A method for detecting harmful substances

Abstract

The present invention relates to a method for quantitatively determining the combined amount of 3-monochloropropanediol (3-mcpd), 3-mcpd esters, glycidol and glycidyl esters suspected of being present in an original sample.

Description

Field of the Invention

[0001] The present invention relates to the detection of harmful substances in edible foods such as edible oils. Background Art

[0002] The listing or discussion of a previously published document in this specification should not necessarily be taken as an admission that the document is part of the state of the art or is common general knowledge.

[0003] Chloropropanols, such as 3-monochloropropanediol (3-MCPD) and its derivatives are foodborne contaminants that can be formed during the processing of various fatty acid-rich foods. 3-MCPD is formed in the presence of glycerol, chloride ions, and heat, and their metabolites have been implicated in mechanisms that promote testicular and renal toxicity. In addition, fatty acid esters of 3-MCPD are also commonly found in various food types and food ingredients, especially refined edible oils.

[0004] Similar to 3-MCPD esters, glycidol or glycidyl fatty acid esters (GE) may also be detected in significant concentrations in refined edible oils. GEs have been identified as a new class of food processing contaminants and they contain terminal epoxide groups in combination with various fatty acids. GEs have also been implicated in the formation of carcinogenic lesions.

[0005] Due to the inherent risks posed by these contaminants, the International Agency for Research on Cancer (IARC) has classified 3-MCPd and Glycidol as "possibly carcinogenic to humans" (Group 2B) and "probably carcinogenic to humans" (Group 2A), respectively. In addition, regulatory agencies and current industry roadmaps have aimed to reduce the levels of 3-MCPd and GE to less than 2ppm and 1ppm, respectively, by September 2019.

[0006] Therefore, in order to meet the strict requirements of various regulatory agencies, it is necessary to obtain a quantitative method that is easy to quantify such pollutants. The current method for quantifying 3-mcpd, glycidol and esters thereof relates to the use of gas chromatography / mass spectrometer (GC / MS), as described in the official method approved by the American Oil Chemists' Society (AOCS official method Cd 29a-13, 29b-13 and 29c-13). Such method requires tedious and tedious sample preparation, and needs to prepare the standard substance for calibration instrument. In addition, GC / MS is used to relate to high cost, and needs the personnel with professional and technical knowledge to operate.

[0007] In view of the above, current test methods for determining the presence of 3-mcpd and glycidol (and its derivatives) remain problematic. Therefore, new robust detection methods are needed that allow for rapid and sensitive quantification of 3-mcpd and / or glycidol in a sample. More importantly, the method must be accurate, economical, and easy to perform. Summary of the invention

[0008] In a first aspect of the present invention, there is provided a method for quantitatively determining the combined amount of 3-monochloropropanediol (3-mcpd), 3-mcpd esters, glycidol and glycidyl esters suspected of being present in an original sample, the method comprising:

[0009] (a) measuring optical properties of a combined sample comprising a reaction product of a suitable compound containing a pyridine ring and a first sample comprising one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters obtained from an original sample suspected of comprising one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters, and quantitatively determining the combined amount of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters by comparing the measured optical properties to one or more standards or calibration curves.

[0010] In an embodiment of the present invention, the method may further include the following steps:

[0011] (b) quantitatively determining the amount of 3-monochloropropanediol and 3-mcpd esters in an original sample comprising the reaction product of a suitable compound containing a pyridine ring and a second sample comprising 3-monochloropropanediol and / or 3-mcpd esters obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol and glycidyl esters by measuring the optical properties of the 3-monochloropropanediol sample, wherein the second sample has been treated to remove any glycidol and glycidyl esters present in the original sample. As will be appreciated, the amount of glycidyl esters and / or glycidol in the original sample can be determined by subtracting the amount of 3-monochloropropanediol and / or 3-mcpd esters determined in step (b) from the combined amount determined in step (a).

[0012] In embodiments of the above aspects and embodiments, suitable compounds containing a pyridine ring may be independently selected from:

[0013] (i) Compounds of formula I:

[0014]

[0015] in:

[0016] R 1Indicates NO2, CN, SO3R 4 、CO2R 5 ,CONR 6 R 7 ;

[0017] R 2 and R 3 independently represents halogen, C 1-4 Alkyl or OR 8 ;

[0018] R 4 To R 7 Independently represents C 1-10 Alkyl; and

[0019] R 8 Represents C 1-4 alkyl;

[0020] (ii) a compound of formula II:

[0021]

[0022] in:

[0023] X stands for HC 1-4 Alkyl, –COOH or COOR 9a ;

[0024] Y represents H, C 1-4 Alkyl, OR 9b or NR 10 R 11 、SO3R 12 、CN、NO2、CO2R 13 ,CONR 14 R 15 ;

[0025] Z means H, COR 16 or -(CH2) n Ar;

[0026] W represents H, CH2OR 17 ;

[0027] V stands for H, C 1-4 Alkyl, –COOH or COOR 18 ;

[0028] R 9a and R 9b To R 18 Indicates H or C 1-4 alkyl;

[0029] Ar represents an aromatic ring system; and

[0030] n is 1 to 10; or

[0031] (iii) a compound of formula III:

[0032]

[0033] Among them, A and BB represent H, C 1-4 Alkyl, OR 19 or NR 20 R 21 、SO3R 22 、CN、NO2、CO2R 23 ,CONR 24 R 25 ;as well as

[0034] R 19 To R 25 Indicates H or C 1-4 Alkyl, provided that when A is H, then BB is not H, and when BB is H, then A is not H. For example, suitable compounds containing a pyridine ring may be selected from:

[0035] Or more specifically, 4-(4-nitrobenzyl)pyridine.

[0036] In embodiments of the above aspects and embodiments, the combined sample may be obtained by:

[0037] (ai) reacting a suitable compound containing a pyridine ring with a first sample obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters in the presence of an aqueous solvent mixture to provide a reaction sample;

[0038] (bi) developing color in the sample by adding a color developer to provide a color-developed sample;

[0039] (ci) separating the reaction product of a suitable compound containing a pyridine ring and one or more of 3-mcpd, 3-mcpd ester, glycidol and glycidyl ester by mixing an organic solvent with the color-developed sample, then separating the organic solvent and the aqueous solvent and collecting the organic layer as the combined sample.

[0040] In an embodiment of the present invention, the aqueous solvent mixture of step (ai) may include water and a buffer, wherein the pH of the aqueous solvent mixture is 4 to 7, optionally, wherein the buffer is a sodium citrate buffer. In a further embodiment of the present invention, the developer in step (bi) may be an aqueous solution of an inorganic base, optionally, wherein:

[0041] (ia) the developer may be an aqueous solution of potassium carbonate; and / or

[0042] (ib) the concentration of the developer may be 500 nM to 1 M; and / or

[0043] (ic) An aqueous solution of an inorganic base may be added in an amount sufficient to provide a colored sample having a pH of at least 11.

[0044] In a further embodiment of the present invention, the organic solvent added to the color development sample in step (ci) can be selected from one or more of acetophenone, diethyl ether, toluene, and ethyl acetate, optionally, wherein the organic solvent is ethyl acetate.

[0045] In an embodiment of the invention involving determining the amount of 3-monochloropropanediol and 3-MCPD esters in a raw sample from which glycidol and glycidyl esters have been separated, the 3-monochloropropanediol sample may be obtained by:

[0046] (aii) reacting a suitable compound containing a pyridine ring with a second sample obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol and glycidyl esters in the presence of an aqueous solvent mixture to provide a reaction sample, wherein the second sample has been treated to remove any glycidol and glycidyl esters present in the original sample;

[0047] (bii) developing color of the reaction sample by adding a color developer to provide a color-developed sample;

[0048] (cii) separating the reaction product of a suitable compound containing a pyridine ring with 3-monochloropropanediol and / or 3-mcpd ester by mixing an organic solvent with the color-developed sample, then separating the organic solvent and the aqueous solvent and collecting the organic layer as the 3-monochloropropanediol sample.

[0049] In an embodiment of the present invention, the aqueous solvent mixture of step (aii) may include water and a buffer, wherein the pH of the aqueous solvent mixture is 4 to 7, and optionally, the buffer is a sodium citrate buffer. In a further embodiment of the present invention, the developer in step (bii) may be an aqueous solution of an inorganic base, and optionally, wherein: (iia) the developer is an aqueous solution of potassium carbonate; and / or (iib) the concentration of the developer is 500nM to 1M. In a further embodiment of the present invention, the organic solvent added to the color development sample in step (cii) may be selected from one or more of acetophenone, diethyl ether, toluene, and ethyl acetate, and optionally, the organic solvent is ethyl acetate.

[0050] In an embodiment of the present invention, the first sample may be obtained by:

[0051] (aiii) adding an organic solvent to the original sample to form a dissolved sample;

[0052] (biii) adding a base dissolved in a primary alcohol having 3 to 5 carbon atoms to the dissolved sample to form an alkaline sample;

[0053] (ciii) adding an acid dissolved in water to the alkaline sample and mixing the sample to form a mixed sample;

[0054] (diii) separating the mixed sample into an organic solvent layer and an aqueous layer, and collecting only the aqueous layer; and

[0055] (eiii) adding a buffer to the aqueous layer to form the first sample.

[0056] In an embodiment of the present invention, the organic solvent added to the original sample in step (aiii) may be isooctane and / or chloroform. In an embodiment of the present invention, in step (biii):

[0057] (iiia) the base may be a hydroxide (e.g. sodium hydroxide or potassium hydroxide), and / or the primary alcohol having 3 to 5 carbon atoms is 1-butanol, optionally wherein the concentration of the potassium hydroxide in the primary alcohol having 3 to 5 carbon atoms is 1 to 5 wt / vol%; or

[0058] (iiib) The alkoxide may be a metal 1-butoxide (eg, sodium 1-butoxide or potassium 1-butoxide).

[0059] In an embodiment of the present invention, the acid dissolved in water in step (ciii) is not hydrochloric acid, optionally, wherein:

[0060] (A) the acid is acetic acid or H2SO4; and / or

[0061] (B) The concentration of the acid in water is 1 to 3 vol%.

[0062] In an embodiment of the present invention, the buffer in step (eiii) may be a sodium citrate buffer, and the buffer provides the first sample with a pH of 4 to 7.

[0063] In yet a further embodiment of the present invention involving determination of the amount of 3-monochloropropanediol and 3-MCPD esters in an initial sample from which glycidol and glycidyl esters are separated, said second sample may be obtained by:

[0064] (aa) adding an organic solvent to an original sample to form a dissolved sample;

[0065] (bb) reacting any glycidol and glycidyl esters present in the dissolved sample with an acid and a primary alcohol having 3 to 5 carbon atoms to form a reacted sample;

[0066] (cc) adding a base dissolved in a primary alcohol having 3 to 5 carbon atoms to the reacted sample to form a neutralized sample, or adding an alkoxide base to the reacted sample to form a neutralized sample, wherein the alkoxide has 3 to 5 carbon atoms;

[0067] (dd) adding an acid dissolved in water to an alkaline sample and mixing the sample to form a mixed sample;

[0068] (ee) separating the mixed sample into an organic solvent layer and an aqueous layer, and collecting only the aqueous layer; and

[0069] (ff) adding a buffer to the aqueous layer to form the second sample.

[0070] In an embodiment of the present invention, the organic solvent added to the original sample in step (aa) may be isooctane and / or chloroform. In an embodiment of the present invention, the base in step (cc) may be a hydroxide (e.g., potassium hydroxide or sodium hydroxide), and / or the primary alcohol having 3 to 5 carbon atoms is 1-butanol, optionally, wherein the concentration of the potassium hydroxide in the primary alcohol having 3 to 5 carbon atoms is 1 to 5 wt / vol%.

[0071] In an embodiment of the present invention, the acid dissolved in water in step (dd) is not hydrochloric acid, optionally, wherein:

[0072] (Ai) the acid is acetic acid or H2SO4; and / or

[0073] (Bi) The concentration of the acid in water is 1 to 3 vol%.

[0074] In an embodiment of the present invention, the buffer in step (ff) may be a sodium citrate buffer, and the buffer provides the first sample with a pH of 4 to 7.

[0075] In an embodiment of the present invention, the acid and the primary alcohol having 3 to 5 carbon atoms used in step (aa) are H2SO4 and 1-propanol, respectively.

[0076] In embodiments of the present invention, the measured optical property may be selected from absorbance, transmittance or reflectance, optionally wherein the measured optical property is absorbance.

[0077] In an embodiment of the present invention, the original sample may be edible oil, edible fat or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Depicted are calibration curves prepared using standard samples comprising: (a) a mixture of 3-mcpd, glycidol, and its esters; and (b) 3-mcpd and its esters.

[0079] Figure 2 Depicted are sample preparation and detection of the combined amount of 3-mcpd, 3-mcpd esters, glycidol, and / or glycidyl esters.

[0080] Figure 3 Depicted are the transesterification of 3-mcpd ester and glycidyl ester to 3-mcpd and glycidol, respectively.

[0081] Figure 4 Depicted is the reaction of 3-mcpd and glycidol with 4-(4-nitrobenzyl)pyridine (NBP).

[0082] Figure 5 Sample preparation and detection of 3-mcpd and / or 3-mcpd esters are depicted.

[0083] Figure 6 The reactions of glycidol and glycidyl esters with acids / alcohols are depicted. DETAILED DESCRIPTION

[0084] As mentioned above, current test methods for determining the presence of 3-monochloropropanediol and glycidol (and its derivatives) remain problematic. Therefore, the present invention seeks to address these issues with a new detection method that is sensitive, quantitative and quick to run.

[0085] First, it is necessary to know whether the composition contains any 3-monochloropropanediol and glycidol (and its derivatives). Therefore, a method is provided for quantitatively determining the total amount of 3-monochloropropanediol (3-mcpd), 3-mcpd esters, glycidol and glycidyl esters suspected of being present in the original sample, the method comprising:

[0086] (a) measuring optical properties of a combined sample comprising a reaction product of a suitable compound containing a pyridine ring and a first sample comprising one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters obtained from an original sample suspected of comprising one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters, and quantitatively determining the combined amount of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters by comparing the measured optical properties to one or more standards or calibration curves.

[0087] In embodiments herein, the words "include / include / contain" may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the words "include / include / contain" may also refer to situations in which only the listed components / features are intended to be present (e.g., the words "include / include / contain" may be replaced with the phrases "consisting of" or "consisting essentially of"). It is expressly contemplated that both the broader and narrower interpretations may apply to all aspects and embodiments of the present invention. In other words, the words "include / include / contain" and their synonyms may be replaced with the phrases "consisting of" or the phrases "consisting essentially of" or their synonyms, and vice versa.

[0088] As will be appreciated, the methods described above work by means of SN2 reactions between the chlorine atoms of 3-mcpd and / or glycidol (and its derivatives) to form new compounds with sufficient conjugation to provide suitable optical properties for analysis. Further details of how optical properties can be captured and analyzed are provided in the experimental section below, where absorbance is the optical property measured. However, it should be understood that other optical properties, such as transmittance or reflectance, can be used instead using standard techniques or methods similar to those described in the examples of absorbance.

[0089] As used herein, the term "derivative of 3-mcpd" refers to an ester of 3-mcpd. As used herein, the term "3-mcpd ester" refers to a compound in which one or both alcohol groups on 3-monochloropropanediol have been reacted with a carboxylic acid (e.g., RCO2H) or an ester thereof (e.g., RCO2R') to form an ester group. There is no particular limitation on the carboxylic acid / ester group used to form the ester with the alcohol (e.g., R can have any suitable value and can be C 1-50 An alkyl group, a phenyl group or a heterocyclic group, which may be substituted or unsubstituted. The R' group may be any suitable group, such as C 1-5 Alkyl group.

[0090] As used herein, the term "derivative of glycidol" refers to an ester of glycidol. As used herein, the term "glycidyl ester" refers to a compound in which an alcohol group on glycidol has been reacted with a carboxylic acid (e.g., RCO2H) or an ester thereof (e.g., RCO2R') to form an ester group. As above, there is no particular limitation on the carboxylic acid / ester group used to form the ester with the alcohol.

[0091] In an embodiment of the present invention, the combined sample used in step (a) can be obtained by:

[0092] (ai) reacting a suitable compound containing a pyridine ring with a first sample obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters in the presence of an aqueous solvent mixture to provide a reaction sample;

[0093] (bi) developing color in the sample by adding a color developer to provide a color-developed sample; and

[0094] (ci) separating the reaction product of a suitable compound containing a pyridine ring and one or more of 3-mcpd, 3-mcpd ester, glycidol and glycidyl ester by mixing an organic solvent with the color-developed sample, then separating the organic solvent and the aqueous solvent and collecting the organic layer as the combined sample.

[0095] In the above step (ai), the aqueous solvent mixture may include water and a buffer, wherein the pH of the aqueous solvent mixture is 4 to 7. Any suitable buffer may be used. For example, the buffer may be a sodium citrate buffer.

[0096] In the above-mentioned steps (bi), coloring agent can be any suitable inorganic alkali aqueous solution. The example of suitable inorganic alkali aqueous solution includes but is not limited to potassium carbonate aqueous solution. The concentration of developer in the aqueous solution can have any suitable concentration, and for example concentration can be 500nM to 1M. As will be appreciated, developer is added to the aqueous solution of step (ai) in an amount sufficient to provide the desired effect, and this amount can be determined by the technician according to conventional trial and error or based on their knowledge and expertise in this field. For example, inorganic alkali aqueous solution can be added in an amount sufficient to provide a color developing sample with a pH value of at least 11.

[0097] In the above-mentioned step (ci), the organic solvent added to the color developing sample can be selected from one or more of acetophenone, diethyl ether, toluene, ethyl acetate, and optionally, the organic solvent is ethyl acetate. As will be appreciated, the solvent can be selected individually or selected to work in coordination with each other. For example, when diethyl ether and / or ethyl acetate are used as organic solvents, an immiscible organic layer can be formed and separated from the aqueous layer.

[0098] In an embodiment of the present invention, the first sample can be obtained by:

[0099] (aiii) adding an organic solvent to the original sample to form a dissolved sample;

[0100] (biii) adding a base dissolved in a primary alcohol having 3 to 5 carbon atoms, or adding an alkoxide having 3 to 5 carbon atoms, to the dissolved sample to form an alkaline sample;

[0101] (ciii) adding an acid dissolved in water to the alkaline sample and mixing the sample to form a mixed sample;

[0102] (diii) separating the mixed sample into an organic solvent layer and an aqueous layer, and collecting only the aqueous layer; and

[0103] (eiii) adding a buffer to the aqueous layer to form the first sample.

[0104] In the above step (aiii), any suitable organic solvent may be used. Examples of suitable organic solvents that may be used include, but are not limited to, isooctane, chloroform, and combinations thereof.

[0105] In the above-mentioned step (biii), any suitable base can be used. Examples of suitable bases include, but are not limited to, hydroxide bases (e.g., sodium hydroxide or potassium hydroxide). Examples of suitable primary alcohols include, but are not limited to, 1-butanol. As will be appreciated, the resulting product of this reaction is a metal alkoxide, and thus, a suitable metal alkoxide that can be used herein can be a metal 1-butoxide salt (sodium 1-butoxide or potassium 1-butoxide).

[0106] In the above step (ciii), any suitable acid may be used, provided that the acid is not hydrochloric acid. Suitable acids include, but are not limited to, acetic acid or H2SO4. Any suitable concentration of acid may be used, for example, the concentration of the acid in water may be 1 to 3 vol% (i.e., the concentration of the acid dissolved in water is 1 to 3 vol%, and the solution is then added to the alkaline sample).

[0107] In the above step (eiii), the buffer can be any suitable buffer that provides a suitable pH value for the first sample. For example, the buffer can be a sodium citrate buffer and / or the buffer can provide the first sample with a pH of 4 to 7.

[0108] As will be appreciated, the process described above allows one to understand the total amount of 3-mcpd, glycidol and its derivatives in the sample to be analyzed. However, the above method does not provide a detailed understanding of the amount of contaminants derived from 3-mcpd and the amount derived from glycidol. Therefore, in order to obtain this information, the process may further include the following steps:

[0109] (b) quantitatively determining the amount of 3-monochloropropanediol and 3-mcpd esters in an original sample by measuring the optical properties of the 3-monochloropropanediol sample, said 3-monochloropropanediol sample comprising the reaction product of a suitable compound containing a pyridine ring and a second sample comprising 3-monochloropropanediol and / or 3-mcpd esters obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol and glycidyl esters, wherein said second sample has been treated to remove any glycidol and glycidyl esters present in said original sample.

[0110] The above method allows one to calculate the amount of 3-mcpd and its derivatives in a sample. As will be appreciated, the amount of glycidyl esters and / or glycidol in the original sample can be determined by subtracting the amount of 3-monochloropropanediol and / or 3-mcpd esters determined in step (b) from the total amount determined in step (a). This allows one to determine the total amount of contaminants and the amount of contaminants derived from 3-mcpd and glycidol.

[0111] In an embodiment of the present invention, the 3-monochloropropanediol sample used in step (b) can be obtained by:

[0112] (aii) reacting a suitable compound containing a pyridine ring with a second sample obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol and glycidyl esters in the presence of an aqueous solvent mixture to provide a reaction sample, wherein the second sample has been treated to remove any glycidol and glycidyl esters present in the original sample;

[0113] (bii) developing color of the reaction sample by adding a color developer to provide a color-developed sample;

[0114] (cii) separating the reaction product of a suitable compound containing a pyridine ring with 3-monochloropropanediol and / or 3-mcpd ester by mixing an organic solvent with the color-developed sample, then separating the organic solvent and the aqueous solvent and collecting the organic layer as the 3-monochloropropanediol sample.

[0115] In the above step (aii), the aqueous solvent mixture may include water and a buffer, wherein the pH of the aqueous solvent mixture is 4 to 7, and optionally, wherein the buffer is a sodium citrate buffer.

[0116] In the above-mentioned step (bii), the developer can be an inorganic alkali aqueous solution. The example of a suitable inorganic alkali aqueous solution includes but is not limited to potassium carbonate aqueous solution. The developer can have any suitable concentration in the aqueous solution, and for example, the concentration can be 500nM to 1M. As will be appreciated, the developer is added to the aqueous solution of step (ai) in an amount sufficient to provide the desired effect, and the amount can be determined by the technician according to conventional trial and error or based on their knowledge and expertise in this field. For example, an inorganic alkali aqueous solution can be added in an amount sufficient to provide a color developing sample with a pH value of at least 11.

[0117] In the above-mentioned step (cii), the organic solvent added to the color developing sample can be selected from one or more of acetophenone, diethyl ether, toluene, ethyl acetate, and optionally, the organic solvent is ethyl acetate. As will be appreciated, the solvent can be selected individually or selected to work in coordination with each other. For example, when diethyl ether and / or ethyl acetate are used as organic solvents, an immiscible organic layer can be formed and separated from the aqueous layer.

[0118] In an embodiment of the present invention, the second sample can be obtained by:

[0119] (aa) adding an organic solvent to an original sample to form a dissolved sample;

[0120] (bb) reacting any glycidol and glycidyl esters present in the dissolved sample with an acid and a primary alcohol having 3 to 5 carbon atoms to form a reacted sample;

[0121] (cc) adding a base dissolved in a primary alcohol having 3 to 5 carbon atoms to the reacted sample to form a neutralized sample, or adding an alkoxide base to the reacted sample to form a neutralized sample, wherein the alkoxide has 3 to 5 carbon atoms;

[0122] (dd) adding an acid dissolved in water to an alkaline sample and mixing the sample to form a mixed sample;

[0123] (ee) separating the mixed sample into an organic solvent layer and an aqueous layer, and collecting only the aqueous layer; and

[0124] (ff) adding a buffer to the aqueous layer to form the second sample.

[0125] In the above step (aa), any suitable organic solvent may be used. Examples of suitable organic solvents that may be used include, but are not limited to, isooctane, chloroform, and combinations thereof.

[0126] In the above step (bb), any suitable acid and alcohol may be used. For example, the acid may be H2SO4 and the alcohol may be 1-propanol.

[0127] In the above step (cc), any suitable base may be used. Examples of suitable bases include, but are not limited to, hydroxide bases (e.g., sodium hydroxide or potassium hydroxide). Examples of suitable primary alcohols include, but are not limited to, 1-butanol. As will be appreciated, the resulting product of this reaction is a metal alkoxide, and thus, a suitable metal alkoxide that may be used herein may be a metal 1-butoxide salt (sodium 1-butoxide or potassium 1-butoxide).

[0128] In the above step (dd), any suitable acid may be used, provided that the acid is not hydrochloric acid. Suitable acids include, but are not limited to, acetic acid or H2SO4. Any suitable concentration of the acid may be used, for example, the concentration of the acid in water may be 1 to 3 vol% (i.e., the concentration of the acid dissolved in water is 1 to 3 vol%, and the solution is then added to the alkaline sample).

[0129] In the above step (ff), the buffer can be any suitable buffer that provides a suitable pH value for the first sample. For example, the buffer can be a sodium citrate buffer and / or the buffer can provide the first sample with a pH of 4 to 7.

[0130] As will be appreciated, the methods described above require the use of suitable compounds containing a pyridine ring to provide the desired reaction products for analysis. The compounds in question may be independently selected from:

[0131] (i) Compounds of formula I:

[0132]

[0133] in:

[0134] R 1 Indicates NO2, CN, SO3R 4 、CO2R 5 ,CONR 6 R 7 ;

[0135] R 2 and R 3 independently represents halogen, C 1-4 Alkyl or OR 8 ;

[0136] R 4 To R 7 Independently represents C 1-10 Alkyl; and

[0137] R 8 Represents C 1-4 alkyl;

[0138] (ii) a compound of formula II:

[0139]

[0140] in:

[0141] X stands for HC 1-4 Alkyl, –COOH or COOR 9a ;

[0142] Y represents H, C 1-4 Alkyl, OR 9b or NR 10 R 11 、SO3R 12 、CN、NO2、CO2R 13 ,CONR 14 R 15 ;

[0143] Z means H, COR 16 or -(CH2) n Ar;

[0144] W represents H, CH2OR 17 ;

[0145] V stands for H, C 1-4 Alkyl, –COOH or COOR 18 ;

[0146] R 9a and R 9b To R 18 Indicates H or C 1-4 alkyl;

[0147] Ar represents an aromatic ring system; and

[0148] n is 1 to 10; or

[0149] (iii) Compounds of formula III:

[0150]

[0151] Among them, A and BB represent H, C 1-4 Alkyl, OR 19 or NR 20 R 21 、SO3R 22 、CN、NO2、CO2R 23 ,CONR 24 R 25 ;as well as

[0152] R 19 To R 25 Indicates H or C 1-4Alkyl, provided that when A is H, then BB is not H, and when BB is H, then A is not H. For example, suitable compounds containing a pyridine ring may be selected from:

[0153] Or more specifically, 4-(4-nitrobenzyl)pyridine.

[0154] As used herein, the term "Ar" or "aryl" includes C 6-14 (such as C 6-13 (For example, C 6-10 )) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, wherein at least one ring is aromatic. The point of attachment of the aryl group may be via any atom of the ring system. However, when the aryl groups are bicyclic or tricyclic, they are attached to the rest of the molecule via the aromatic ring. 6-10 Aryl groups include phenyl, naphthyl, and the like, such as 1,2,3,4-tetrahydronaphthyl, indanyl, indenyl, and fluorenyl. Embodiments of the invention that may be mentioned include those wherein the aryl group is phenyl.

[0155] In embodiments of the present invention, the measured optical property may be selected from absorbance, transmittance or reflectance, optionally wherein the measured optical property is absorbance.

[0156] As will be appreciated, the original sample may be any suitable sample suspected of containing 3-mcpd and / or glycidol and its derivatives.Examples of suitable original samples include, but are not limited to, edible oils, edible fats, or combinations thereof.

[0157] Further aspects and embodiments of the invention are described below with reference to the following non-limiting examples.

[0158] Example

[0159] Device

[0160] The equipment required to perform the test includes:

[0161] Vortexer;

[0162] Boiling water bath;

[0163] Heating plate;

[0164] Centrifuge;

[0165] Ice box;

[0166] Spectrophotometer or colorimeter (with a minimum resolution of 0.01 nm); and

[0167] Pipette.

[0168] Material

[0169] Isooctane (analytical grade, ≥99%), chloroform (analytical grade, ≥99%), H2SO4 (≥99%), 1-propanol (analytical grade, ≥99%), KOH (85% and above), 1-butanol (analytical grade, ≥99%), sodium citrate (≥99%), ethanol (≥95%), glycerol (≥99%), 4-(4-nitrobenzyl)pyridine (98% and above), K2CO3 (≥99%), ethyl acetate (analytical grade, ≥99%), acetophenone (analytical grade, ≥99%), toluene (analytical grade, ≥99%), acetone (analytical grade, ≥99%) and ethylene glycol (≥99%) were obtained from commercial sources and used directly in the preparation of the following solvents or reagents without further purification:

[0170] Solvent for dissolving samples: isooctane or chloroform

[0171] Reagent 1: H2SO4 in 1-propanol, working concentration is 1-5μL H2SO4 in 1-propanol (1mL)

[0172] Reagent 2: KOH in 1-butanol, working concentration is 1-5% KOH in 1-butanol (w / v)

[0173] Reagent 3: H2SO4 in deionized water, working concentration is 1-3% H2SO4 in deionized water

[0174] Reagent 4: Sodium citrate buffer, with a pH of 4-7

[0175] Reagent A: 4-(4-nitrobenzyl)pyridine (NBP) dissolved in a mixture of ethanol:glycerol at a working concentration of 2-10% NBP (w / v). The percentage of ethanol in the ethanol:glycerol mixture can be in the range of 10-100% (v / v). Ethanol can be replaced by other solvents such as acetone or other polar solvents, and glycerol can be replaced by ethylene glycol.

[0176] Reagent B: K2CO3 dissolved in deionized water, working concentration 500nM-1M

[0177] Reagent C: ethyl acetate, acetophenone, diethyl ether or toluene

[0178] Constructing a calibration curve using standard samples

[0179] Typically, the calibration curve can be obtained by the following steps:

[0180] 1. Six or more samples containing standard products (3-mcpd and its esters; or a mixture of 3-mcpd, glycidol and its esters) whose concentrations were predetermined by GC / MS were used to construct a calibration curve.

[0181] For quantification of the total concentration of 3-mcpd, glycidol and their esters (in Example 1), the sample should contain a total concentration of 3-mcpd, glycidol and their esters of 0.2 to 8 ppm, with various sample concentrations distributed throughout the range (e.g., 0.2, 0.5, 1, 3, 5, 8 ppm).

[0182] For quantification of 3-mcpd and its esters (in Example 2), the sample should contain 3-mcpd and its esters concentrations between 0.1 and 5 ppm, with various sample concentrations distributed throughout the range (e.g., 0.1, 0.5, 1, 2, 3, 5 ppm).

[0183] 2. The samples were then subjected to the sample preparation and detection steps as described in Example 1 or 2, and their absorbance was measured.

[0184] 3. These absorbance values ​​obtained for the standard samples are then used to construct a calibration curve, such as Figure 1 a and b (for a combined sample of 3-mcpd, glycidol and its esters; and for 3-mcpd and its esters, respectively). The concentration of 3-mcpd and its esters, or the total concentration of 3-mcpd, glycidol and its esters, was then determined from the respective calibration curves.

[0185] Example 1. Detection of the total amount of 3-monochloropropanediol (3-mcpd), 3-mcpd esters, glycidol and / or glycidyl esters

[0186] The apparatus and reagents listed above are used to detect the total amount of 3-monochloropropanediol (3-mcpd), 3-mcpd esters, glycidol and / or glycidyl esters in a sample. The method allows for the simultaneous detection of 3-mcpd, glycidol and their respective ester derivatives. The detection process is divided into two parts: sample preparation (2) and detection (4), such as Figure 2 The sample preparation involves the conversion of 3-mcpd esters and glycidyl esters to 3-mcpd (22) and glycidol (24), respectively, while the detection step involves the use of 4-(4-nitrobenzyl)-pyridine (NBP) and a base to give a color change upon reaction with 3-mcpd or glycidol.

[0187] The test method has been applied to food samples including RBDPO (refined, bleached and deodorized palm oil), palm olein, interesterified palm oil, sunflower oil, canola oil, solid stearin, cream margarine for cakes, confectionery fat for chocolate products and rice bran oil.

[0188] Sample preparation (2)

[0189] Typically, 0.1-5g (± 0.1%) of each sample (for batches of four samples) is weighed into a tube. For this embodiment, 3g ± 3mg of sample is weighed into each tube respectively. Then 3mL of solvent is added to each sample and mixed until the sample is completely dissolved. If the sample is solid, it can first be completely melted by heating it in an oven / water bath (e.g., at 30-80°C, depending on the melting point of the solid), and then solvent is added to the sample. After adding the solvent, and if the sample is not completely dissolved, the mixture can be heated in an oven at 30-80°C (depending on the melting point of the sample and the boiling point of the solvent-if a temperature higher than 60°C is required, isooctane is used), until the sample is completely dissolved and the solution appears clear.

[0190] Then, 4 mL of reagent 2 (20) was added to each sample (10), mixed and allowed to stand for 5-15 min. Reagent 2 was added to convert 3-mcpd ester (13) and / or glycidyl ester (14) into 3-mcpd (22) and / or glycidol (24), respectively (e.g. Figure 3 ) as shown in the reaction scheme in .

[0191] Thereafter, 3.5 mL of Reagent 3 (30) was added to each sample, mixed and vortexed to neutralize the alkaline conditions ( Figure 2 ). The sample was then centrifuged at 2,000 or higher rcf for at least 1 min to obtain two layers. The organic layer was discarded and the aqueous layer was transferred to a new tube. Care was taken to ensure that the organic layer did not contaminate the aqueous layer sample. This was followed by the addition of 15 μL of Reagent 4 (40) to the sample to ensure that the sample was neutralized ( Figure 2 ). This results in a prepared sample 50 which is directly used in the subsequent detection step.

[0192] Sample testing(4)

[0193] To detect the amount of 3-mcpd and / or glycidol in the prepared sample 50 (from the previous preparation step), add 1 mL of reagent A (60) to each tube and vortex thoroughly. Then incubate the tube in a boiling water bath for 15-40 min to allow the reaction of NBP with 3-mcpd and glycidol to complete (e.g. Figure 4The tubes were then removed from the water bath and rapidly cooled in ice. Thereafter, 0.5 mL of Reagent B (70) and 1 mL of Reagent C (80) were added to each tube and vortexed thoroughly ( Figure 2 ). The tube is then centrifuged to obtain two layers. The organic layer (90) containing NBP reacted with 3-mcpd or glycidol (75) is transferred to an absorption cell and the absorbance is measured at about 530-560 nm using a spectrophotometer.

[0194] The combined concentration of 3-mcpd, 3-mcpd esters, glycidol and / or glycidol esters can be determined from a calibration curve generated using standard samples containing various concentrations of 3-mcpd, glycidol and their esters ( Figure 1 a).

[0195] Example 2. Detection of the amount of 3-monochloropropylenediol (3-mcpd) and / or 3-mcpd esters

[0196] The amount of 3-monochloropropanediol (3-mcpd) and / or 3-mcpd ester in the sample was detected using the apparatus and reagents listed above. Similar to Example 1, the detection process was divided into two parts: sample preparation (6) and detection (8), as shown in Figure 5 The method is essentially the same as that in Example 1, but it includes an additional step in the sample preparation stage to remove epoxy functional groups from glycidol and glycidyl esters that may be present in the sample. This effectively inactivates glycidol and glycidyl esters so that they do not react with NBP to give a color change in the detection step.

[0197] The test method has been applied to food samples including RBDPO (refined, bleached and deodorized palm oil), palm olein, interesterified palm oil, sunflower oil, canola oil, solid stearin, cream margarine for cakes, confectionery fat for chocolate products and rice bran oil.

[0198] Sample preparation (6)

[0199] Typically, 0.1-5g (± 0.1%) of each sample (for a batch of six samples) is weighed into a tube. For this embodiment, 3g ± 3mg of sample is weighed into each tube respectively. Then 3mL of solvent is added to each sample and shaken until the sample is completely dissolved. If the sample is solid, it can first be completely melted by heating it in an oven / water bath (e.g., at 30-80°C, depending on the melting point of the solid), and then solvent is added to the sample. After adding the solvent, and if the sample is not completely dissolved, the mixture can be heated in an oven at 30-80°C (depending on the melting point of the sample and the boiling point of the solvent--if a temperature higher than 60°C is required, isooctane is used), until the sample is completely dissolved and the solution appears clear.

[0200] First, 1 mL of Reagent 1 (12) was added to each sample (5), mixed and incubated in a water bath at 40-70°C for 20-30 min. Adding Reagent 1 is an additional step (compared to Example 1) and is important for removing epoxy functional groups from glycidol (24) and glycidyl esters (14) that may be present in the sample (in Figure 6 ) is shown in the reaction scheme in .

[0201] Then, 4 mL of reagent 2 (20) was added to each sample (10), mixed and incubated at room temperature for 10-15 min. Reagent 2 was added to convert 3-mcpd ester and other esters into 3-mcpd and respective alcohols (e.g. Figure 2 ) as shown in the reaction scheme in .

[0202] Thereafter, a sufficient amount (~3.4 mL) of Reagent 3 (30) was added to each sample, mixed, and vortexed to neutralize the alkaline conditions ( Figure 5 ). The sample was then centrifuged at 2,000 or higher rcf for at least 1 min to obtain two layers. The organic layer was discarded and the aqueous layer was transferred to a new tube. Care was taken to ensure that the organic layer did not contaminate the aqueous layer sample. This was followed by the addition of 15 μL of Reagent 4 (40) to the sample to ensure that the sample was neutralized ( Figure 5 ). This yields a prepared sample 55 which is directly used in the subsequent detection step.

[0203] Sample testing(8)

[0204] To detect the amount of 3-mcpd in the prepared sample 55 (from the previous preparation step), 1 mL of reagent A (60) was added to each tube and vortexed thoroughly. The tubes were then incubated in a boiling water bath for 15-40 min to allow the reaction of NBP with 3-mcpd and glycidol to proceed completely (eg, Figure 3The tubes were then removed from the water bath and rapidly cooled in ice. Thereafter, 0.5 mL of Reagent B (70) and 1 mL of Reagent C (80) were added to each tube and vortexed thoroughly ( Figure 5 ). The tube is then centrifuged to obtain two layers. The organic layer (95) containing NBP reacted with 3-mcpd (75) is transferred to an absorption cell and the absorbance is measured at about 530-560 nm using a spectrophotometer.

[0205] The concentration of 3-mcpd and / or 3-mcpd esters can be determined from a calibration curve generated using standard samples containing various concentrations of 3-mcpd and its esters ( Figure 1 a).

[0206] Comparison of the concentrations of 3-mcpd and / or 3-mcpd esters (in various samples) determined by the current method with those determined by GC / MS showed that the current method was able to achieve comparable accuracy and sensitivity as the conventional GC / MS method (Table 1).

[0207] Table 1. Comparison of various concentrations of 3-mcpd and / or 3-mcpd esters determined by the current method with concentrations determined by GC / MS.

[0208]

[0209] Example 3. Detection of the amount of glycidol and / or glycidyl esters

[0210] The amount of glycidol and / or glycidyl esters in a sample can be determined by subtracting the concentration of 3-mcpd and / or 3-mcpd esters (in Example 2) from the combined concentration of 3-mcpd, 3-mcpd esters, glycidol and / or glycidyl esters (in Example 1) as follows:

[0211] =The total concentration of glycidol and / or 3-mcpd, 3-mcpd esters, glycidol and / or glycidyl esters or the degree of glycidyl esterification (as determined in Example 1) -

[0212] Concentration of 3-mcpd and / or 3-mcpd ester (as determined in Example 2)

[0213] Comparison of the concentrations of glycidol and / or glycidyl esters (in various samples) determined by the current method with those determined by GC / MS showed that the current method was able to achieve comparable accuracy and sensitivity to conventional GC / MS methods (Table 2).

[0214] Table 2. Comparison of various concentrations of glycidol and / or glycidyl esters determined by the current method with concentrations determined by GC / MS.

[0215]

[0216] Example 4. Comparison of current test methods with the official method of the American Oil Chemists Association

[0217] The currently claimed method for quantifying 3-mcpd and glycidol was compared with the American Oil Chemists Society (AOCS) official methods Cd-29a-13, Cd-29b-13, and Cd-29c-13 (referred to as Methods A, B, and C, respectively), and the advantages are summarized in Table 3 below.

[0218] Table 3. Summary of the advantages of the current method compared to the official AOCS method.

[0219]

Claims

1. A method for quantitatively determining the total amount of 3-monochloropropanediol (3-mcpd), 3-mcpd esters, glycidol and glycidyl esters suspected of being present in an original sample, the method comprising: (a) measuring an optical property of a combined sample comprising a reaction product of a suitable compound containing a pyridine ring and a first sample comprising one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters obtained from an original sample suspected of comprising one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters, and quantitatively determining the combined amount of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters by comparing the measured optical property to one or more standards or calibration curves, Wherein, the suitable compound containing a pyridine ring is independently selected from: 4-(4-Nitrobenzyl)pyridine and a compound of formula I: in: R 1 Indicates NO2, CN, SO3R 4 、CO2R 5 ,CONR 6 R 7 ; R 2 and R 3 independently represents halogen, C 1-4 Alkyl or OR 8 ; R 4 To R 7 Independently represents C 1-10 Alkyl; and R 8 Represents C 1-4 alkyl.

2. The method according to claim 1, further comprising: (b) quantitatively determining the amount of 3-monochloropropanediol and 3-mcpd esters in an original sample by measuring the optical properties of the 3-monochloropropanediol sample, said 3-monochloropropanediol sample comprising the reaction product of a suitable compound containing a pyridine ring and a second sample comprising 3-monochloropropanediol and / or 3-mcpd esters obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol and glycidyl esters, wherein said second sample has been treated to remove any glycidol and glycidyl esters present in said original sample.

3. The method according to claim 2, wherein: The amount of glycidyl esters and / or glycidol in the original sample is determined by subtracting the amount of 3-monochloropropanediol and / or 3-mcpd esters determined in step (b) from the combined amount determined in step (a).

4. The method according to claim 1, wherein: The suitable compound containing a pyridine ring is 4-(4-nitrobenzyl)pyridine.

5. The method according to claim 1, wherein: The combined sample is obtained by: (ai) reacting a suitable compound containing a pyridine ring with a first sample obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol, and glycidyl esters in the presence of an aqueous solvent mixture to provide a reaction sample; (bi) developing color in the sample by adding a color developer to provide a color-developed sample; as well as (ci) separating the reaction product of a suitable compound containing a pyridine ring and one or more of 3-mcpd, 3-mcpd ester, glycidol and glycidyl ester by mixing an organic solvent with the color-developed sample, then separating the organic solvent and the aqueous solvent and collecting the organic layer as the combined sample.

6. The method according to claim 5, wherein: The aqueous solvent mixture comprises water and a buffer, wherein the pH of the aqueous solvent mixture is 4 to 7.

7. The method according to claim 6, wherein: The buffer is sodium citrate buffer.

8. The method according to claim 5, wherein: The developer is an inorganic alkali aqueous solution.

9. The method according to claim 8, wherein: (ia) the developer is an aqueous solution of potassium carbonate; and / or (ib) the concentration of the developer is 500 nM to 1 M; and / or (ic) adding the aqueous inorganic base solution in an amount sufficient to provide the color-developed sample with a pH value of at least 11.

10. The method according to claim 5, wherein: The organic solvent added to the color developing sample is selected from one or more of acetophenone, diethyl ether, toluene and ethyl acetate.

11. The method according to claim 5, wherein: The organic solvent added to the color development sample is ethyl acetate.

12. The method according to claim 2, wherein: The 3-monochloropropanediol sample was obtained by: (aii) reacting a suitable compound containing a pyridine ring with a second sample obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd esters, glycidol and glycidyl esters in the presence of an aqueous solvent mixture to provide a reaction sample, wherein the second sample has been treated to remove any glycidol and glycidyl esters present in the original sample; (bii) developing color of the reaction sample by adding a color developer to provide a color-developed sample; (cii) separating the reaction product of a suitable compound containing a pyridine ring with 3-monochloropropanediol and / or 3-mcpd ester by mixing an organic solvent with the color-developed sample, then separating the organic solvent and the aqueous solvent and collecting the organic layer as the 3-monochloropropanediol sample.

13. The method according to claim 12, wherein: The aqueous solvent mixture comprises water and a buffer, wherein the pH of the aqueous solvent mixture is 4 to 7.

14. The method according to claim 13, wherein: The buffer is sodium citrate buffer.

15. The method according to claim 12, wherein: The developer is an inorganic alkali aqueous solution.

16. The method of claim 15, wherein: (iia) the developer is an aqueous solution of potassium carbonate; and / or (iib) The concentration of the color developing agent is 500 nM to 1 M.

17. The method according to claim 12, wherein: The organic solvent added to the color developing sample is selected from one or more of acetophenone, diethyl ether, toluene and ethyl acetate.

18. The method according to claim 12, wherein: The organic solvent added to the color development sample is ethyl acetate.

19. The method according to claim 1, wherein: The first sample is obtained by: (aiii) adding an organic solvent to the original sample to form a dissolved sample; (biii) adding a base dissolved in a primary alcohol having 3 to 5 carbon atoms, or adding an alkoxide having 3 to 5 carbon atoms, to the dissolved sample to form an alkaline sample; (ciii) adding an acid dissolved in water to the alkaline sample and mixing the sample to form a mixed sample; (diii) separating the mixed sample into an organic solvent layer and an aqueous layer, and collecting only the aqueous layer; as well as (eiii) adding a buffer to the aqueous layer to form the first sample.

20. The method according to claim 19, wherein: The organic solvent added to the original sample is isooctane and / or chloroform.

21. The method of claim 19, wherein: (iiia) the base is a hydroxide, and / or the primary alcohol having 3 to 5 carbon atoms is 1-butanol; or (iiib) The alkoxide is a metal 1-butoxide.

22. The method according to claim 21, wherein: The hydroxide is sodium hydroxide or potassium hydroxide.

23. The method according to claim 22, wherein: The concentration of the potassium hydroxide in the primary alcohol having 3 to 5 carbon atoms is 1 to 5 wt / vol%.

24. The method according to claim 21, wherein: The metal 1-butoxide salt is sodium 1-butoxide or potassium 1-butoxide.

25. The method of claim 19, wherein: The acid dissolved in water is not hydrochloric acid.

26. The method of claim 25, wherein: (A) the acid is acetic acid or H2SO4; and / or (B) The concentration of the acid in water is 1 to 3 vol%.

27. The method of claim 19, wherein: The buffer is a sodium citrate buffer, and the buffer provides the first sample with a pH of 4 to 7.

28. The method according to claim 2, wherein: The second sample was obtained by: (aa) adding an organic solvent to an original sample to form a dissolved sample; (bb) reacting any glycidol and glycidyl esters present in the dissolved sample with an acid and a primary alcohol having 3 to 5 carbon atoms to form a reacted sample; (cc) adding a base dissolved in a primary alcohol having 3 to 5 carbon atoms to the reacted sample to form a neutralized sample, or adding an alkoxide base to the reacted sample to form a neutralized sample, wherein the alkoxide has 3 to 5 carbon atoms; (dd) adding an acid dissolved in water to the neutralized sample and mixing the sample to form a mixed sample; (ee) separating the mixed sample into an organic solvent layer and an aqueous layer, and collecting only the aqueous layer; as well as (ff) adding a buffer to the aqueous layer to form the second sample.

29. The method according to claim 28, wherein: The organic solvent added to the original sample is isooctane and / or chloroform.

30. The method of claim 28, wherein: The base is a hydroxide, and / or the primary alcohol having 3 to 5 carbon atoms is 1-butanol.

31. The method according to claim 30, wherein: The hydroxide is potassium hydroxide or sodium hydroxide.

32. The method according to claim 31, wherein: The concentration of the potassium hydroxide in the primary alcohol having 3 to 5 carbon atoms is 1 to 5 wt / vol%.

33. The method of claim 28, wherein: The acid dissolved in water in step (dd) is not hydrochloric acid.

34. The method of claim 33, wherein: (a) the inorganic acid is acetic acid or H2SO4; and / or (b) The concentration of the acid in water is 1 to 3 vol%.

35. The method of claim 28, wherein: The buffer is a sodium citrate buffer, and the buffer provides the second sample with a pH of 4 to 7.

36. The method of claim 28, wherein: The acid and the primary alcohol having 3 to 5 carbon atoms used in step (bb) are H2SO4 and 1-propanol, respectively.

37. The method of claim 1, wherein: The optical property measured is selected from absorbance, transmittance or reflectance.

38. The method of claim 1, wherein: The optical property measured is absorbance.

39. The method according to any one of claims 1 to 38, wherein: The original sample is edible oil, edible fat or a combination thereof.

Images