive for prednisolone, while providing information about the stress status of the animal.

Development of an Offline Bidimensional High-Performance Liquid Chromatography Method for Analysis of Stereospecific Triacylglycerols in Cocoa Butter Equivalents

Acyl migration is a serious problem in enzymatic modification of fats and oils, particularly in production of cocoa butter equivalent (CBE) through enzymatic acidolysis reaction, which leads to the formation of non-symmetrical triacylglycerols (TAGs) from symmetrical TAGs. Non-symmetrical TAGs may affect the physical properties of final products and are therefore often undesired. Consequently, an accurate method is needed to determine positional isomer TAGs during the production of CBE. A bidimentional high-performance liquid chromatography (HPLC) method with combination of non-aqueous reversed-phase HPLC and silver ion HPLC joining with an evaporative light scattering detector was successfully developed for the analysis of stereospecific TAGs. The best separation of positional isomer standards was obtained with a heptane/acetone mobile-phase gradient at 25 °C and 1 mL/min. The developed method was then used in multidimensional determination of the TAG positional isomers in fat and oil blends and successfully identified the TAGs and possible isomers in enzymatically acidolyzed CBE.