Method for the (partially) automated analysis of mineral oil contamination in foodstuffs during sample preparation

Abstract

A method for the (partially) automated analysis of mineral oil contaminants in foodstuffs is disclosed, which comprises the following steps in sample preparation: contacting and dissolving the foodstuff with a solution of an excess of a hydrocarbon solvent and an alcohol at a temperature between 20° C. and the boiling point of the solvent mixture; extracting, after cooling, and if alcohol was used in the previous step, initiating phase separation by adding water or a mixture of water and alcohol; separating the organic phase with an excess of at least 30% hydrogen peroxide and a compound of general formula (I) R 1 -COOH(I) in the presence of a small amount of inorganic acid at a temperature above 20°C to 70°C for 5 minutes to 2 hours, isolating the reagent by adding an excess of water, and using the hydrocarbon solvent extract for subsequent analysis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to German Application No. 102021125598.8, filed October 2, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a method for the analysis of mineral oil contaminants in foodstuffs. [Background technology]

[0003] Mineral oil pollution in the sense of the present invention refers to two different groups of compounds found in mineral oil: MOSH (mineral saturated hydrocarbons) are saturated mineral oil hydrocarbons, mainly kerosene and naphthenes, and MOAH (mineral aromatic hydrocarbons) are aromatic mineral oil hydrocarbons, mainly (polycyclic) aromatic hydrocarbons. MOSH accumulate in the human body, while MOAH are suspected of possibly containing carcinogenic substances.

[0004] Both types of contamination, which may originate from product packaging, lubricants or similar, have been routinely tested in foods for over 10 years and are the subject of constant discussion. The following standards and DGF publications (German Standards) describe the current state of the art regarding sample preparation and analysis.

[0005] In EN 16995-Vegetable oils and foodstuffs based on vegetable oils - Determination of MOSH and MOAH with online HPLC-GC-FID (as of June 2017), the direct determination of MOSH and MOAH in oils / fats soluble in n-hexane is described in section 9.1. In the presence of significant amounts of bio-based n-alkanes, section 9.3 prescribes an additional pre-purification via a silica gel ALOX column for the determination of MOSH. In this case, MOAH remains on the chromatographic column and must then be quantified independently of MOSH in a separate analysis.

[0006] Furthermore, in section 9.4, for the quantification of MOAH in the presence of significant amounts of biogenic olefinic sample components, purification by epoxidation using chloroperbenzoic acid (CPBA) (77% purity) performed at room temperature with initial cooling is proposed. The achievable quantification limit of this test method is 10 mg per kilogram of food for MOSH and MOAH. Combined with manual and error-prone sample preparation, this method is considered insufficient and not suitable for practical use.

[0007] The German Standard Method for the Analysis of Fats, Fat Products, Surfactants and Related Substances - Method C-VI 22, 26th update edition, German Society for Fat Science 2020 (hereafter referred to as DGF 2020) describes on page 11, under point 7, a procedure to increase the sensitivity (quantification limit of 1 mg / kg) by improving the sample preparation. For this, the sample is further purified via a silica gel column clean-up (step 7.4) after saponification (step 7.2). Separation of the biogenic n-alkanes via ALOX (step 7.3) is optional. The olefinic components are subsequently removed using epoxidation using chloroperbenzoic acid (CPBA) (77% purity) in ethanol at 40 °C and, if necessary, subsequent concentration of the solvent (step 7.5). This procedure also requires a preparative column chromatography before the actual analysis in order to reliably quantify MOSH and MOAH.

[0008] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or could reasonably be expected to be combined with other prior art by a person of ordinary skill in the art. Summary of the Invention

[0009] The present invention is based on the task of providing a method for the (partially) automated analysis of mineral oil contamination in foodstuffs, including edible fats and oils, which is technically simpler than the methods previously discussed from 2017 and 2020, in that it does not yet use column chromatography during sample preparation and, moreover, uses basic chemicals available in any laboratory for epoxidation (hydrogen peroxide, formic acid, mineral acids, etc.) instead of special fine chemicals that may be blind.

[0010] In a first aspect of the present disclosure, there is provided a method of preparing a sample for analysis of mineral oil contamination in foodstuffs, the method being at least partially automated, the method comprising: (i) contacting and dissolving or extracting the food material with a solution of a hydrocarbon solvent and, optionally, an excess of an alcohol, at a temperature between about 20° C. and the boiling point of the solvent mixture; (ii) cooling the solution and, if alcohol was used in the previous step, initiating phase separation by the addition of water or a mixture of water and alcohol; (iii) treating the organic phase containing the dissolved or extracted foodstuff with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R 1 -COOH (I), In the formula, R 1 represents H or the general subformula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 are, independently of each other, H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; (iv) partitioning the solution by adding water to provide an aqueous phase and a reacted organic phase; (v) separating the reacted organic phase for analysis of mineral oil contamination in the foodstuff; (vi) optionally, analyzing the sample.

[0011] In embodiments, the ratio of hydrocarbon solvent to alcohol (if present) may be about 1:x (v / v), where x is the volume of alcohol to the volume hydrocarbon solvent, and x is a value from 0 to 10. This ratio may be expressed herein as (1:x, x:0-10, v / v).

[0012] In an embodiment, prior to the epoxidation step (iii), the method may further comprise evaporating the organic phase and adding an amount of a halogenated aliphatic or aromatic hydrocarbon required to dissolve the sample.

[0013] Alternatively, in an embodiment, the epoxidation step (iii) may be carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon, and the method further comprises, after step (iii), evaporating the organic phase and exchanging the solvent with a hydrocarbon solvent.

[0014] The hydrocarbon solvent may be any suitable hydrocarbon solvent capable of extracting mineral oil from the food material. Thus, the hydrocarbon solvent is typically a non-polar hydrocarbon, such as a hydrocarbon containing 4 to 8 carbon atoms, preferably 5 or 6 carbon atoms. In an embodiment, the hydrocarbon solvent is hexane or pentane, or a combination thereof. In an embodiment, the hydrocarbon solvent is selected from n-hexane, n-pentane, iso-pentane, iso-hexane, or a combination thereof. In an embodiment, the hydrocarbon solvent is n-hexane. The hydrocarbon solvent may be used in step (i), optionally together with an alcohol. Typically, the alcohol is ethanol, although any suitable alcohol may be used. In an embodiment, the ethanol is at least 96% (by volume) ethanol. In an embodiment, the ethanol is absolute ethanol.

[0015] In an embodiment, a method is provided as described herein, which is fully automated.

[0016] The aforementioned epoxidation step (iii) generates the corresponding peracid from the compound of general formula (I) in situ in the presence of hydrogen peroxide in an acid-catalyzed manner. The alkanoic acid may be formic acid (R 1 =H), acetic acid or substituted acetic acids can also be used. Examples are mono-, di-, or trifluoroacetic acid, mono-, di-, or trichloroacetic acid, glycolic acid, propionic acid, 2-methylpropanoic acid, neopentanoic acid, in addition to acetic acid. Hydrochloric acid, phosphoric acid, nitric acid, and sulfuric acid can be used as inorganic acids.

[0017] According to one embodiment of the present disclosure, the food material comprises an alkaline hydrolysable component in an organic phase, and after step (i) and before step ii, the alkaline hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at a temperature between about 20°C and about 70°C; - Extracting the non-saponifiable components by adding a hydrocarbon solvent; - Optionally, by one or more repeated extractions with a hydrocarbon solvent,

[0018] In embodiments, the alkaline metal hydroxide solution is a sodium hydroxide solution or a potassium hydroxide solution, other strong bases are alkaline earth hydroxides such as barium hydroxide, calcium hydroxide, and strontium hydroxide, or organic bases such as alkylamines.

[0019] In embodiments, methods are provided herein that do not involve a prior clean-up step on silica gel to remove the matrix prior to the epoxidation step.

[0020] The method may be performed on any suitable quantified amount of sample. Starting the process with a known amount of sample is important to quantify the mineral oil content. In some cases, the method of the present invention allows for the analysis of smaller amounts of sample due to improved detection of MOSH / MOAH contaminants, but sufficient sample is required to ensure that the amount of MOSH / MOAH contaminants, if present, is above the detection limit of the selected analytical technique (e.g., LC-GC-FID). In some embodiments, a maximum sample of about 10 milliliters (mL), about 9 mL, about 8 mL, about 7 mL, about 6 mL, about 5 mL, about 4 mL, about 3 mL, about 2 mL, or about 1 milliliter (mL) or less may be used. In embodiments, the minimum sample volume may be at least about 1 microliter (μL), about 10 μL, about 50 μL, about 75 μL, about 100 μL, about 150 μL, about 200 μL, about 300 μL, about 400 μL, about 500 μL, about 600 μL, about 700 μL, about 800 μL, about 900 μL, or about 950 μL. In embodiments, the sample volume may be from any of these minimum volumes to any of these maximum volumes, e.g., from about 1 μL to about 10 mL, or from about 500 μL to about 2 mL. One of skill in the art will be able to select appropriate amounts of reagents and / or solvents depending on the mass / volume of the initial sample aliquot.

[0021] According to one embodiment of the present disclosure, the food material comprises an acidic hydrolysable component in an organic phase, and after step (i) and before step (ii), the acidic hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous hydrochloric acid or other strong acid at about 20°C to about 70°C; - Extracting the non-hydrolyzed components by adding a hydrocarbon solvent; - Optionally, by one or more repeated extractions with a hydrocarbon solvent,

[0022] In embodiments, the other strong acid is an inorganic acid, such as phosphoric acid, sulfuric acid, or nitric acid.

[0023] According to one embodiment of the present disclosure, the foodstuff is a dry foodstuff or a foodstuff present in an aqueous phase, and prior to step (i), mineral oil contaminants are removed from the foodstuff by: contacting the food material with an excess of an organic non-aqueous solvent; - separating the solvent containing the extracted mineral oil impurities from the foodstuff using any suitable means.

[0024] In one embodiment, extraction of mineral oil components from food materials is carried out using a solution of a hydrocarbon solvent and an excess of alcohol (1:x, x:0-10, v / v) at a temperature of or about 20° C. to the boiling point of the solvent mixture.

[0025] In an embodiment, the alcohol is ethanol. In an embodiment, the ethanol is at least 96% (by volume) ethanol. In an embodiment, the ethanol is absolute ethanol.

[0026] Preferably, any solvents and / or reagents used in the methods described herein are free of MOSH / MOAH contaminants.

[0027] The MOSH / MOAH content of the solvents and / or reagents may be quantified by similar methods described for the methods of the present disclosure, such as gas chromatography (e.g., using an MXT-1 column - Siltek treated stainless steel, 15 m x 0.25 mm x 0.25 μm, Restek, Bellefonte, PA, USA), or any other suitable technique, including, for example, LC-GC-FID, as described herein. Thus, the MOSH / MOAH content of any solvent and / or reagent used in the methods described herein may be below the detection limit of the analytical technique intended to be used to analyze the MOSH / MOAH content of the foodstuff sample.

[0028] In some embodiments, the methods include an initial step of determining the MOSH / MOAH content of solvents and reagents prior to their use, and optionally purifying the solvents and / or reagents to remove the MOSH / MOAH content prior to their use in the methods of the present disclosure. The solvents and / or reagents may be purified by techniques known in the art, such as distillation, chromatography, etc.

[0029] In one embodiment of the present disclosure, saponification of the organic phase is carried out at about 20° C. to about 70° C. using an excess of concentrated alkaline metal hydroxide solution in the form of sodium hydroxide solution and / or potassium hydroxide solution.

[0030] In one embodiment of the present disclosure, the halogenated hydrocarbon chloroform is added to the organic phase prior to the epoxidation step (iii).

[0031] In one embodiment of the present invention, the epoxidation, step (iii), comprises contacting the organic phase with a mixture of 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated phosphoric acid and / or sulfuric acid at a temperature of about 20° C. to about 70° C. for about 10 minutes to about 60 minutes.

[0032] According to one embodiment of the present disclosure, the foodstuff is selected from natural fats, protein-containing foodstuffs, carbohydrate-rich foodstuffs, alcohol-containing foodstuffs, alkaloid-containing foodstuffs, vegetables and vegetable products, fruits and fruit products, spices or herbs, drinking water, soft drinks, functional foodstuffs, dietary supplements, dietary foodstuffs, novel foods, vitamins, minerals, enzymes, lipids, amino acids, additives, or mixtures of the aforementioned substances, and / or foodstuff class products. According to one embodiment of the present disclosure, the natural fat is fruit pulp fat seed fat, or animal fat, or a combination thereof.

[0033] According to another embodiment of the present disclosure, the fat containing food product is milk or dairy products, chocolate, chocolate products, cocoa, margarine products, mixed fat products, infant formulas and follow-on formulas, or mixtures of the aforementioned products.

[0034] In a second aspect of the present disclosure, there is provided a system, comprising: a receptacle for a sample of a food material for analysis of the mineral oil content of the food material, the receptacle being in fluid communication with the first well, the second well, the third well, and the fourth well; a first well adapted for introduction into the receptacle of a hydrocarbon solvent and optionally an alcohol at a temperature between about 20° C. and the boiling point of the solvent mixture; a second well adapted for introducing an excess of at least 30% hydrogen peroxide into the receptacle; a third well, comprising a compound of general formula (I), R 1 -COOH (I), In the formula, R 1 represents H or the general subformula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 a third well adapted for introduction of a compound, wherein each of said compounds is independently H, F, Cl, OH, or methyl, into said receptacle in the presence of a catalytic amount of an inorganic acid at a temperature between about 20° C. and about 70° C.; a fourth well adapted to introduce water into the receptacle; a separator for separating the reacted organic phase from the receptacle and transporting the reacted organic phase to an analyzer; a controller for controlling the addition of the medium contained in each of the first, second, third and fourth wells to the receptacle and for controlling the temperature of the receptacle; - optionally an analyzer capable of detecting mineral oil in the reacted organic phase.

[0035] In some embodiments, the system includes a shaker for shaking the receptacle, which may also be under the control of the controller, hi some embodiments, the system may be adapted to automatically transfer the receptacle to the shaker.

[0036] In some embodiments, the separator comprises a centrifuge. The centrifuge may be controlled by the controller. In some embodiments, the system may be adapted to automatically transfer the receptacle to the centrifuge.

[0037] In a third aspect of the present disclosure, there is provided a use of the system described herein for carrying out the methods described herein.

[0038] In a further aspect, there is provided MOSH and MOAH analysis of a sample, wherein the sample is prepared according to the methods and embodiments of the first aspect described herein and the sample is analyzed.

[0039] In a further aspect, there is provided MOSH and MOAH analysis of a sample, wherein the sample is prepared and analyzed using the system of the second aspect and embodiment described herein.

[0040] In embodiments, the samples are analyzed by LC-GC-FID, or the samples are pre-separated using HPLC and analyzed using GC-MS or GCXGC-MS.

[0041] As used herein, unless the context otherwise requires, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude additional additives, components, elements or steps.

[0042] It is understood that when an embodiment is defined as "comprising," the narrower positions of "consists essentially of" and "consists of" are also disclosed. The term "consists essentially of" or variations such as "consisting essentially of" indicate that the embodiment includes all listed components or steps, and may include other unlisted components or steps that do not materially affect the basic properties or function of the embodiment. The term "consists of" or variations such as "consisting of" are intended to indicate that the embodiment is defined to exclude additional additives, components, or steps.

[0043] Further aspects of the disclosure and further embodiments of the aspects described in the previous paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0044] [Figure 1] Figure 2 shows the MOAH chromatogram of LC-GC-FID of palm oil after epoxidation (RBD - refined, bleached, deodorized) according to DGF standard method and EN 16995:2017. [Diagram 2] LC-GC-FID chromatograms of the MOAH fraction of palm olein after sample preparation according to example 1 of the present invention (top chromatogram) and prior art, comparative examples [EN 16995, DGF-2020]. Right panel: time less than 20 min. [Diagram 3] LC-GC chromatograms of the MOAH fraction of a 1:1 mixture of olive and sunflower oils after sample preparation with the addition of mineral oil to simulate contamination. The upper chromatogram shows the preparation according to DGF 2020, a comparative example, and the lower curve shows the method according to Example 2 of the present invention. [Figure 4]FIG. 1 shows MOAH chromatogram overlays of LC-GC-FID of epoxidized RBD palm oil showing remaining polyunsaturations depending on the epoxidation technique used. [Diagram 5] FIG. 1 shows the MOAH chromatogram of EIE palm oil after epoxidation highlighting the peak clusters found in multiple refined palm oil fractions. [Figure 6] Figure 6 shows an exploded view of the highlighted peak clusters from Figure 5. Monocyclic aromatic steroid hydrocarbons tendently identified in refined palm oil according to mass spectra and elution sequence from the literature. [Figure 7A] 1 shows the mass spectrum obtained for monocyclic aromatic hydrocarbons derived from sitosterol. [Figure 7B] 1 shows the mass spectrum obtained for monocyclic aromatic hydrocarbons derived from sitosterol. [Figure 7C] 1 shows the mass spectrum obtained for monocyclic aromatic hydrocarbons derived from sitosterol. [Figure 8] FIG. 1 shows a diagram of aromatic hydrocarbon structures and their half-life times for epoxidation with performic acid in chloroform. [Figure 9] The workflow for MOSH / MOAH sample preparation and analysis is shown. [Figure 10A] LC-GC-FID chromatograms of reagent blanks of MOSH (10A) and MOAH (10B) are shown. [Figure 10B] LC-GC-FID chromatograms of reagent blanks of MOSH (10A) and MOAH (10B) are shown. [Figure 11A] Figure 2 shows LC-GC-FID MOAH chromatograms of cocoa butter (A), sunflower oil (B), spiked rapeseed oil (C), spiked olive oil (D), spiked sunflower oil (E), and spiked palm oil (F) samples from the developmental studies of DGF Standard Method C-VI 22. [Figure 11B]Figure 2 shows LC-GC-FID MOAH chromatograms of cocoa butter (A), sunflower oil (B), spiked rapeseed oil (C), spiked olive oil (D), spiked sunflower oil (E), and spiked palm oil (F) samples from the developmental studies of DGF Standard Method C-VI 22. [Figure 11C] Figure 2 shows LC-GC-FID MOAH chromatograms of cocoa butter (A), sunflower oil (B), spiked rapeseed oil (C), spiked olive oil (D), spiked sunflower oil (E), and spiked palm oil (F) samples from the developmental studies of DGF Standard Method C-VI 22. [Figure 11D] Figure 2 shows LC-GC-FID MOAH chromatograms of cocoa butter (A), sunflower oil (B), spiked rapeseed oil (C), spiked olive oil (D), spiked sunflower oil (E), and spiked palm oil (F) samples from the developmental studies of DGF Standard Method C-VI 22. [Figure 11E] Figure 2 shows LC-GC-FID MOAH chromatograms of cocoa butter (A), sunflower oil (B), spiked rapeseed oil (C), spiked olive oil (D), spiked sunflower oil (E), and spiked palm oil (F) samples from the developmental studies of DGF Standard Method C-VI 22. [Figure 11F] Figure 2 shows LC-GC-FID MOAH chromatograms of cocoa butter (A), sunflower oil (B), spiked rapeseed oil (C), spiked olive oil (D), spiked sunflower oil (E), and spiked palm oil (F) samples from the developmental studies of DGF Standard Method C-VI 22. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0046] definition For purposes of interpreting this specification, terms used in the singular include the plural and vice versa. For example, "a" means one or more, unless otherwise specified.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test this disclosure, the preferred materials and methods are described below.

[0048] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of this disclosure, and the disclosure is in no way limited to the methods and materials described.

[0049] The term "measurement" as used herein should be interpreted in its broadest sense, including quantitative and qualitative measurements. It should be understood that unless otherwise specified, measurements are performed at ambient conditions, typically at room temperature and pressure, e.g., about 20-25°C, at about 1 atmosphere (atm).

[0050] As used herein, the term "and / or" means "and", or "or", or both.

[0051] The term "(s)" following a noun contemplates both the singular and the plural.

[0052] Reference to a numerical range disclosed herein (e.g., 1-10) is also intended to incorporate reference to all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), as well as any range of rational numbers within that range (e.g., 2-8, 1.5-5.5, and 3.1-4.7), and thus all subranges of all ranges expressly disclosed herein are expressly disclosed herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited are to be considered as expressly set forth in this application in a similar manner.

[0053] Various features of the present invention are described with reference to specific values ​​or ranges of values. These values ​​are intended to relate to the results of various suitable measurement techniques and should therefore be interpreted as including the error range inherent in any particular measurement technique. Some of the values ​​referred to herein are indicated by the term "about" to at least partially account for this variability. When the term "about" is used to describe a value, it can mean an amount within ±10%, ±5%, ±1%, or ±0.1% of that value.

[0054] As used herein, the term "excess" is intended to mean either a volume excess or a molar excess, unless the context requires otherwise. If not clear based on the context, references herein to an excess should be interpreted as a volume excess.

[0055] ingredients According to one embodiment of the present disclosure, the foodstuff is selected from natural fats, protein-containing foodstuffs, carbohydrate-rich foodstuffs, alcohol-containing foodstuffs, alkaloid-containing foodstuffs, vegetables and vegetable products, fruits and fruit products, spices or herbs, drinking water, soft drinks, functional foodstuffs, dietary supplements, dietary foodstuffs, novel foods, vitamins, minerals, enzymes, lipids, amino acids, additives, or mixtures of the aforementioned substances, and / or food class products. Examples of protein foods are meat, meat products including sausages, meat extracts, broth seasonings, gelatin, fish, crustaceans, shellfish and mollusks, fish products, eggs, egg products, vegetable protein products including those derived from soybeans and lupins. Examples of carbohydrate-rich foods include sugar, sugar alcohols, sugar confectionery, honey, cereals, bread and bakery products, baking agents, baking powder, pasta, starch. Examples of alcohol-containing foods are wine, sparkling wine, beer, brandy as an alcohol additive in confectionery and baked goods. Examples of alkaloid-containing foods are coffee, tea, cocoa, and chocolate. Examples of vegetables and vegetable products are fresh vegetables such as potatoes, tomatoes, cabbage vegetables, legumes, mushrooms, and vegetable durables such as frozen, canned, dehydrated, fermented, and pickled vegetables. Examples of fruits and fruit products include fresh fruits, dried fruits, candied fruit confections, jams, jellies, marmalades, fruit juices, and fruit nectars. Examples of spices or herbs include spices, spice blends, soy sauces, essences, salts, vinegars, and fruit acids of fruits, seeds, flowers, roots, barks, leaves, and herbs, in addition to fresh produce. Examples of soft drinks are mineral waters, sweet soft drinks, non-alcoholic soft drinks, sodas, and isotonic drinks. Examples of functional foods are products enriched with omega-3 fatty acids, ACE vitamins, or beta-glucans. Examples of additives are antioxidants, emulsifiers, thickeners, stabilizers, humectants, and flavorings.

[0056] According to one embodiment of the disclosure, the natural fat, in particular the refined fat, is a fat that can be used as a fat for foodstuffs in the sense of the present invention, for example vegetable fats, for example fruit fats, for example palm oil, olive oil, avocado oil, seed fats, for example coconut fat, salt fat, palm kernel fat, babassu fat, bay leaf fat, nutmeg butter, ukufuba fat, zika fat, cocoa butter, shea butter, Borneo fat, cottonseed oil, kapok oil, okra oil, corn seed oil, pumpkin seed oil, corn germ oil, cereal oils, sunflower oil, sesame oil, linseed oil, perilla oil, hemp oil, camellia oil, safflower oil, Niger oil, grapeseed oil, poppy seed oil, beech nut oil, hazelnut oil, soybean oil, peanut oil, beet oil, tung oil, oiticica oil, boreko oil, parinarium oil, ricinus oil, chaulmgra oil, hydnocarpus oil, gorli oil, Fats, especially refined fats, in the sense of the present invention are further understood to be terrestrial animal fats, such as pig fat, beef tallow, mutton tallow, horse fat, goose fat, chicken fat, or marine animal oils, such as whale oil, fish oil, fish and whale liver oil, palm oil, etc. Mixtures of the aforementioned fats are also possible here. Preferred fats in the sense of the present invention are palm fat, shea fat, coconut fat and salt fat in all marketable refinement stages, as well as vegetable fats and oils, as well as edible oils and edible fats, which include, by way of example, butter, margarine, speciality margarine. Special fats (plate fats, deep-frying fats), separating oils, mayonnaise and salad dressings.

[0057] The general detection of MOSH / MOAH content in foods is difficult due to the presence of other components in the oil, such as polyunsaturates, with signals that overlap with MOSH / MOAH in certain oils. Palm oil in particular is known to have a difficult matrix that makes the detection of MOSH / MOAH content difficult. Surprisingly, the inventors have found that the method of the present disclosure can detect MOSH / MOAH content in foods containing palm oil with improved sensitivity.

[0058] In some embodiments, the foodstuff comprises palm oil.

[0059] According to another embodiment of the present disclosure, the fat-containing food product is milk or milk products, chocolate, chocolate products, cocoa, margarine products, mixed fat products, infant formulas and follow-on formulas, or a mixture of the aforementioned products. Dairy products are, for example, condensed milk, dry milk products, sour milk products, cream, milk-based dessert products, butter, cheese, ice cream. Chocolate products are, for example, pralines, drinking chocolate, chocolate powder, hollow chocolate, chocolate sprinkles. Margarine products are, for example, household margarine, semi-fat margarine, baking margarine. Infant formulas and follow-on formulas are, for example, infant formulas in dry form based on dry milk powder.

[0060] automation As used herein, the phrase "at least partially automated" refers to a process or method in which at least two or more (or up to all) of the steps of the process or method can be performed automatically without the need for manual intervention.

[0061] In embodiments, partially automated refers to two automated steps. In embodiments, partially automated refers to three automated steps, or four automated steps, or five automated steps, or six automated steps. In embodiments, partially automated refers to all but one step being automated, or all but two steps being automated, or all but three steps being automated, or all but five steps being automated, or all but six steps being automated.

[0062] In embodiments, some of the automated steps are sequential steps. In embodiments, a series of automated steps may be followed by a manual processing step followed by two or more further automated steps.

[0063] As used herein, the phrase "fully automated process" refers to a process in which none of the steps require manual human handling other than providing a sample at the start of the process.

[0064] As described herein, the present disclosure relates to an at least partially automated method for analyzing MOAH and MOSH content in mineral oils, preferably edible oils.

[0065] Pre-extraction Optionally, the method can be applied when the food product is a dry food product and a pre-extraction step is required to obtain a edible oil, which can be done by any suitable means known in the art.

[0066] Suitable means include solid-liquid extraction in a Soxhlet extractor. Similarly, the common methods for fat extraction according to Weibull-Stoldt, Rose-Gottlieb or Schmid-Bondzynski-Ratzlaff are suitable for extracting non-polar mineral oil components, depending on the matrix of the sample.

[0067] Extraction Step In an embodiment of the present disclosure, the method includes an extraction step.

[0068] The extraction step is (i) contacting and dissolving or extracting the food material with a solution of a hydrocarbon solvent and, optionally, an excess of an alcohol, at a temperature between about 20° C. and the boiling point of the solvent mixture; (ii) cooling the solution.

[0069] In an embodiment, where an alcohol is used in step (i), the method further comprises initiating phase separation by the addition of water or a mixture of water and alcohol.

[0070] In embodiments, the volume:volume ratio of the hydrocarbon solvent to the alcohol may be from 1:x, where x is the volume of the alcohol relative to the hydrocarbon solvent, and x is a value from 0 to 10.

[0071] Saponification According to one embodiment of the present disclosure, the food material comprises an alkaline hydrolysable component in an organic phase, and after step (i) and before step ii, the alkaline hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at a temperature between about 20°C and about 70°C; - Extracting the non-saponifiable components by adding a hydrocarbon solvent; - Optionally, by one or more repeated extractions with a hydrocarbon solvent,

[0072] In an embodiment, the extraction with a hydrocarbon solvent is repeated 1 to 10 times.

[0073] In embodiments, the extraction step is repeated 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 times.

[0074] The hydrocarbon solvent may be any suitable hydrocarbon solvent capable of extracting mineral oil from the food material. Thus, the hydrocarbon solvent is typically a non-polar hydrocarbon, such as a hydrocarbon containing 4 to 8 carbon atoms, preferably 5 or 6 carbon atoms. In an embodiment, the hydrocarbon solvent is hexane or pentane, or a combination thereof. In an embodiment, the hydrocarbon solvent is selected from n-hexane, n-pentane, iso-pentane, iso-hexane, or a combination thereof. In an embodiment, the hydrocarbon solvent is n-hexane. Optionally, a saponification step may be performed after the extraction solution has cooled (step (ii) described herein).

[0075] In an embodiment, the alkaline metal hydroxide solution is a sodium hydroxide solution or a potassium hydroxide solution.

[0076] In embodiments, the other strong base is an alkaline earth hydroxide, such as barium hydroxide, calcium hydroxide, and strontium hydroxide, or an organic base, such as an alkylamine.

[0077] In an embodiment, saponification of the organic phase is carried out using an excess of concentrated alkaline metal hydroxide solution in the form of sodium hydroxide solution and / or potassium hydroxide solution at a temperature of about 20°C to about 70°C.

[0078] Advantageously, the saponification step is not limited by the volume of the sample and can be scaled as needed for the sample. One reason the sample volume can be increased is to increase the sensitivity of the measurement.

[0079] In an embodiment, the method does not involve a clean-up step on the silica gel to remove the matrix prior to the epoxidation step.

[0080] In prior art MOSH / MOAH analysis, HPLC cleanup on bare silica gel helped to remove the matrix, but this is not practical when the amount of sample increases beyond the limits of the HPLC column used. Choosing a larger HPLC column creates problems with the transfer volume to the GC. Advantageously, saponification can handle larger sample volumes. Advantageously, this step can also be automated.

[0081] acidic hydrolysis According to one embodiment of the present disclosure, the food material comprises an acidic hydrolysable component in an organic phase, and after step (i) and before step ii, the acidic hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous hydrochloric acid or other strong acid at about 20°C to about 70°C; - Extracting the non-hydrolyzed components by adding a hydrocarbon solvent; - Optionally, by one or more repeated extractions with a hydrocarbon solvent,

[0082] In an embodiment, the extraction with a hydrocarbon solvent is repeated 1 to 10 times.

[0083] In embodiments, the extraction step is repeated 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 times.

[0084] The hydrocarbon solvent can be any suitable hydrocarbon solvent capable of extracting mineral oil from foodstuff. Thus, the hydrocarbon solvent is typically a non-polar hydrocarbon, such as a hydrocarbon containing 4 to 8 carbon atoms, preferably 5 or 6 carbon atoms. In an embodiment, the hydrocarbon solvent is hexane or pentane, or a combination thereof. In an embodiment, the hydrocarbon solvent is selected from n-hexane, n-pentane, iso-pentane, iso-hexane, or a combination thereof. In an embodiment, the hydrocarbon solvent is n-hexane.

[0085] In embodiments, the other strong acid may be an inorganic acid, such as phosphoric acid, sulfuric acid, or nitric acid.

[0086] Epoxidation Step In an embodiment, the method includes an epoxidation step, which comprises: The organic phase containing the dissolved or extracted foodstuff is treated with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R 1 -COOH (I), In the formula, R 1 represents H or the general subformula (II), (R 2 )(R 3 )C(R 4 )- (II), In the formula, R 2 , R 3 , and R 4are, independently of each other, H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; partitioning the solution by adding water to provide an aqueous phase and a reacted organic phase; and separating the reacted organic phase for analysis of mineral oil contamination in the foodstuff.

[0087] The hydrocarbon solvent can be any suitable hydrocarbon solvent capable of extracting mineral oil from foodstuff. Thus, the hydrocarbon solvent is typically a non-polar hydrocarbon, such as a hydrocarbon containing 4 to 8 carbon atoms, preferably 5 or 6 carbon atoms. In an embodiment, the hydrocarbon solvent is hexane or pentane, or a combination thereof. In an embodiment, the hydrocarbon solvent is selected from n-hexane, n-pentane, iso-pentane, iso-hexane, or a combination thereof. In an embodiment, the hydrocarbon solvent is n-hexane.

[0088] In an embodiment, the epoxidation step is carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon.

[0089] In an embodiment, prior to the epoxidation step, the method includes evaporating the organic phase and adding a halogenated aliphatic or aromatic hydrocarbon. The amount of halogenated aliphatic or aromatic solvent may be the amount necessary to dissolve the sample.

[0090] If the epoxidation step (iii) is carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon, the method may further comprise evaporating the halogenated aliphatic or aromatic hydrocarbon and adding a hydrocarbon solvent after epoxidation to exchange the halogenated solvent for a preferred hydrocarbon solvent for analysis. Typically the hydrocarbon solvent is hexane, although any of the hydrocarbon solvents described for the sample preparation step can be used.

[0091] In embodiments, solvent exchange is carried out by any suitable means known in the art, such as by application of vacuum or gas flow in conjunction with heat and / or shaking, or a combination thereof.

[0092] In embodiments, the halogenated aliphatic hydrocarbon may be a chlorinated aliphatic hydrocarbon such as (but not limited to) chloromethane (monochloromethane, dichloromethane, chloroform, and carbon tetrachloride), chloroethane, vinyl chloride, 1,1-dichloroethylene, 1,2-dichloroethylene, trichloroethylene and perchloroethylene, chloropropane, chlorobutane, chlorobutene, fluorinated aliphatic fluoromethane, fluoroethane, fluoroethylene, fluoropropane, fluorobutane, and fluorobutene.

[0093] In a preferred embodiment, the halogenated aliphatic hydrocarbon is chloromethane, preferably dichloromethane or chloroform, most preferably chloroform.

[0094] In embodiments, the halogenated aromatic hydrocarbons may be chlorinated aromatic hydrocarbons such as chlorobenzenes, including (but not limited to) monochlorobenzene, dichlorobenzene, trichlorobenzene, tetrachlorobenzene, pentachlorobenzene, and hexachlorobenzene, and fluorinated aromatic hydrocarbons such as fluorobenzenes, including monofluorobenzene, difluorobenzene, trifluorobenzene, tetrafluorobenzene, pentafluorobenzene, and hexafluorobenzene.

[0095] In an embodiment, the solution of halogenated aliphatic or aromatic hydrocarbons does not include meta-chloroperoxybenzoic acid (mCPBA).

[0096] In some embodiments, the organic phase containing the extracted or dissolved food material further comprises a keeper solvent. The keeper solvent preferably does not contain unsaturated C-C bonds (e.g., olefins) that are reactive to epoxidation conditions. The keeper solvent is intended to keep the ingredients of the food material in solution to aid in the solvent exchange step, and is therefore preferred, for example, in methods involving solvent exchange between a hydrocarbon solvent and a halogenated solvent. Any suitable keeper solvent that is unreactive to epoxidation conditions can be used. Suitable keeper solvents include sebacic acid and the like. The amount of keeper solvent is not limited, provided that there is a minimum amount to keep the extracted or dissolved food material in solution, which minimum amount depends on the specificity of the extracted or dissolved food material.

[0097] In accordance with a preferred embodiment of the present invention, epoxidation is carried out by contacting the organic phase with a mixture of about 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated phosphoric acid and / or sulfuric acid at a temperature of about 20° C. to about 70° C. for about 10 minutes to about 60 minutes.

[0098] In an embodiment, the epoxidation is carried out by contacting the organic phase with a mixture of about 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated sulfuric acid at a temperature of about 20° C. to about 70° C. for about 10 minutes to about 60 minutes.

[0099] In an embodiment, the epoxidation reaction is carried out in the absence of phosphoric acid.

[0100] In embodiments, the formic acid is concentrated formic acid. In embodiments, the formic acid is neat formic acid (e.g., 100% concentration). Importantly, the acid should be free of MOSH / MOAH impurities.

[0101] In embodiments, the acetic acid is concentrated acetic acid. In embodiments, the formic acid is neat formic acid (e.g., 100% concentration). Importantly, the acid should be free of MOSH / MOAH impurities.

[0102] As used herein, a "catalytic amount" as used in reference to an acid present in an epoxidation reaction step is an amount sufficient to increase the rate of the reaction, e.g., an amount of about 5% to about 10%.

[0103] In an embodiment, the concentrated sulfuric acid has a purity of at least 95%.

[0104] In an embodiment, the concentrated phosphoric acid has a purity of at least 85%. In an embodiment, the catalytic amount of concentrated phosphoric acid and / or sulfuric acid is about 5% to about 10%. In a preferred embodiment, the catalytic amount is about 10%.

[0105] In an embodiment, the epoxidation is carried out in the presence of a catalytic amount of concentrated sulfuric acid. In an embodiment, the epoxidation reaction does not include phosphoric acid.

[0106] The temperature of the epoxidation reaction will vary depending on the analyte (size, chemical composition, etc.). In some embodiments, the minimum temperature of the epoxidation reaction may be at least about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, or about 50°C. In some embodiments, the maximum temperature of the epoxidation reaction may be about 70°C, about 65°C, about 60°C, about 55°C, or about 50°C or less. The temperature of the epoxidation step may be from any of these minimum temperatures to any of these maximum temperatures, provided that the maximum temperature is higher than the minimum temperature. For example, in some embodiments, the temperature of the epoxidation step is from about 30°C to about 55°C, or from about 40°C to about 60°C.

[0107] The required reaction time will depend in part on the temperature selected, and the nature of the analyte and the presence of biological interferences. Typically, the epoxidation reaction is allowed to run for about 10 minutes to about 60 minutes.

[0108] In embodiments, the sample volume for analysis can be up to 400 μL. Advantageously, increasing the sample amount increases the sensitivity of the analysis. Typically, prior art methods are limited to a maximum sample volume of 100 μL.

[0109] In embodiments, the hydrogen peroxide used in the epoxidation step, step (iii), is at least 35% hydrogen peroxide, or at least 40% hydrogen peroxide, or at least 45% hydrogen peroxide, or at least 50% hydrogen peroxide.

[0110] One problem with prior art methods of MOSH / MOAH analysis is the introduction of biological interferences remaining after the epoxidation step using mCPBA. These contaminants hinder the identification and quantification of MOAHs (see FIG. 1). Advantageously, the disclosed method ameliorates these effects and allows for better identification of MOAHs in samples.

[0111] Separation Step The method of the present disclosure comprises: partitioning the solution by adding water to provide an aqueous phase and a reacted organic phase; and For analysis of mineral oil contamination in foodstuffs, a separation step is further included to separate the reacted organic phase.

[0112] Further analysis The methods of the present disclosure relate to methods of preparing samples for analysis of mineral oil contaminants.

[0113] The prepared samples can be analyzed by suitable means known in the art, for example LC-GC-FID (online combined liquid chromatography gas chromatography flame ionization detection). Alternatively, the obtained extracts are pre-separated using HPLC and then analyzed by GC-MS or GCXGC-MS.

[0114] In an embodiment, the sample is evaporated to dryness before adding an aliquot of n-hexane. The current calibration curves for MOSH / MOAH are developed for n-hexane samples, so in a preferred method, any alternative solvent may be removed and replaced with n-hexane prior to analysis. Evaporation may be accomplished by any of the solvent exchange techniques known in the art and / or described herein. In at least partially automated methods of the present disclosure, this may be performed in an autosampler to automate this step as well.

[0115] When using the analytical methods described herein, a more accurate MOSH / MOAH content can be quantified and based on these results, it will be understood whether the foodstuff is recommended for sale based on the recommended MOSH / MOAH limits for the foodstuff.

[0116] In embodiments, methods are provided for preparing samples as described herein and analyzing the samples using LC-GC-FID.

[0117] system Also described herein is a system for carrying out the partially automated methods for analyzing mineral oil contaminants in foodstuffs described herein.

[0118] Accordingly, a second aspect is a system comprising: a receptacle for a sample of a food material for analysis of the mineral oil content of the food material, the receptacle being in fluid communication with the first well, the second well, the third well, and the fourth well; a first well adapted for introduction into the receptacle of a hydrocarbon solvent and optionally an alcohol at a temperature between about 20° C. and the boiling point of the solvent mixture; a second well adapted for introducing an excess of at least 30% hydrogen peroxide into the receptacle; a third well, comprising a compound of general formula (I), R 1 -COOH (I), In the formula, R 1represents H or the general subformula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 a third well adapted for introduction of a compound, wherein each of said compounds is independently H, F, Cl, OH, or methyl, into said receptacle in the presence of a catalytic amount of an inorganic acid at a temperature between about 20° C. and about 70° C.; a fourth well adapted to introduce water into the receptacle; a separator for separating the reacted organic phase from the receptacle and transporting the reacted organic phase to an analyzer; a controller for controlling the addition of the medium contained in each of the first, second, third and fourth wells to the receptacle and for controlling the temperature of the receptacle; - optionally an analyzer capable of detecting mineral oil in the reacted organic phase.

[0119] In embodiments, the system may further comprise a fifth well adapted to introduce a catalytic amount of an inorganic acid into the receptacle. Typically, the inorganic acid is added together with the compound of general formula (I), but in some embodiments, the system comprises this optional fifth well for its separate addition. The separate addition of the catalytic acid may assist and control the epoxidation reaction conditions within the system.

[0120] In an embodiment, the system may further comprise a sixth well adapted to introduce a halogenated aliphatic or aromatic hydrocarbon into the receptacle prior to the addition of hydrogen peroxide and / or the compound of general formula (I).

[0121] In some embodiments, the system includes a seventh well for providing an alcohol to the receptacle, separate from the hydrocarbon solvent.

[0122] In an embodiment, the first, second, third, and fourth, and optional fifth, sixth, and seventh wells each comprise an inlet for filling each well with the required solvent and / or reagent (e.g., the first well comprises a first inlet, the second well comprises a second inlet, the third well comprises a third inlet, the fourth well comprises a fourth inlet, the fifth well comprises a fifth inlet, the sixth well comprises a sixth inlet, and the seventh well comprises a seventh inlet). Each inlet may be adapted for manual filling with a desired volume of solvent or may be in communication with an external source of the required solvent and / or reagent. The supply of each solvent and / or reagent from each well to the receptacle is controlled by the controller. To facilitate this, each well may comprise a pump for controlling the supply of its contents to the receptacle. The pump may be a metering pump capable of delivering a desired amount of solvent and / or reagent to the receptacle, or in embodiments in which the well is filled with a fixed amount of solvent and / or reagent, the pump may be operable to eject the entire contents of the well into the receptacle.

[0123] The receptacle may be adapted to control the temperature of its contents. Thus, in an embodiment, the receptacle may comprise a thermocouple. In other embodiments, the receptacle comprises an inlet and an outlet through which the contents of the receptacle may be transported through a heat exchanger to control the temperature of the contents of the receptacle. The heat exchanger may be adapted to heat and / or cool the contents of the receptacle.

[0124] In some embodiments, the system may include a shaker for shaking the receptacle, which may also be controllable by the controller, hi some embodiments, the system may be adapted to automatically transfer the receptacle to the shaker.

[0125] In some embodiments, the separator comprises a centrifuge. The centrifuge may be controlled by the controller. In some embodiments, the system may be adapted to automatically transfer the receptacle to the centrifuge.

[0126] In some embodiments, the separator includes an autosampler for transporting the reacted organic phase to an analyzer.

[0127] In some embodiments, the system further comprises a solvent evaporation unit. The solvent evaporation unit is preferred when the method includes a step of exchanging a hydrocarbon solvent with a halogenated aliphatic or aromatic hydrocarbon solvent. The solvent evaporation unit may be any suitable for selectively removing a hydrocarbon solvent (e.g., hexane and / or pentane) from compounds present in the sample and / or for selectively removing a halogenated aliphatic or aromatic hydrocarbon from compounds present in the sample. Thus, the solvent evaporation unit may be one in which the hydrocarbon solvent is removed under reduced pressure or gas flow (e.g., nitrogen or argon), optionally with the application of heat and / or agitation (e.g., shaking or rotating). In some embodiments, the solvent evaporation unit is integrated with a receptacle. In other embodiments, the receptacle may be automatically transferred to the solvent evaporation unit. The solvent evaporation unit may be controllable by a controller when the removal of a volatile solvent is required in the methods described herein.

[0128] The controller is as follows: The organic phase containing the dissolved or extracted food material is then mixed with an excess of at least 30% hydrogen peroxide from the second well and a compound of general formula (I) from a third well, R 1 -COOH (I), In the formula, R 1 represents H or the general subformula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 are, independently of each other, H, F, Cl, OH, or methyl, preferably in the presence of a catalytic amount of an inorganic acid, at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; dividing the solution by adding water from a fourth well to provide an aqueous phase and a reacted organic phase; and operating a separator to separate the reacted organic phase for analysis of mineral oil contamination in the foodstuff; Optionally, the sample may be adapted to be analyzed in an analyzer.

[0129] In some embodiments, the controller may perform the following steps prior to the epoxidation step: (i) contacting and dissolving or extracting the food material in a receptacle with an excess of a solution of a hydrocarbon solvent from a first well and optionally an alcohol from a second well, the hydrocarbon solvent and optional alcohol being provided at a temperature between about 20° C. and the boiling point of the solvent mixture; (ii) cooling the solution in the receptacle and, if alcohol was used in the previous step, initiating phase separation by adding water or a mixture of water and alcohol, from the fourth well; The controller may preferably be adapted to perform steps (i) and (ii) above when the sample is an edible oil. For other foodstuffs, it may be preferable to perform steps (i) and (ii) manually before providing the sample to the at least partially automated system.

[0130] In some embodiments, the controller is also adapted to operate the solvent evaporation unit to substantially remove the hydrocarbon solvent from the dissolved or extracted food material in the receptacle, and preferably to add a quantity of a halogenated aliphatic or aromatic hydrocarbon from the sixth well.

[0131] In some embodiments, the controller is adapted to add a quantity of a halogenated aliphatic or aromatic hydrocarbon from the sixth well to the receptacle prior to the addition of hydrogen peroxide and / or the compound of general formula (I).

[0132] In some embodiments, the controller is also adapted to operate the solvent evaporation unit to substantially remove the halogenated aliphatic or aromatic hydrocarbon from the receptacle and add the hydrocarbon solvent from the first well.

[0133] In some embodiments, the controller is also adapted to operate a solvent evaporation unit to substantially evaporate the sample to dryness, and optionally add a hydrocarbon solvent from the first well.

[0134] In embodiments, the analyzer may be any device suitable for the detection of MOSH / MOAH in the hydrocarbon phase. Thus, in embodiments, the analyzer may comprise an LC-GC-FID or an HPLC coupled with a GC-MS or a GCXGC-MS.

[0135] The controller may be adapted to control the supply of solvents and / or reagents from the optional fifth and / or sixth and / or seventh wells to the receptacle.

[0136] In some embodiments, the system may include a column for further purification of the hydrocarbon phase collected from the receptacle before reaching the analyzer. The column may be any suitable column, such as an alumina, silica, or size exclusion gel. In a preferred embodiment, the system does not include a silica gel column (other than the column included in the analyzer that utilizes a silica chromatography component).

[0137] A third aspect provides the use of the system of the second aspect for carrying out the methods described herein.

[0138] Description of the embodiments Various embodiments of aspects of the present disclosure are described below.

[0139] 1. A method for preparing a sample for analysis of mineral oil contamination in foodstuffs, comprising: (i) contacting and dissolving or extracting the food material with an excess of a (1:x, x:0-10, v / v) solution containing n-hexane and optionally ethanol at a temperature between about 20° C. and the boiling point of the solvent mixture; - (ii) cooling the solution and, if ethanol was used in the previous step, initiating phase separation by adding water or a mixture of water and ethanol (1:x, x:0-10, v / v); - (iii) treating the organic phase produced in the previous step with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R 1 -COOH (I) In the formula, R 1 represents H or partial formula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 are each independently H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; -(iv) partitioning the solution by adding an excess of water to provide an aqueous phase and an n-hexane phase; and -(v) isolating the n-hexane phase for analysis of mineral oil contamination in foodstuffs; -(vi) optionally, analyzing the sample. 2. The method according to embodiment 1, characterized in that, prior to the epoxidation step (iii), the organic phase is evaporated and a halogenated aliphatic or aromatic hydrocarbon is added in an amount sufficient to dissolve the sample. 3. The method according to embodiment 1, characterized in that the epoxidation step (iii) is carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon, and the method further comprises, after step (iii), evaporating the organic phase and exchanging the solvent with n-hexane. 4. A method for preparing a sample for analysis of mineral oil contamination in foodstuffs, said method being at least partially automated, said method comprising: (i) contacting and dissolving or extracting the food material with an excess of a (1:x, x:0-10, v / v) solution containing n-hexane and optionally ethanol at a temperature between about 20° C. and the boiling point of the solvent mixture; - (ii) cooling the solution and, if ethanol was used in the previous step, initiating phase separation by adding water or a mixture of water and ethanol (1:x, x:0-10, v / v); - (iii) treating the organic phase produced in the previous step with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R 1 -COOH (I) In the formula, R 1 represents H or partial formula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 are each independently H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; -(iv) partitioning the solution by adding an excess of water to provide an aqueous phase and an n-hexane phase; and -(v) isolating the n-hexane phase for analysis of mineral oil contamination in foodstuffs; -(vi) optionally, analyzing the sample. 5. Prior to the epoxidation step (iii), the organic phase is evaporated and a halogenated aliphatic or aromatic hydrocarbon is added in an amount sufficient to dissolve the sample, or 2. The method according to embodiment 1, wherein the epoxidation step (iii) is carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon, and the method further comprises, after step (iii), evaporating the organic phase and exchanging the solvent with n-hexane. 6. The method according to embodiment 4 or 5, characterized in that at least two steps, or at least three steps, or at least four steps, or at least five steps, or at least six steps of the method are automated and performed without operator involvement. 7. The method according to embodiment 4 or 5, characterized in that the method is fully automated. 8. The food material comprises an alkaline hydrolysable component in an organic phase, and after step (ii) and before step (iii), the alkaline hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at a temperature between about 20°C and about 70°C; - extracting the non-saponifiable components by adding n-hexane; Optionally, the saponification is by one or more repeated extractions with n-hexane. 9. The food material comprises an alkaline hydrolysable component in an organic phase, and after step (ii) and before step (iii), the alkaline hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at a temperature between about 20°C and about 70°C; - extracting the non-saponifiable components by adding n-hexane; 8. The process according to any one of the preceding embodiments, characterized in that the saponification is carried out by, optionally, repeating the extraction with n-hexane one or more times. 10. The food material is a dry food material or a food material present in an aqueous phase, and prior to step (i), mineral oil impurities are removed from the food material by: contacting the food material with an excess of an organic non-aqueous solvent; - separating the solvent containing the extracted mineral oil impurities from the foodstuff using suitable means. 11. The process according to any one of the preceding embodiments, characterized in that the dissolution / extraction of mineral oil components in the food product is carried out using a (1:x, x:0-10, v / v) solution of n-hexane and an excess of 96% by volume or absolute ethanol at a temperature between about 20°C and the boiling point of the solvent mixture. 12. The process according to any one of embodiments 8 to 11, characterized in that the saponification of the organic phase is carried out using an excess of concentrated alkaline metal hydroxide solution in the form of a sodium hydroxide solution and / or a potassium hydroxide solution at a temperature between about 20 ° C and about 70 ° C. 13. The process according to any one of the preceding embodiments, wherein the halogenated hydrocarbon chloroform is added to the organic phase before the epoxidation, step (iii), is started. 14. The epoxidation, step (iii), 14. The process according to any one of the preceding embodiments, comprising contacting the organic phase with a mixture of 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated phosphoric acid and / or sulfuric acid at a temperature of about 20° C. to about 70° C. for about 10 minutes to about 60 minutes. 15. The method according to any one of the preceding embodiments, characterized in that the foodstuff is a substance or product that is intended or can reasonably be expected to be ingested by humans in a processed, partially processed or unprocessed state, and is in particular selected from natural fats, protein-containing foods, carbohydrate-rich foods, alcohol-containing foods, alkaloid-containing foods, vegetables and vegetable products, fruits and fruit products, spices or herbs, drinking water, soft drinks, functional foods, dietary supplements, dietary foods, novel foods, vitamins, minerals, enzymes, lipids, amino acids, additives, or mixtures of the aforementioned substances, and / or products of the food classes. 16. The method according to any one of the preceding embodiments, characterized in that the natural fat is a fruit fat or a seed fat or an animal fat, or a mixture of the aforementioned fats, preferably edible oils or fats. 17. The method according to any one of the preceding embodiments, wherein the fat-containing food product is milk or dairy products, chocolate, chocolate products, cocoa, margarine products, mixed fat products, infant formulas and follow-on formulas, or mixtures of the aforementioned products. 18. A method for preparing a sample for analyzing mineral oil contamination in foodstuffs, comprising: (i) contacting and dissolving or extracting the food material with an excess of a (1:x, x:0-10, v / v) solution containing n-hexane and optionally ethanol at a temperature between about 20° C. and the boiling point of the solvent mixture; - (ii) cooling the solution and, if ethanol was used in the previous step, initiating phase separation by adding water or a mixture of water and ethanol (1:x, x:0-10, v / v); - (iii) treating the organic phase produced in the previous step with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R 1 -COOH (I) In the formula, R 1 represents H or partial formula (II), (R 2 )(R 3)C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4 are each independently H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; -(iv) partitioning the solution by adding an excess of water to provide an aqueous phase and an n-hexane phase; and -(v) isolating the n-hexane phase for analysis of mineral oil contamination in foodstuffs; (vi) optionally analyzing the sample. 19. A method for preparing a sample for analysis of mineral oil contamination in foodstuffs, the method being at least partially automated, the method comprising: (i) contacting and dissolving or extracting the food material with an excess of a (1:x, x:0-10, v / v) solution containing n-hexane and optionally ethanol at a temperature between about 20° C. and the boiling point of the solvent mixture; - (ii) cooling the solution and, if ethanol was used in the previous step, initiating phase separation by adding water or a mixture of water and ethanol (1:x, x:0-10, v / v); - (iii) treating the organic phase produced in the previous step with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R 1 -COOH (I) In the formula, R 1 represents H or partial formula (II), (R 2 )(R 3 )C(R 4 )- (II) In the formula, R 2 , R 3 , and R 4are each independently H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; -(iv) partitioning the solution by adding an excess of water to provide an aqueous phase and an n-hexane phase; and -(v) isolating the n-hexane phase for analysis of mineral oil contamination in foodstuffs; (vi) optionally analyzing the sample. 20. The method according to embodiment 18 or 19, wherein prior to the epoxidation step (iii), the organic phase is evaporated and a halogenated aliphatic or aromatic hydrocarbon is added in an amount necessary to dissolve the sample. 21. The method of embodiment 18 or 19, wherein the epoxidation step (iii) is carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon, and the method further comprises, after step (iii), evaporating the organic phase and exchanging the solvent with n-hexane. 21. The method according to any one of embodiments 19 to 21, wherein the method is fully automated. 22. A method according to any one of embodiments 19 to 21, wherein at least two steps, or at least three steps, or at least four steps, or at least five steps, or at least six steps of the method are performed without operator involvement. 23. The method according to any one of embodiments 19 to 21, wherein the method is fully automated. 24. The food material comprises an alkaline hydrolysable component in an organic phase, and after step (Ii) and before step iii, the alkaline hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at a temperature between about 20°C and about 70°C; - extracting the non-saponifiable components by adding n-hexane; 24. The method according to any one of embodiments 18 to 23, wherein the saponification is optionally carried out by one or more repeated extractions with n-hexane. 25. The food material comprises an alkaline hydrolysable component in an organic phase, and after step (ii) and before step (iii), the alkaline hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at a temperature between about 20°C and about 70°C; - extracting the non-saponifiable components by adding n-hexane; 24. The process according to any one of embodiments 18 to 23, wherein the saponification is optionally carried out by one or more repeated extractions with n-hexane. 26. The food material is a dry food material or a food material present in an aqueous phase, and prior to step (i), mineral oil impurities are removed from the food material by: contacting the food material with an excess of an organic non-aqueous solvent; - separating the solvent containing the extracted mineral oil impurities from the food material using suitable means. 27. The process according to any one of embodiments 18 to 26, wherein dissolution / extraction of mineral oil components in the food product is carried out using a (1:x, x:0-10, v / v) solution of n-hexane and an excess of 96% by volume or absolute ethanol at a temperature of about 20°C to the boiling point of the solvent mixture. 28. The method according to any one of embodiments 18 to 27, wherein the saponification of the organic phase is carried out using an excess of concentrated alkaline metal hydroxide solution in the form of sodium hydroxide solution and / or potassium hydroxide solution at a temperature between about 20°C and about 70°C. 29. The process of any one of embodiments 18 to 28, wherein the halogenated hydrocarbon chloroform is added to the organic phase before the epoxidation, step (iii), is initiated. 30. The epoxidation, step (iii), 30. The process of any one of embodiments 18 to 29, comprising contacting the organic phase with a mixture of 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated phosphoric acid and / or sulfuric acid at a temperature of about 20° C. to about 70° C. for about 10 minutes to about 60 minutes. 31. The method according to any one of the preceding embodiments, wherein the foodstuff is a substance or product that is intended or can reasonably be expected to be ingested by humans in a processed, partially processed or unprocessed state, and is in particular selected from natural fats, protein-containing foods, carbohydrate-rich foods, alcohol-containing foods, alkaloid-containing foods, vegetables and vegetable products, fruits and fruit products, spices or herbs, drinking water, soft drinks, functional foods, dietary supplements, dietary foods, novel foods, vitamins, minerals, enzymes, lipids, amino acids, additives, or mixtures of the aforementioned substances, and / or products of the food classes. 32. The method according to embodiment 31, wherein the natural fat is a fruit fat or a seed fat or an animal fat, or a mixture of the aforementioned fats, preferably edible oils or fats. 33. The method according to any one of embodiments 18 to 31, wherein the fat-containing foodstuff is milk or dairy products, chocolate, chocolate products, cocoa, margarine products, mixed fat products, infant formulas and follow-on formulas, or mixtures of the aforementioned products. 34. The method of any one of embodiments 18-31, wherein the fat-containing foodstuff comprises palm oil. 35. A method for the (partially) automated analysis of mineral oil contamination in foodstuffs, comprising the steps of: - contacting and dissolving the food with an excess of n-hexane and ethanol (1:x, x:0-10, v / v) at a temperature between 20°C and the boiling point of the solvent mixture, i.e., extracting; - after cooling and, if ethanol was used in the previous step, initiation of phase separation by adding water or a mixture of water and ethanol (1:x, x:0-10, v / v), - The organic phase is treated with an excess of at least 30% hydrogen peroxide and a solvent having general formula (I) R 1 -COOH (I), wherein R 1 is H or the general formula (II) (R 2 )(R 3 )C(R 4 )-(II), wherein R 2 , R3 , and R 4 are each independently H, F, Cl, OH, or methyl, in the presence of a small amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours, with saponification; - separation of the reagents by adding an excess of water; - using the n-hexane extract for subsequent analysis, A process characterized in that the epoxidation step is preceded by evaporation of the organic phase and addition of the required amount of halogenated aliphatic or aromatic hydrocarbon to the solution, or the epoxidation step is followed by evaporation of the organic phase and exchange of the solvent with n-hexane in the presence of the halogenated aliphatic or aromatic hydrocarbon. 36. The process according to embodiment 35, characterized in that for foodstuffs having alkaline hydrolysable components in the organic phase, saponification is carried out after the first step by intimate contact with an excess of concentrated aqueous alkaline metal hydroxide solution or other strong base at above 20°C to 70°C, and subsequent extraction is carried out at least once by adding n-hexane. 37. The process according to embodiment 35, characterized in that for foodstuffs having acidic hydrolyzable components in the organic phase, the hydrolysis is carried out after the first step by intimate contact with an excess of concentrated aqueous hydrochloric acid or other strong acid at above 20°C to 70°C, and a subsequent extraction is carried out at least once by adding n-hexane. 38. The method according to embodiment 35, characterized in that in the case of dry foodstuffs or foodstuffs present in an aqueous phase, prior to the first step of sample preparation, extraction of mineral oil impurities from the foodstuff is carried out in a manner known per se using an excess of an organic non-aqueous solvent. 39. The process according to embodiment 35, characterized in that the dissolution / extraction of mineral oil components in the food product is carried out using a (1:x, x:0-10, v / v) solution of n-hexane and an excess of 96% by volume or absolute ethanol at a temperature between 20°C and the boiling point of the solvent mixture. 40. The process according to embodiment 36, characterized in that the saponification of the organic phase is carried out using an excess of concentrated alkaline metal hydroxide solution in the form of sodium hydroxide solution and / or potassium hydroxide solution at 20°C to 70°C. 41. The process according to embodiment 35, wherein the halogenated hydrocarbon chloroform is added to the organic phase before epoxidation is initiated. 42. The process according to embodiment 35, wherein the epoxidation is carried out by contacting the organic phase with a mixture of 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated phosphoric acid and / or sulfuric acid at a temperature of above 20° C. to 70° C. for 10 minutes to 60 minutes. 43. The method according to any one of embodiments 35 to 42, characterized in that the food is a substance or product that is intended or can reasonably be expected to be ingested by humans in a processed, partially processed or unprocessed state, and is in particular selected from natural fats, protein-containing foods, carbohydrate-rich foods, alcohol-containing foods, alkaloid-containing foods, vegetables and vegetable products, fruits and fruit products, spices or herbs, drinking water, soft drinks, functional foods, dietary supplements, dietary foods, novel foods, vitamins, minerals, enzymes, lipids, amino acids, additives, or mixtures of the aforementioned substances, and / or products of the food classes. 44. The method according to embodiment 43, characterized in that the natural fat is fruit fat or seed fat or animal fat, or a mixture of the aforementioned fats, preferably edible oils or fats. 45. The method according to embodiment 43, wherein the fat-containing food is milk or dairy products, chocolate, chocolate products, cocoa, margarine products, mixed fat products, infant formulas and follow-on formulas, or mixtures of the aforementioned products. EXAMPLES

[0140] The present invention will now be described by way of non-limiting examples. Those skilled in the art of the invention will understand that many modifications may be made thereto without departing from the spirit and scope of the invention.

[0141] Example 1 As an example, 1 gram of palm olein (iodine value 62-64) was weighed into a 20 mL screw-cap glass vial with a blank septum and dissolved in 10 mL of n-hexane / ethanol (1:1, v / v). 10 μL of internal standard was added (Restek GmbH, #31070, w=300 ng / μL). Subsequently, 2 mL of aqueous potassium hydroxide solution (50%, w / v) was added. The sample was saponified by the autosampler at 65 °C and 750 rpm for 20 min. After adding 4 mL of water, the sample was automatically centrifuged (2 min, 3500 rpm). The resulting upper phase was transferred via syringe to a new screw-cap glass vial. Another 5 mL of n-hexane was added and the sample was shaken. After subsequent centrifugation (2 min, 3500 rpm), the upper phases were combined. The organic phase was reduced in volume to approximately 1 mL with shaking at 250 rpm (65 °C) under a vacuum of 200 mbar. This was followed by the addition of 2 mL of chloroform and 70 μL of H3PO4 (Sigma Aldrich, 85 wt%, aqueous solution), 700 μL of formic acid (Sigma Aldrich, 98-100%), and 700 μL of H2O2 (Bernd kraft, 50%, aqueous solution). The extract was epoxidized for 30 min at 65 °C with vigorous shaking at 750 rpm. The reaction was stopped by adding 7.5 mL of water. To better transfer the organic lower phase, 2.5 mL of n-hexane was added for phase inversion. Thus, the upper phase could be transferred to a new screw-cap glass vial. After adding a keeper (50 μL of bis(2-ethylhexyl)-sebacate, Sigma Aldrich, >90%), the organic phase was carefully concentrated under vacuum (200 mbar, 250 rpm, 65 °C) and made up with 1.5 mL of n-hexane. To remove the last suspended solids, the solution was centrifuged again (2 min, 3500 rpm).

[0142] Up to 400 μL of the extract thus obtained could be injected into the LC-GC-FID system. The hexane extract injected into the HPLC column was separated on a silica gel phase by a normal phase gradient (hexane / DCM) into the substance classes MOSH and MOAH and the polar components. The HPLC column now completely takes over the task of the silica gel cleanup from the previous sample instructions. The MOSH and MOAH fraction cuts each constituted a volume of 600 μL, which was compressed by a large-capacity on-column transfer interface using partial simultaneous solvent evaporation and early vapor ejection. The two GC-compatible fractions obtained were separated on a suitable capillary column according to their boiling points and detected by flame ionization.

[0143] At the same time, the flow direction of the HPLC column was reversed (backflushed) to effectively remove polar residues from the column. The whole process took approximately 35 min before the column was ready for injection of the next sample.

[0144] If purification via alumina is required for the MOSH fraction, the corresponding fraction is passed from the silica gel column through an Al2O3 column before being transferred to the gas chromatograph. Thus, no manual ALOX-cleanup is required.

[0145] Example 2 Example 1 was repeated, but instead of refined palm olein, 1 g of an oil mixture of extra native olive oil and refined sunflower oil (1:1, v / v) was reprocessed. The work-up can be 100% similar, but allows for simplification if necessary.

[0146] If the edible oil contains only olefins with medium or high electron density, as is often the case for natural edible oils, the solvent change to halogenated hydrocarbons can be omitted, i.e., epoxidation can be carried out directly from the (saponified) hexane phase. This procedure ensures the acceleration of the test method presented herein.

[0147] Example 3 - Sample preparation according to DGF The fat according to Example 1 was subjected to sample preparation according to DGF as follows: 3 g of sample was weighed into a 40 mL screw-capped centrifuge tube. 30 mL of a mixture of n-hexane and ethanol (1:1, v / v) and 10 μL of ISTD (w=300 ng / μL) were added and shaken. 10 mL was transferred to a sample vial and 3 mL of potassium hydroxide solution (33 g / 100 g water) was added. The solution was saponified in a water bath at 60 °C for 30 min with shaking. Subsequently, 5 mL each of n-hexane and 5 mL of a mixture of ethanol and water (1:1, v / v) were added, the mixture was shaken again and after phase separation the lower phase was discarded.

[0148] Since palm olein contains only small amounts of bio-derived n-alkanes, ALOX purification was not necessary. The top phase from the saponification was transferred directly to a clean-up column (3 g silica gel + 1 g Na2SO4) and the hydrocarbons (MOSH+MOSH) were eluted from the column using 15 mL of a solvent mixture of n-hexane and DCM (7:3, v / v).

[0149] After adding two drops of bis(2-ethylhexyl)maleate, the mixture was concentrated in vacuo at 40° C. and brought to a volume of 1 mL with n-hexane, while keeping all internal standards.

[0150] To the extract thus obtained, 1 mL of ethanolic CPBA solution (100 mg / mL) was added and the sample was placed on a shaker for 20 min at 40 °C, for example at 500 rpm. Then, 500 μL of ethanol and 2 mL of a solution (Na2S2O3, 5 g / 100 mL + NaHCO3, 5 g / 100 mL of aqueous solution) were used so that excess CPBA would not react. The sample vial was shaken for about 1 min at about 750 rpm so that excess CPBA would not react. The upper hexane phase was transferred to a fresh sample vial and dried with a spatula tip amount of sodium sulfate. Now, the dried solution was injected into the LC-GC-FID system with an injection volume of 60-90 μL. To allow reproducible evaluation at low levels close to the quantification limit of 1 mg / kg, the solvent was adjusted to a more suitable volume of about 300 μL at 40 °C while maintaining the internal standard.

[0151] Figure 2 shows LC-GC-FID chromatograms of the MOAH fraction of palm olein after sample preparation according to Example 1 of the invention (top chromatogram) compared to Example 3 [EN 16995, DGF-2020]. In the right panel: for times less than 20 min, the lower chromatograms 2 and 3 show significant unresolved signal peaks that could be misinterpreted as MOAH, while these peaks are significantly reduced in the top chromatogram [according to the invention]. (All three methods were performed with identical amounts of sample injected for comparison). Since unresolved signal humps typically represent mineral oil contamination, a significantly lower false positive identification rate can be expected when using the test method presented herein. Coupled with the simplified sample preparation and increased injection volume (400 μL vs. 60-90 μL), the identification confidence close to the limit of quantification is also increased.

[0152] Figure 3 shows the LC-GC chromatograms of the MOAH fraction of a 1:1 mixture of olive and sunflower oils after sample preparation with the addition of mineral oil to simulate contamination. The upper chromatogram shows the preparation according to the comparative example of DGF 2020, while the lower curve shows the method according to example 2 of the invention. The MOAH content is 2.5 mg / kg. The unresolved signal hump (or unresolved signal peak) between 12 and 16 min corresponds to this amount. Also, for this "simple" oil, the new method shows improved chromatography. The whole chromatogram, especially the unresolved signal hump (or unresolved signal peak), shows fewer interfering individual peaks or peak clusters. (For comparison, both methods were carried out in such a way that the amount of sample injected was the same).

[0153] Example 4 Materials and Methods sample Extra virgin olive oil was obtained from a local supermarket and used for method development and validation. In addition, edible oil samples were available from a collaborative study carried out in 2020, organized by the DGF during the validation of standard method C-VI 22 (20). They consisted of cocoa butter, sunflower oil, spiked rapeseed oil, spiked olive oil, spiked sunflower oil, and spiked palm oil. EIE (enzymatic interesterification) and RBD palm oil were personal gifts.

[0154] Chemicals and Solutions n-Hexane was from Th. Geyer GmbH & Co. KG (CHEMSOLUTE, Renningen, Germany). Internal standards (ISTDs) for MOH quantification (cat. no. 31070-n-undecane, n-tridecane, bicyclohexyl, α-cholestane, n-pentylbenzene, 1-methylnaphthalene, 2-methylnaphthalene, 1,3,5-tri-tert-butylbenzene, perylene), retention time standards (cat. no. 31076), and EPA-PAH standards (cat. no. 31011) were supplied by Restek (Bellefonte, PA, USA). Industrial gear oil (Omala S2 GX68) and naphthenic process oil (Gravex 913) for spiking experiments were obtained from Shell Deutschland GmbH (Hanburg, Germany). Bulk aluminum oxide (90 active basic, 0.063-0.200 mm), bis(2-ethylhexyl) sebacate (for synthesis), chloroform (HPLC Plus, ≥99.9%), dichloromethane (Suprasolv), ethanol (Suprasolv), β-carotene (≥93% UV), 3,5-cholestadiene (≥93% HPLC), dibenzofuran (DBF, 98%), dibenzothiophene (DBT, 98%), formic acid (EMPROVE ESSENTIAL, 98-100%), meta-chloroperoxybenzoic acid (≦77%), phosphoric acid (85 wt. % in HO), isooctane (Suprasolv), potassium hydroxide (EMSURE, ≧85%), sodium metabisulfite (ReagentPlus, ≧99%), squalene (≧98%), 1,13-tetradecadiene (90%), and 1-octadecene (analytical standard) were from Merck (Steinheim, Germany). Sodium sulfate was from Fluka (Buchs, Switzerland). Hydrogen peroxide (50%, w / v) was obtained from Berndt Kraft GmbH (Duisburg, Germany). Water was supplied by a Milli-Q water purification system (Merck, Darmstadt, Germany).

[0155] 3A Epoxidation of model substances for kinetic studies To a 1 mL mixture of the model substance used in n-hexane or chloroform (20 μg / mL, w / v), an autosampler added 200 μL of formic acid (acidified with 10% H3PO4, v / v) and 200 μL of H2O2 aqueous solution to the sample. The vials were placed in a shaker and shaken at a speed of 750 rpm (revolutions per minute) for 5, 10, 15, 20, 25, and 30 minutes at 65 °C. During the reaction, significant amounts of oxygen and carbon dioxide were formed by side reactions, which increased the pressure inside the autosampler vial. Therefore, the vials were tightly closed.

[0156] Then, 2.5 mL of water was added to quench the reaction and induce phase separation. The vial was shaken at 750 rpm for 1 min. The vial was then centrifuged at 2000 rpm for 1 min to obtain a clear organic phase. 500 microliters of the n-hexanoic acid upper phase was transferred to a 2 mL autosampler vial pre-filled with a spatula tip of sodium sulfate. The dried organic phase was subjected to LC-GC-FID.

[0157] Sample Preparation Workflow Saponification One gram of fat or oil was weighed into a 20 mL autosampler vial. Five microliters of ISTD (300 μg / mL, w / v) and 10 mL of n-hexane / ethanol (1:1, v / v) were added. After adding 2 mL of aqueous KOH (1 g / L, w / v), the vial was tightly closed. Saponification was carried out at 65° C. for 20 min with steady shaking at 750 rpm. Then, 4 mL of water was added and the vial was vigorously shaken at 750 rpm for 1 min. After centrifugation at 3000 rpm for 1 min, the clear upper n-hexane phase was transferred to a fresh 20 mL autosampler vial. A second extraction was carried out by adding 5 mL of fresh n-hexane to the saponified sample, shaking and centrifugation. Both hexane phases were combined to give a final volume of about 8.5 mL.

[0158] Epoxidation Fifty microliters of bis(2-ethylhexyl) sebacate was added to the n-hexane extract of the saponified sample, and the solvent was evaporated by vacuum (65°C, 200 mbar) to a volume of approximately 1 mL. An autosampler then added 2 mL of CHCl3, 400 μL of formic acid (acidified with 10% H3PO4, v / v), and 400 μL of aqueous H2O2 to the sample. The vial was placed in a shaker and shaken at 750 rpm for 20 min at 65°C.

[0159] A mixture of 3.5 mL of n-hexane, 1 mL of ethanol, and 7 mL of water was then added to quench the reaction and induce phase separation. The added n-hexane and ethanol were used for phase inversion, allowing the organic layer to be aspirated from the top of the vial. The vial was shaken at 750 rpm for 1 minute. The vial was then centrifuged at 4000 rpm for 1 minute to obtain a clear organic phase.

[0160] Six milliliters of the upper organic phase was transferred to a 10 mL autosampler vial pre-filled with a spatula tip of sodium sulfate and gently evaporated to dryness (65° C., 200 mbar). Finally, the residue was dissolved in 1.5 mL of n-hexane, shaken at 750 rpm for 1 min and centrifuged at 4000 rpm for 5 min. The clear organic phase was subjected to LC-GC-FID.

[0161] Excess H2O2 in the aqueous phase of a 20 mL autosampler vial was destroyed by slowly adding 2 mL of aqueous sodium metabisulfite solution (300 g / L).

[0162] LC-GC-FID Method LC-GC-FID experiments were carried out on a CHRONECT Workstation MOSH / MOAH from Axel Semrau (Sprockhovel, Germany), which consisted of a 1260 Infinity II HPLC system (binary and quaternary pumps and variable wavelength detectors from Agilent Technologies, Waldbronn, Germany), an Agilent 7890B gas chromatograph equipped with two flame ionization detectors, and a CHRONECT Robotics platform based on a PAL3 autosampler (CTC Analytics AG, Zwingen, Switzerland).

[0163] The HPLC eluent was directed from the HPLC to the GC using four rotary switching valves (VICI AG International, Schenkon, Switzerland). The gas chromatograph was equipped with two on-column interfaces (Y-interfaces) and a solvent vapor outlet.

[0164] Typically, 400 μL of prepared sample (equivalent to 267 mg of edible oil or fat) was injected onto an Allure Si HPLC column (250 mm × 2.1 mm, 5 μm, 60 Å, Restek, Bellefonte, PA, USA) without additional column temperature control. The mobile phase consisted of n-hexane and dichloromethane. Starting with 100% n-hexane at 300 μL / min, the mobile phase was changed to 70% n-hexane after injection. This was held until 7.5 min. For removal of biogenic n-alkanes, MOSH was eluted (600 μL) from 2.0 to 4.0 min through a manually packed aluminum oxide HPLC column (125 mm × 2.1 mm, sorbent was activated at 500 °C for 16 h prior to use). After elution of the MOAH fraction (4.1–6.1 min, 600 μL), the column was backflushed with dichloromethane at 500 μL / min for 9 min. The column was then reconditioned with n-hexane at 500 μL / min for 15 min. Concurrently, the aluminum oxide HPLC column was backflushed with isooctane at 1 mL / min for 10 min, followed by reconditioning with n-hexane at 1 mL / min for 10 min. The MOAH elution window was verified by UV detection at 230 nm. 1,3,5-tri-tert-butylbenzene (TBB) and perylene (Per) marked the beginning and end of this window.

[0165] LC-GC transfer occurred by retention gap technique and partially simultaneous solvent evaporation (PCSE). An uncoated, deactivated precolumn (non-activated stainless steel, 10 m × 0.53 mm, Axel Semrau, Sprockhovel, Germany) was followed by a steel T-piece union (modified butt-to-butt connector, Trajan Scientific and Medical, Ringwod, Australia) connecting to the solvent vapor outlet and a separation column (MXT-1, Siltek treated stainless steel, 15 m × 0.25 mm × 0.25 μm, Restek, Bellefonte, PA, USA) coated with 100% dimethylpolysiloxane film.

[0166] From the HPLC, the MOSH and MOAH fractions were transferred to the GC at carrier gas inlet pressures (hydrogen) of 95 and 55 kPa, respectively, and an oven temperature of 60° C. The solvent vapor outlet was opened 0.5 min before the elution of each fraction and closed 0.4 min after the fraction was transferred. The recovery of undecane and n-pentylbenzene was metered under these conditions. At the same time, the carrier gas inlet pressure was set to 150 kPa and maintained throughout the analysis. The oven temperature was programmed from 60° C. (8 min) to 370° C. (6.5 min, total run time 30.00 min) at 20° C. / min. The FID base temperature was set at 380° C. The gas flows of air, hydrogen, and nitrogen were set at 300, 30, and 25 mL / min, respectively.

[0167] Data processing was performed in Clarity 8.5 (DataApex, Prague, Czech Republic). Quantification was based on bicyclohexyl (cyclohexylcyclohexane, Cycy) for MOSH and TBB for MOAH used as ISTD. MOxH content was calculated according to the following equation:

number

[0168] For identification, MOSH or MOAH fractions were collected and analyzed by GC-MS (GCMS-QP2020 NX, Shimadzu Europa GmbH, Duisburg, Germany). The temperatures of the ion source and transfer line were set at 250 and 320 °C, respectively. Data acquisition was performed in full scan mode (50–750 amu) at a rate of 3 spectra / s with EI ionization at 70 eV. Data processing was performed with GCMSsolution 4.52.

[0169] Figure 4 shows the MOAH chromatogram overlay of the LC-GC-FID of epoxidized RBD palm oil showing residual polyunsaturations. Not only did performic acid remove more of the interfering mCPBA, but it also showed virtually no contamination from percid. Epoxidation of this sample with mCPBA in ethanol showed the expected retardation of reaction rate compared to dichloromethane.

[0170] One of the problems with the present method for analyzing MOSH / MOAH in edible oils is the inconsistent quality of mCPBA, which leads to concerns regarding contaminants. Through GC-MS investigations, it was found that these contaminants originate from the synthesis of peracids. They consist mainly of polychlorinated benzenes and biphenyls (PCBs). Although washing of mCPBA with n-hexane has already removed the majority, disturbing residues may still be present, making additional cleanup mandatory. Particular care should be taken with pure mCPBA, as it tends to be sensitive to shocks. For this reason, alternative epoxidation reagents were checked. Performic acid and peracetic acid were further analyzed, as these compounds are widely used in the epoxidation of edible oils, for example to prepare epoxidized soybean oil. Both peracids are unstable under ambient conditions and therefore need to be generated in situ from hydrogen peroxide and formic or acetic acid, respectively. Strong inorganic acids, such as H3PO4 or H2SO4, act as catalysts to increase the rate of formation.

[0171] To that end, a challenging matrix such as RBD palm oil, known to have persistent interferences, was treated with both reagents for 30 min at 65° C. As expected, performic acid showed higher and faster removal of polyunsaturates than peracetic acid.

[0172] Surprisingly, the removal was even higher compared to mCPBA. In FIG. 4, a chromatogram overlay is shown showing that the MOAH fraction of RBD palm oil after epoxidation with performic acid contained fewer chromatographic interferences. It is also evident for this particular sample that epoxidation with mCPBA in ethanol is inferior to dichloromethane. Without wishing to be bound by theory, it is believed that this may be due to the polarity of the solvent causing a slowdown in the reaction rate. For these reasons, performic acid was chosen for further investigation of the nature of the epoxidation within the scope of this study.

[0173] Five model substances similar to typical bio-based olefins were selected and epoxidized with performic acid. Where appropriate, comparisons were made with conventional epoxidation with mCPBA. The epoxidation follows second-order kinetics according to the following equation:

number

[0174] If peracid is added in excess and its concentration is considered constant, the reaction follows pseudo first order and therefore the upper equation can be simplified from which important parameters such as the reaction rate constant (k1) and the half-life time (t1 / 2) can be derived.

number

[0175] Table 1 summarizes the results of these experiments. The epoxidation of squalene and β-carotene is much faster than that of 3,5-cholestadiene. Without wishing to be bound by theory, it is proposed that this is due to their conjugated π-electron systems. The half-life times for the epoxidation of 1,13-tetradecadiene and 1-octadecene were significantly higher. We propose that this may be due to the effect of the number and position of double bonds on the reaction rate. In addition, the effect of solvent polarity can be seen when comparing the results of mCPBA epoxidation in ethanol and dichloromethane.

[0176] While epoxidations carried out with mCPBA in dichloromethane showed the highest reaction rates for most compounds, this was not the case for terminal olefins when compared to performic acid in chloroform. This system was the only one capable of epoxidizing terminal olefins in a reasonable time and was therefore chosen for the desired sample preparation procedure.

[0177] 3B Explanation of MOAH chromatogram by GC-MS Next, the chromatographic signals after epoxidation were investigated. After HPLC separation, the MOAH fractions were collected and subjected to GC-MS analysis. Even though most of the signals could not be safely identified, a very prominent peak cluster was found in almost all refined palm oil fractions analyzed (highlighted in Figure 5 and enlarged in Figure 6). Interestingly, this was absent in virgin palm oil, implying its formation during the refining process.

[0178] Mass spectra indicated the inheritance of these compounds from plant sterols. The discovery of highly abundant m / z 211 and m / z 253 implied the presence of aromatic steroid compounds. Without wishing to be bound by theory, it is proposed that dehydration leads to the formation of stellatrienes. Subsequent rearrangement of the double bonds with aromatization of one ring moiety may result in monocyclic aromatic steroid hydrocarbons (see Figures 7A-C). It is proposed that these compounds cannot be removed by epoxidation since they share the same aromatic structure as the majority of MOAHs, but must be distinguished from the rest in other ways, such as by careful visual inspection of the LC-GC-FID chromatograms by GCxGC.

[0179] According to the definition, the classification of MOAHs is based solely on their chromatographic elution profile on bare silica gel and the practice of collecting the eluate containing the highly alkylated MACs and perylenes. The following substance classes typically found in (unrefined) mineral oils are eluted within this fraction: Monocyclic, bicyclic, tricyclic, and polycyclic aromatic compounds (partially hydrogenated) with various degrees of alkylation (MACs, BACs, TACs, and PACs), sulfur- or oxygen-containing aromatic heterocycles such as PASHs or PAOHs (nitrogen-containing heterocycles, i.e., PANHs, do not elute in the same window), MACs, and to some extent BACs, are the most prominent substance classes found in the MOAH. Higher conjugated aromatic systems are found to a much lesser extent.

[0180] To simply test which materials are affected by epoxidation, a model mixture was constructed containing MOH ISTD, EPA PAH, dibenzofuran, and dibenzothiopene. This mixture was subjected to epoxidation. In addition, two commercial mineral oils were epoxidized. The results are summarized in Table 2. [Table 2]

[0181] Firstly, it is noteworthy that, despite a few exceptions, all compounds show half-life times much higher than olefins (see Table 2 and Figure 8). MAC and DBF (as representatives of PAOH) are not significantly affected by epoxidation. Even in chloroform, which lacks a competing sample matrix, the half-life time of TBB is longer than 90 min. PASH is oxidized to sulfoxides and finally to sulfones in a short time. For BAC, TAC, and PAC, oxidation products such as phthalates or quinones have been reported in the literature. Losses do not strictly correlate with ring size or ionization energy, but are highly dependent on the solvent used. Chloroform not only allows the epoxidation of terminal olefins, but also accelerates the oxidation of aromatics.

[0182] As can be further seen in Table 2 for the naphthalene class (BAC), the losses of methylated naphthalane and acenaphthene are higher than the parent compounds, indicating that hyperconjugation by alkylation has a significant effect. The high loss of acenaphthylene is caused by its prominent double bond, which is susceptible to oxidation.

[0183] In the TAC class, anthracene is lost to a much higher extent than phenanthrene.

[0184] It can be summarized that the majority of the quantified MOAH content, i.e., MAC and BAC, is mainly unaffected by epoxidation, while harsh epoxidation conditions are unavoidable to remove olefins that would otherwise prevent reliable MOAH quantification.

[0185] Lowering the limit of quantification One of the simplest ways to improve the sensitivity of an analytical method is to increase the amount of sample analyzed. Obviously, by increasing the sample amount, the amount of undesirable sample by-products (or matrix) also increases. For the analysis of common hydrocarbons, saponification is a valuable tool to remove most of the matrix. Typically, the unsaponifiables of a typical edible oil or fat make up only 1-2% of the total weight.

[0186] While in the early stages of MOSH / MOAH analysis, HPLC cleanup of bare silica gel helped to remove the matrix, this approach becomes impractical when the sample volume increases beyond the limits of the HPLC column used. The choice of a larger HPLC column leads to problems regarding the transfer volume to the GC. Therefore, saponification is the simplest solution to tackle this obstacle, even if it leads to a more complicated sample preparation. Conveniently, this step can be automated.

[0187] EN16995:2017 used a sample amount of 20 mg and was validated with a LOQ of 10 mg / kg, so it is reasonable to assume that about 200 mg is needed to obtain 1 mg / kg (if other interferences do not prevent quantification). The DGF standard method only uses 100 mg of sample to facilitate manual work in the laboratory. In cases where the MOAH content is very low, a second evaporation step is mandatory to increase the sample amount to about 200-300 mg. In the current study, it was the aim to develop a method that works with a sample size of at least 200-300 mg, preferably 400 mg.

[0188] Typically, potassium hydroxide in an excess of ethanol is used to saponify primarily triglycerides at high temperatures. Other hydroxides or alcohols often result in the formation of insoluble fatty acid salts. The unsaponifiables are then extracted with a non-polar solvent such as n-hexane. This approach is also applicable to MOSH / MOAH detection.

[0189] Low recoveries of ISTDs after saponification and extraction of unsaponifiables have been reported in the past. Apparently, the large amount of soap formed during saponification can encapsulate some of the ISTDs, especially the BAC. The impact on MOAH has not been documented, but it is fair to assume that similar compounds are also partially lost. Own tests revealed losses of as much as 20% of methylnaphthalene, while other standards were fully recovered. The addition of a second n-hexane extraction solved this problem.

[0190] method design Epoxidation with performic acid in chloroform showed the best removal of interferences and was therefore used in conjunction with saponification. Solvent switching before HPLC injection was not considered an issue since the entire process was fully automated on the autosampler used. This made user-related issues during the evaporation step irrelevant. The overall process is presented in Figure 9.

[0191] Keeper solvent was pre-added to aid in hexane removal and solvent switching to chloroform. The traditionally used keeper, bis(2-ethylhexyl) maleate, could not be used because its olefinic double bond interferes with epoxidation. However, the corresponding sebacate was found to be suitable for the purpose.

[0192] Manual silica gel column chromatography was omitted because it was found that epoxidation with performic acid under highly acidic conditions showed three important advantages compared to conventional methods. First, polar sample by-products did not retard epoxidation. Second, washing of the n-hexane phase after saponification was not necessary because the residual fatty acid soaps were neutralized and finally removed by HPLC. Finally, the epoxides formed were rapidly cleaved by aqueous acid. The resulting diols show higher retention on bare silica gel. Thus, in the absence of triglycerides, the HPLC column used to remove these compounds could be used, and for samples with a low proportion of unsaponifiables, manual column chromatography was unnecessary. Visual inspection of the MOAH chromatograms verified the absence of polar by-products typical of oils and fats. In any case, in case of matrix breakthrough due to column aging, flushing with a polar eluent was a suitable method to restore the performance of the HPLC column.

[0193] As a final modification to the original method, the maximum injection volume into the HPLC was increased from 100 μL to 400 μL by using an enlarged sample loop and slight adjustments to the HPLC and GC transfer conditions. This modification made the final evaporation step during sample preparation more robust because the otherwise required volume of 375 μL could be increased to 1500 μL, allowing the use of 10 mL autosampler vials. Otherwise, special vials with conical inserts would have been required, or the evaporation would have had to be performed in two steps with intermediate liquid transfer to a smaller vial size.

[0194] Validation of the method The proposed sample preparation involving saponification, concentration and epoxidation was tested in terms of reproducibility on olive oil from a local supermarket. Eight measurements within a three week period led to MOSH and MOAH measurements of 14.9±1.0mg / kg and 2.1±0.1mg / kg, respectively. According to the latest collaborative test, the relative standard deviation under reproducible conditions was less than 7%, fully complying with the 25% tolerance.

[0195] According to the DGF standard method, the blank level should be less than 1 / 3 of the desired LOQ. The proposed method was subjected to a reagent blank, revealing that the blank values ​​of MOSH and MOAH were less than 0.2 mg / kg and 0.1 mg / kg, respectively (see Figures 10A and 10B). Therefore, the blank values ​​are independent of the desired LOQ of at least 1 mg / kg.

[0196] Accuracy and recovery experiments were performed on known samples previously used in collaborative studies within the DGF standard method development. Table 3 summarizes the results obtained and shows that acceptable comparisons were generally obtained within a z-score range of ±2. [Table 3]

[0197] However, some negative MOAH z-scores were obtained for cocoa butter, spiked rapeseed oil and palm oil. Whether this is related to unintentional losses of MOAH or to an overestimation of MOAH by the participants of the collaborative study could not be fully clarified. The corresponding chromatograms are shown in Figures 11A-11F. The results for the MOAH of sunflower oil and rapeseed oil, which are below the desired LOQ, show how important it is to inject as large a sample volume as possible with the lowest achievable contamination. Otherwise, reliable integration will not be possible.

[0198] For MOSH, samples containing large amounts of biogenic n-alkanes exhibit z-scores outside the desired range, which may be due to variability in the efficiency of the aluminum oxide cleanup.

[0199] conclusion The results indicate that removal of persistent biological interferences should be preferred over the milder reaction conditions of epoxidation. Epoxidation with performic acid in chloroform proved to be the most effective reagent for removing bioderived π-electron deficient olefins, representing an important achievement in MOSH and MOAH sample preparation. Although MOAH losses of TAC and PAC cannot be completely prevented, they are quantitatively irrelevant due to the low abundance of these compounds in refined mineral oils. Major classes such as MAC and BAC are affected by epoxidation to a much lesser extent.

[0200] A fully automated method was established in combination with a streamlined workflow involving saponification, concentration, and increased injection volume into the HPLC. Quantitative results on collaborative test samples validate the accuracy of the proposed method, making it suitable for the analysis of edible oils and fats in a routine environment.

Claims

1. 1. A method for preparing a sample for analyzing mineral oil contamination in foodstuffs, said method being at least partially automated, said method comprising: (i) contacting and dissolving or extracting the food material with a solution of a hydrocarbon solvent and optionally an excess amount of an alcohol at a temperature between 20°C and the boiling point of the solvent mixture; (ii) cooling and, if an alcohol was used in the previous step, initiating phase separation by addition of water or a mixture of water and said alcohol; (iii) treating the organic phase containing the dissolved or extracted food material with an excess of at least 30% hydrogen peroxide and a compound of general formula (I), R １ -COOH (I) In the formula, R １ represents H or the general subformula (II), (R ２ )(R ３ )C(R ４ )- (II) In the formula, R ２ , R ３ , and R ４ are each independently H, F, Cl, OH, or methyl, in the presence of a catalytic amount of an inorganic acid at a temperature of about 20° C. to about 70° C. for about 5 minutes to about 2 hours; (iv) partitioning the solution by adding excess water to provide an aqueous phase and a reacted organic phase; (v) separating the reacted organic phase for the analysis of mineral oil contamination in the foodstuff; (vi) optionally analyzing the sample.

2. Before the epoxidation step (iii), the organic phase is evaporated and a halogenated aliphatic or aromatic hydrocarbon is added in an amount sufficient to dissolve the sample; or 10. The process of claim 1, wherein the epoxidation step (iii) is carried out in the presence of a halogenated aliphatic or aromatic hydrocarbon.

3. 10. The method of claim 1, wherein the method further comprises, after step (iii), evaporating the organic phase and exchanging the solvent with n-hexane.

4. 10. The method of claim 1, wherein the hydrocarbon solvent is hexane or pentane, or a combination thereof.

5. 10. The method of claim 1, wherein the hydrocarbon solvent is a solvent selected from n-hexane, n-pentane, iso-pentane, iso-hexane, or a combination thereof.

6. The method of claim 1, wherein the hydrocarbon solvent is n-hexane.

7. The method of claim 1 , wherein the alcohol is ethanol.

8. The method of claim 1 , wherein the method is fully automated.

9. the food material comprises an alkaline hydrolysable component in the organic phase, and after step (ii) and before step (iii), the alkaline hydrolysable component is contacting the dissolved or extracted food material with an excess of concentrated aqueous alkaline metal hydroxide or other strong base at above about 20°C to about 70°C; - extracting the non-saponifiable components by adding said hydrocarbon solvent; - optionally repeating said extraction with said hydrocarbon solvent one or more times.

10. the food material comprises an acidic hydrolysable component in the organic phase, and after step (ii) and before step (iii), the acidic hydrolysable component is - contacting the dissolved or extracted food material with an excess of concentrated aqueous hydrochloric acid or other strong acid at about 20°C to about 70°C; - Extracting the non-hydrolyzed components by adding a hydrocarbon solvent; - optionally repeating said extraction with said hydrocarbon solvent one or more times.

11. The food material is a dry food material or a food material present in an aqueous phase, and prior to step (i) and prior to step (ii), mineral oil impurities are removed from the food material by: - contacting the food material with an excess amount of an organic non-aqueous solvent; - separating the solvent containing the extracted mineral oil impurities from the food material using suitable means.

12. 10. The method of claim 1, wherein the dissolution and / or extraction of mineral oil components in the food product is carried out using an excess solution of a hydrocarbon solvent and an alcohol, wherein the volume:volume ratio of the hydrocarbon solvent to ethanol is 1:x, where x is a number between 0 and 10 at a temperature or between about 20° C. and the boiling point of the solvent mixture.

13. 10. The process of claim 9, wherein the saponification of the organic phase is carried out using an excess of concentrated alkaline metal hydroxide solution in the form of sodium hydroxide solution and / or potassium hydroxide solution at a temperature of from about 20°C to about 70°C.

14. 10. The method of claim 1, wherein the halogenated hydrocarbon chloroform is added to the organic phase before the epoxidation, step (iii), is initiated.

15. The epoxidation, step (iii), 10. The method of claim 1, comprising contacting the organic phase with a mixture of 50% hydrogen peroxide and concentrated formic acid and / or acetic acid in the presence of a catalytic amount of concentrated phosphoric acid and / or sulfuric acid at a temperature of from about 20° C. to about 70° C. for from about 10 minutes to about 60 minutes.

16. 2. The method of claim 1, wherein the foodstuff is a substance or product that is intended to be or can reasonably be expected to be ingested by humans in a processed, partially processed or unprocessed state, and is selected in particular from natural fats, protein-containing foods, carbohydrate-rich foods, alcohol-containing foods, alkaloid-containing foods, vegetables and vegetable products, fruits and fruit products, spices or herbs, drinking water, soft drinks, functional foods, dietary supplements, dietary foods, novel foods, vitamins, minerals, enzymes, lipids, amino acids, additives, or mixtures of the aforementioned substances and / or products of the food class.

17. 17. The method according to claim 16, wherein the natural fat is a fruit fat or a seed fat or an animal fat, or a mixture of the aforementioned fats, preferably edible oils or fats.

18. 18. The method of claim 17, wherein the fat-containing food product is milk or dairy products, chocolate, chocolate products, cocoa, margarine products, mixed fat products, infant formula and follow-on formula, or mixtures of the foregoing products.

19. 2. The method of claim 1, wherein step (vi) is carried out by a coupled liquid and gas chromatogram (LC-GC-FID), preferably equipped with an FID detector.

20. 1. A system for carrying out the partially automated method for analyzing mineral oil contaminants in foodstuffs, comprising: a receptacle for a sample of a food material for analysis of the mineral oil content of said food material, said receptacle being in fluid communication with the first well, the second well, the third well, and the fourth well; the first well adapted for introducing into the receptacle a hydrocarbon solvent and optionally an alcohol at a temperature between about -20°C and the boiling point of the solvent mixture; - said second well adapted to introduce an excess of hydrogen peroxide of at least 30% into said receptacle; a third well, which is a compound of general formula (I), R １ -COOH (I), In the formula, R １ represents H or the general subformula (II), (R ２ )(R ３ )C(R ４ )- (II) In the formula, R ２ , R ３ , and R ４ are, independently of each other, H, F, Cl, OH, or methyl, and a third well adapted for introduction into said receptacle at a temperature of about 20° C. to about 70° C., preferably in the presence of a catalytic amount of an inorganic acid; - said fourth well adapted to introduce water into said receptacle; a separator that separates the reacted organic phase from the receptacle and transports the hydrocarbon phase to an analyzer; a controller for controlling the addition of the solvent contained in each of the first, second, third and fourth wells to the receptacle and for controlling the temperature of the receptacle; - optionally an analyzer capable of detecting mineral oil in said reacted organic phase.

21. The system of claim 20, wherein the controller is adapted to perform the method of any one of claims 1 to 19.

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