Field of Invention

This invention relates to the detection of harmful substances in edible foodstuffs, such as edible oils.

Background

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Chloropropanols, like 3-monochloropropane diol (3-mcpd), and their derivatives are foodborne contaminants that can be formed during the processing of various fatty acid rich foodstuffs. 3- mcpd are formed in the presence of glycerol, chloride ions and heat, and their metabolites have been linked to mechanisms that promote testicular and renal toxicity. In addition, the fatty acid esters of 3-mcpd are also commonly found in various food types and food ingredients, in particular refined edible oils.

Similar to 3-mcpd esters, glycidols or glycido fatty acid esters (GEs) may also be detected in significant concentrations in refined edible oils. GEs have been identified as a new class of food-processing contaminant and they contain a terminal epoxide group, with various fatty acid compositions. Furthermore, GEs have also been linked to the formation of carcinogenic lesions.

Due to the inherent risk posed by these contaminants, 3-mcpd and glycidol have been categorised as “possible human carcinogens” (Group 2B) and “probably carcinogenic to humans” (Group 2A), respectively, by the International Agency for Research on Cancer (IARC). In addition, regulatory bodies and current industry roadmaps have aimed to reduce the levels of 3-mcpd and GEs to less than 2 ppm and 1 ppm, respectively, by September 2019.

Therefore, to meet the stringent requirement enforced by various regulatory bodies, it is essential to have a quantification method that is easily accessible to quantify such contaminants. The current method for quantifying 3-mcpd, glycidols and their respective esters involves the use of gas chromatography/mass spectrometer (GC/MS), as described in the approved official methods by the American Oil Chemists’ Society (in AOCS official methods Cd 29a-13, 29b-13 and 29c-13). Such methods requires long and tedious sample preparation and requires the preparation of standards for calibrating the instrument. In addition, the use of GC/MS involves high cost and require personnel with specialised technical knowledge to operate.

Given the above, there remain problems with the current testing methods used to establish the presence of 3-mcpd and glycidol (and derivatives thereof). Therefore, there is a need for a new robust detection method that allows rapid and sensitive quantification of 3-mcpd and/or glycidol in samples. More importantly, the method has to be accurate, cost-effective and simple to perform.

Summary of Invention

In a first aspect of the invention, there is provided a method of quantitatively determining the combined amount of 3-monochloropropane diol (3-mcpd), 3-mcpd ester(s), glycidol and glycidol ester(s) suspected to be present in an original sample, the method comprising:

(a) measuring an optical property of a combined sample comprising the reaction product of a suitable compound comprising a pyridine ring with a first sample comprising one or more of 3-mcpd, a 3-mcpd ester, glycidol, a glycidol ester obtained from the original sample suspected of containing one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) and quantitatively determining the combined amount of the 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) by comparison of the measured optical property to one or more standards or a calibration curve.

In an embodiment of the invention the method may further comprise the step:

(b) quantitatively determining the amount of 3-monochloropropane diol and 3-mcpd ester(s) in an original sample by measuring an optical property of a 3-monochloropropane diol sample comprising the reaction product of a suitable compound comprising a pyridine ring with a second sample comprising 3-monochloropropane diol and/or a 3-mcpd ester(s) obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s), where the second sample has been treated to remove any glycidol and glycidol ester(s) present in the original sample. As will be appreciated, the amount of glycidol ester and/or glycidol in an original sample may be determined by subtracting the amount of 3-monochloropropane diol and/or 3-mcpd ester(s) determined in step (b) from the combined amount determined in step (a). In embodiments of the above aspect and embodiments, the suitable compound comprising a pyridine ring may be independently selected from:

(i) a compound of formula I:

wherein:

Ft ¹ represents N0 , CN, S0 ₃ R ⁴ , C0 ₂ R ⁵ , CONR ⁶ R ⁷ ;

R ² and R ³ independently represent halo, C _1-4 alkyl or OR ⁸ ;

R ⁴ to R ⁷ independently represent C _{M O} alkyl; and

R ⁸ represents C _1-4 alkyl;

(ii) a compound of formula II:

wherein:

X represents H C1-4 alkyl, -COOH, or COOR ^9a ;

Y represents H, C1-4 alkyl, OR ^9b , or NR ¹⁰ R ¹¹ , SO3R ¹² , CN, N0 ₂ , C0 ₂ R ¹³ , CONR ¹⁴ R ¹⁵ ;

Z represents H, COR ¹⁶ , or -(CH ₂ ) _n Ar;

W represents H, CH ₂ OR ¹⁷ ;

V represents H, C1-4 alkyl, -COOH, or COOR ¹⁸ ;

R ^9a and R ^9b to R ¹⁸ represents H or C _1-4 alkyl; Ar represents an aromatic ring system; and

n is from 1 to 10; or

(iii) a compound of formula III:

wherein A and BB represent H, Ci- ₄ alkyl, OR ¹⁹ , or NR ²⁰ R ²¹ , S0 ₃ R ²² , CN, N0 , C0 ₂ R ²³ , CONR ²⁴ R ²⁵ ; and

R ¹⁹ to R ²⁵ represents H or Ci- 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, the suitable compound comprising a pyridine ring may be selected from:

or, more particularly, 4-(4-nitrobenzyl)pyridine.

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

(ai) reacting a suitable compound comprising a pyridine ring in the presence of an aqueous solvent mixture with a first sample obtained from an original sample suspected to contain one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) to provide a reaction sample;

(bi) developing colour in the sample by adding a colour development agent to provide a colour-developed sample;

(ci) separating the reaction product of the suitable compound comprising a pyridine ring with one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) by mixing an organic solvent with the colour-developed sample, then allowing the organic and aqueous solvents to separate into layers and collecting the organic layer as the combined sample.

In embodiments of the invention, the aqueous solvent mixture of step (ai) may comprise water and a buffering agent, where the pH of the aqueous solvent mixture is from 4 to 7, optionally wherein the buffering agent is sodium citrate buffer. In further embodiments of the invention, the colour development agent in step (bi) may be an aqueous inorganic base, optionally wherein:

(ia) the colour development agent may be aqueous potassium carbonate; and/or

(ib) the concentration of the colour development agent may be from 500 nM to 1 M; and/or

(ic) the aqueous inorganic base may be added in an amount sufficient to provide the colour- developed sample with a pH value of at least 1 1.

In yet further embodiments of the invention, the organic solvent added to the coloured sample in step (ci) may be selected from one or more of acetophenone, diethyl ether, toluene, ethyl acetate, optionally wherein the organic solvent is ethyl acetate.

In embodiments of the invention involving establishing the amount of 3-monochloropropane diol and 3-mcpd ester(s) in an original sample in isolation from glycidol and glycidolester(s), the 3-monochloropropane diol sample may be obtained by:

(aii) reacting a suitable compound comprising a pyridine ring in the presence of an aqueous solvent mixture with a second sample obtained from an original sample suspected to contain one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) to provide a reaction sample, where the second sample has been treated to remove any glycidol and glycidol ester(s) present in the original sample;

(bii) developing the colour of the reaction sample by adding a colour development agent to provide a colour-developed sample;

(cii) separating the reaction product of the suitable compound comprising a pyridine ring with 3-monochloropropane diol and/or 3-mcpd ester(s) by mixing an organic solvent with the colour-developed sample, then allowing the organic and aqueous solvents to separate into layers and collecting the organic layer as the 3-monochloropropane diol sample.

In embodiments of the invention, the aqueous solvent mixture of step (aii) may comprise water and a buffering agent, where the pH of the aqueous solvent mixture is from 4 to 7, optionally wherein the buffering agent is sodium citrate buffer. In further embodiments of the invention, the colour development agent in step (bii) the colour development agent may an aqueous inorganic base, optionally wherein: (iia) the colour development agent is aqueous potassium carbonate; and/or (iib) the concentration of the colour development agent is from 500 nM to 1 M. In yet further embodiments of the invention, the organic solvent added to the coloured sample in step (cii) may be selected from one or more of acetophenone, diethyl ether, toluene, ethyl acetate, optionally wherein the organic solvent is ethyl acetate.

In embodiments of the invention, the first sample may be obtained by:

(aiii) adding an organic solvent to an original sample to form a dissolved sample;

(biii) adding a base dissolved in a primary alcohol having from 3 to 5 carbon atoms to the dissolved sample to form a basic sample;

(ciii) adding an acid dissolved in water to the basic sample and subjecting the sample to mixing to form a mixed sample;

(diii) allowing the mixed sample to separate into an organic solvent layer and an aqueous layer and collecting only the aqueous layer; and

(eiii) adding a buffering agent to the aqueous layer to form the first sample.

In embodiments of the invention, the organic solvent added to the original sample of step (aiii) may be iso-octane and/or chloroform. In embodiments of the invention, in step (biii):

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

(iiib) the alkoxide may be a metal 1 -butoxide (e.g. sodium or potassium 1 -butoxide).

In embodiments of the invention, the acid dissolved in water in step (ciii) is not hydrochloric acid, optionally wherein:

(A) the acid is acetic acid or H ₂ SO ₄ ; and/or

(B) the concentration of the acid in water is from 1 to 3 vol%.

In embodiments of the invention, the buffering agent in step (eiii) may be sodium citrate buffer and the buffering agent provides the first sample with a pH of from 4 to 7.

In yet further embodiments of the invention, involving establishing the amount of 3- monochloropropane diol and 3-mcpd ester(s) in an original sample in isolation from glycidol and glycidol ester(s), the second sample may be obtained by:

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

(bb) reacting any glycidol and glycidol ester(s) present in the dissolved sample with an acid and a primary alcohol having from 3 to 5 carbon atoms to form a reacted sample; (cc) adding a base dissolved in a primary alcohol having from 3 to 5 carbon atoms to the reacted sample to form a neutralised sample, or adding an alkoxide base to the reacted sample to form a neutralised sample, where the alkoxide has from 3 to 5 carbon atoms;

(dd) adding an acid dissolved in water to the basic sample and subjecting the sample to mixing to form a mixed sample;

(ee) allowing the mixed sample to separate into an organic solvent layer and an aqueous layer and collecting only the aqueous layer; and

(ff) adding a buffering agent to the aqueous layer to form the second sample.

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

In embodiments of the invention, the acid dissolved in water in step (dd) is not hydrochloric acid, optionally wherein:

(Ai) the acid is acetic acid or H ₂ SO ₄ ; and/or

(Bi) the concentration of the acid in water is from 1 to 3 vol%.

In embodiments of the invention, the buffering agent in step (ff) may be sodium citrate buffer and the buffering agent provides the first sample with a pH of from 4 to 7.

In embodiments of the invention, the acid and primary alcohol having from 3 to 5 carbon atoms used in step (aa) are H ₂ SO ₄ and 1 -propanol, respectively.

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

In embodiments of the invention, the original sample may be an edible oil, an edible fat or a combination thereof.

Brief Descriptions of Drawings

Fig. 1 Depicts the calibration curves prepared using standard samples containing: (a) a mixture of 3-mcpd, glycidol and their esters; and (b) 3-mcpd and its ester. Fig. 2 Depicts the sample preparation and detection of a combined amount of 3-mcpd, 3-mcpd ester, glycidol and/or glycidol esters.

Fig. 3 Depicts the transesterification of 3-mcpd esters and glycidol esters to 3-mcpd and glycidol respectively.

Fig. 4 Depicts the reaction of 3-mcpd and glycidol with 4-(4-nitrobenzyl)pyridine (NBP).

Fig. 5 Depicts the sample preparation and detection of 3-mcpd and/or 3-mcpd ester.

Fig. 6 Depicts the reaction of glycidol and glycidol esters with acid/alcohol.

Description

As noted above, there remain problems with the current testing methods used to establish the presence of 3-monochloropropane diol and glycidol (and derivatives thereof). As such, the current invention seeks to solve these issues with a new method of detection that is sensitive, quantitative and quick to run.

In the first instance, there is a need to know whether the composition contains any 3- monochloropropane diol and glycidol (and derivatives thereof). Thus, there is provided a method of quantitatively determining the combined amount of 3-monochloropropane diol (3- mcpd), 3-mcpd ester(s), glycidol and glycidol ester(s) suspected to be present in an original sample, the method comprising:

(a) measuring an optical property of a combined sample comprising the reaction product of a suitable compound comprising a pyridine ring with a first sample comprising one or more of 3-mcpd, a 3-mcpd ester, glycidol, a glycidol ester obtained from the original sample suspected of containing one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) and quantitatively determining the combined amount of the 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) by comparison of the measured optical property to one or more standards or a calibration curve.

In embodiments herein, the word“comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word“comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word“comprising” may be replaced by the phrases“consists of” or“consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word“comprising” and synonyms thereof may be replaced by the phrase“consisting of” or the phrase“consists essentially of” or synonyms thereof and vice versa.

As will be appreciated, the method described above functions by way of an SN2 reaction between the chlorine atom of the 3-mcpd and/or glycidol (and their derivatives) to form a new compound that has sufficient conjugation to provide a suitable optical property for analysis. Further details of how the optical property may be captured and analysed is provided in the experimental section below, where absorbance is the measured optical property. However, it will be appreciated that other optical properties, such as transmittance or reflectance may be used instead using standard techniques or by analogy to the methodology described in the examples for absorbance.

When used herein, the term“derivatives of 3-mcpd” refers to esters of 3-mcpd. When used herein, the term“3-mcpd ester” refers to a compound where one or both of the alcohol groups on 3-monochloropropane diol have reacted with a carboxylic acid (e.g. RCO ₂ H) or an ester thereof (e.g. RCO ₂ R’) 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 may have any suitable value and may be a C _1-50 alkyl group, a phenyl group or a heterocyclic group, which groups may be substituted or unsubstituted. The R’ group may be any suitable group, such as a C _1-5 alkyl group.

When used herein, the term“derivatives of glycidol” refers to esters of glycidol. When used herein, the term“glycidol ester” refers to a compound where the alcohol group on the glycidol has 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.

In embodiments of the invention the combined sample used in step (a) may be obtained by: (ai) reacting a suitable compound comprising a pyridine ring in the presence of an aqueous solvent mixture with a first sample obtained from an original sample suspected to contain one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) to provide a reaction sample;

(bi) developing colour in the sample by adding a colour development agent to provide a colour-developed sample; and (ci) separating the reaction product of the suitable compound comprising a pyridine ring with one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) by mixing an organic solvent with the colour-developed sample, then allowing the organic and aqueous solvents to separate into layers and collecting the organic layer as the combined sample.

In step (ai) above, the aqueous solvent mixture may comprise water and a buffering agent, where the pH of the aqueous solvent mixture is from 4 to 7. Any suitable buffering agent may be used. For example, the buffering agent may be sodium citrate buffer.

In step (bi) above, the colour development agent may be any suitable aqueous inorganic base. Examples of a suitable aqueous inorganic bases include, but are not limited to, aqueous potassium carbonate. The concentration of the colour development agent in the aqueous solution may have any suitable concentration, for example the concentration may be from 500 nM to 1 M. As will be appreciated, the colour development agent is added to the aqueous solution of step (ai) in an amount sufficient to provide the desired effect, this amount may be determined by the skilled person in accordance with routine trial and error or based upon their knowledge and expertise in this field. As an example, the aqueous inorganic base may be added in an amount sufficient to provide the colour-developed sample with a pH value of at least 1 1 .

In step (ci) above, the organic solvent added to the coloured sample may be selected from one or more of acetophenone, diethyl ether, toluene, ethyl acetate, optionally wherein the organic solvent is ethyl acetate. As will be appreciated, the solvents may be selected individually or selected to work in concert with one another. For example, when diethyl ether and/or ethyl acetate are used as the organic solvent an immiscible organic layer may form and be separated from the aqueous layer.

In embodiments of the invention, the first sample may be obtained by:

(aiii) adding an organic solvent to an original sample to form a dissolved sample;

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

(ciii) adding an acid dissolved in water to the basic sample and subjecting the sample to mixing to form a mixed sample;

(diii) allowing the mixed sample to separate into an organic solvent layer and an aqueous layer and collecting only the aqueous layer; and

(eiii) adding a buffering agent to the aqueous layer to form the first sample. In step (aiii) above, any suitable organic solvent may be used. Examples of suitable organic solvents that may be used include, but are not limited to iso-octane, chloroform and combinations thereof.

In step (biii) above, any suitable base may be used. Examples of suitable bases include, but are not limited to, a hydroxide base (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 it follows that a suitable metal alkoxide that may be used herein may be a metal 1 -butoxide (sodium or potassium 1 -butoxide).

In step (ciii) above, 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 H ₂ SO ₄ . Any suitable concentration of the acid may be used, for example, the concentration of the acid in water may be from 1 to 3 vol% (i.e. the concentration of the acid dissolved in water is from 1 to 3 vol%, which solution is then added to the basic sample).

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

As will be appreciated, the process described above allows one to obtain an understanding of the total amount of 3-mcpd, glycidol and their derivatives in the sample to be analysed. However, the above method is not capable of providing a break-down into the amount of the contaminants derived from 3-mcpd and the amount derived from glycidol. Thus, in order to obtain this information, the process may further comprise the step:

(b) quantitatively determining the amount of 3-monochloropropane diol and 3-mcpd ester(s) in an original sample by measuring an optical property of a 3-monochloropropane diol sample comprising the reaction product of a suitable compound comprising a pyridine ring with a second sample comprising 3-monochloropropane diol and/or a 3-mcpd ester(s) obtained from an original sample suspected of containing one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s), where the second sample has been treated to remove any glycidol and glycidol ester(s) present in the original sample.

The above method allows one to calculate the amount of 3-mcpd and its derivatives in the sample. As will be appreciated, the amount of glycidol ester and/or glycidol in an original sample may be determined by subtracting the amount of 3-monochloropropane diol and/or 3- mcpd ester(s) determined in step (b) from the combined amount determined in step (a). This allows one to determine the total amount of contaminant and the amounts of contaminant derived from 3-mcpd and glycidol.

In embodiments of the invention, the 3-monochloropropane diol sample used in step (b) may be obtained by:

(aii) reacting a suitable compound comprising a pyridine ring in the presence of an aqueous solvent mixture with a second sample obtained from an original sample suspected to contain one or more of 3-mcpd, 3-mcpd ester(s), glycidol, and glycidol ester(s) to provide a reaction sample, where the second sample has been treated to remove any glycidol and glycidol ester(s) present in the original sample;

(bii) developing the colour of the reaction sample by adding a colour development agent to provide a colour-developed sample;

(cii) separating the reaction product of the suitable compound comprising a pyridine ring with 3-monochloropropane diol and/or 3-mcpd ester(s) by mixing an organic solvent with the colour-developed sample, then allowing the organic and aqueous solvents to separate into layers and collecting the organic layer as the 3-monochloropropane diol sample.

In step (aii) above, the aqueous solvent mixture may comprise water and a buffering agent, where the pH of the aqueous solvent mixture is from 4 to 7, optionally wherein the buffering agent is sodium citrate buffer.

In step (bii) above, the colour development agent may be an aqueous inorganic base. Examples of a suitable aqueous inorganic bases include, but are not limited to aqueous potassium carbonate. The colour development agent in the aqueous solution may have any suitable concentration, for example the concentration may be from 500 nM to 1 M. As will be appreciated, the colour development agent is added to the aqueous solution of step (ai) in an amount sufficient to provide the desired effect, this amount may be determined by the skilled person in accordance with routine trial and error or based upon their knowledge and expertise in this field. As an example, the aqueous inorganic base may be added in an amount sufficient to provide the colour-developed sample with a pH value of at least 1 1 .

In step (cii) above, the organic solvent added to the coloured sample may be selected from one or more of acetophenone, diethyl ether, toluene, ethyl acetate, optionally wherein the organic solvent is ethyl acetate. As will be appreciated, the solvents may be selected individually or selected to work in concert with one another. For example, when diethyl ether and/or ethyl acetate are used as the organic solvent an immiscible organic layer may form and be separated from the aqueous layer.

In embodiments of the invention, the second sample may be obtained by:

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

(bb) reacting any glycidol and glycidol ester(s) present in the dissolved sample with an acid and a primary alcohol having from 3 to 5 carbon atoms to form a reacted sample;

(cc) adding a base dissolved in a primary alcohol having from 3 to 5 carbon atoms to the reacted sample to form a neutralised sample, or adding an alkoxide base to the reacted sample to form a neutralised sample, where the alkoxide has from 3 to 5 carbon atoms;

(dd) adding an acid dissolved in water to the basic sample and subjecting the sample to mixing to form a mixed sample;

(ee) allowing the mixed sample to separate into an organic solvent layer and an aqueous layer and collecting only the aqueous layer; and

(ff) adding a buffering agent to the aqueous layer to form the second sample.

In step (aa) above, any suitable organic solvent may be used. Examples of suitable organic solvents that may be used include, but are not limited to, iso-octane, chloroform and combinations thereof.

In step (bb) above, any suitable acid and alcohol may be used. For example, the acid may be H ₂ SO ₄ and the alcohol may be 1 -propanol.

In step (cc) above, any suitable base be used. Examples of suitable bases include, but are not limited to, a hydroxide base (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 it follows that a suitable metal alkoxide that may be used herein may be a metal 1 -butoxide (sodium or potassium 1 - butoxide).

In step (dd) above, 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 H ₂ SO ₄ . Any suitable concentration of the acid may be used, for example, the concentration of the acid in water may be from 1 to 3 vol% (i.e. the concentration of the acid dissolved in water is from 1 to 3 vol%, which solution is then added to the basic sample). In step (ff) above, the buffering agent may be any suitable buffering agent that provides a suitable pH value for the first sample. For example, the buffering agent may be sodium citrate buffer and/or the buffering agent may provide the first sample with a pH of from 4 to 7. As will be appreciated, the methods described above require the use of a suitable compound comprising a pyridine ring to provide the desired reaction products for analysis. The compound in question may be selected independently from:

(i) a compound of formula I:

wherein:

Ft ¹ represents N0 , CN, S0 ₃ R ⁴ , C0 ₂ R ⁵ , CONR ⁶ R ⁷ ;

R ² and R ³ independently represent halo, C1-4 alkyl or OR ⁸ ;

R ⁴ to R ⁷ independently represent C1-10 alkyl; and

R ⁸ represents C1-4 alkyl;

(ii) a compound of formula II:

wherein:

X represents H C1-4 alkyl, -COOH, or COOR ^9a ; Y represents H, Ci- ₄ alkyl, OR ^9b , or NR ¹⁰ R ¹¹ , S0 ₃ R ¹² , CN, N0 , C0 ₂ R ¹³ , CONR ¹⁴ R ¹⁵ ;

Z represents H, COR ¹⁶ , or -(CH ₂ ) _n Ar;

W represents H, CH ₂ OR ¹⁷ ;

V represents H, Ci- ₄ alkyl, -COOH, or COOR ¹⁸ ;

R ^9a and R ^9b to R ¹⁸ represents H or Ci- ₄ alkyl;

Ar represents an aromatic ring system; and

n is from 1 to 10; or

(iii) a compound of formula III:

wherein A and BB represent H, Ci- ₄ alkyl, OR ¹⁹ , or NR ²⁰ R ²¹ , S0 ₃ R ²² , CN, N0 ₂ , C0 ₂ R ²³ , CONR ²⁴ R ²⁵ ; and

R ¹⁹ to R ²⁵ represents H or Ci- 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, the suitable compound comprising a pyridine ring may be selected from:

,or, more particularly, 4-(4-nitrobenzyl)pyridine.

The term "Ar" or“aryl” when used herein includes Ce-14 (such as C ₆ -i ₃ (e.g. C6-10)) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Ce-io 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 in which aryl is phenyl.

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

As will be appreciated, the original sample may be any suitable sample suspected of containing 3-mcpd and/or glycidol and their derivatives. Examples of suitable original samples include, but are not limited to, an edible oil, an edible fat or a combination thereof.

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

Examples

The apparatus needed to carry out the detection include:

• a vortex;

• a boiling water bath;

• heating plate;

• centrifuge;

• an ice box;

• a spectrophotometer or colourimeter (with minimum 0.01 nm resolution); and

• pipettes.

Materials

Isooctane (analytical grade, ³99%), chloroform (analytical grade, ³99%), H ₂ SO ₄ (³99%), 1- propanol (analytical grade, ³99%), KOH (85% and above), 1 -butanol (analytical grade, ³99%), sodium citrate (³99%), ethanol (³95%), glycerol (³99%), 4-(4-nitrobenzyl)pyridine (98% and above), K ₂ CO ₃ (³99%), ethyl acetate (analytical grade, ³99%), acetophenone (analytical grade, ³99%), toluene (analytical grade, ³99%), acetone (analytical grade, ³99%) and ethylene glycol (³99%) were obtained from commercial sources and used directly without further purification to prepare the following solvents or reagents: • Solvents to dissolve the samples: isooctane or chloroform

• Reagent 1 : H2SO4 in 1 -propanol, at a working concentration of 1 -5 pl_ of H2SO4 in 1 - propanol (1 ml.)

• Reagent 2: KOH in 1 -butanol, at a working concentration of 1 -5% KOH in 1 -butanol (w/v)

• Reagent 3: H ₂ SO ₄ in deionised water, with a working concentration of 1 -3% of H ₂ SO ₄ in deionised water (v/v)

• Reagent 4: sodium citrate buffer, with a pH of 4-7

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

• Reagent B: K ₂ CO ₃ dissolved in deionised water, at a working concentration of 500 nM - 1 M

• Reagent C: ethyl acetate, acetophenone, diethyl ether, or toluene

Constructing a calibration curve using the standard samples

Typically, a calibration curve can be obtained via the following steps:

1. Six or more samples containing the standard product (3-mcpd and its ester; or a mixture of 3-mcpd, glycidol, and their esters), with their concentrations pre-determined by GC/MS, were used for constructing the calibration curve.

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

For the quantification of 3-mcpd and its ester (in Example 2), the samples should contain 3-mcpd and its ester concentrations of 0.1 to 5 ppm, with the various sample concentrations spread across the entire range (e.g. 0.1 , 0.5, 1 , 2, 3, 5 ppm).

2. The samples were then subject to the sample preparation and detection steps as described in Example 1 or 2, with their absorbance measured. 3. These absorbance values as obtained for the standard samples were then used to construct a calibration curve as shown in Fig. 1 a and b (for a combined sample of 3- mcpd, glycidol, and their esters; and for 3-mcpd and its ester respectively). The concentration of 3-mcpd and its ester, or a total concentration of 3-mcpd, glycidol, and their esters were then determined from the respective calibration curves.

Example 1. Detecting the combined amount of 3-monochloropropane diol (3-mcpd), 3- mcpd ester, glycidol and/or glycidol ester

The detection of the combined amount of 3-monochloropropane diol (3-mcpd), 3-mcpd ester, glycidol and/or glycidol ester in the samples was carried out using the apparatus and reagents listed above. This method allows 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), as shown in Fig. 2. The sample preparation involves the conversion of 3-mcpd esters and glycidol esters into 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 colour change when reacted with 3-mcpd or glycidol.

This detection method has been carried out on foodstuff samples which include RBDPO (refined, bleached and deodorised palm oil), palm olein, interesterified palm oil, sunflower oil, canola oil, solid stearin, cream margarine for cakes, confectionary fats for chocolate products and rice bran oil.

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

4 mL of Reagent 2 (20) was then added to each sample (10), mixed and left to stand for 5-15 min. The addition of Reagent 2 is to covert 3-mcpd esters (13) and/or glycidol esters (14) into 3-mcpd (22) and/or glycidol (24) respectively (as shown in the reaction scheme in Fig. 3). Thereafter, 3.5 mL of Reagent 3 (30) was added to each samples, mixed and vortexed to neutralise the alkaline conditions (Fig. 2). The samples were then centrifuged at ref of 2,000 or higher for at least 1 min to give two layers. The organic layer was discarded and the aqueous layer was transferred into a new tube. Care was taken to ensure that the organic layer did not contaminate the aqueous layer sample. This was then followed by the addition of 15 pL of Reagent 4 (40) to the sample to ensure that the sample was neutralised (Fig. 2). This gave the as-prepared samples 50 which were used directly in the subsequent detection step.

To detect the amount of 3-mcpd and/or glycidol in the as-prepared samples 50 (from previous preparation step), 1 mL of Reagent A (60) was added to each tube and vortexed thoroughly. The tubes were then incubated in boiling water bath for 15-40 min to allow the reaction of the NBP with 3-mcpd and glycidol to proceed completely (as shown in the reaction scheme in Fig. 4). The tubes were then removed from the water bath and cooled rapidly in ice. Thereafter, 0.5 mL of Reagent B (70) and 1 mL of Reagent C (80) were added to each tube and vortex thoroughly (Fig. 2). The tubes were then centrifuged to give two layers. The organic layer (90) containing the NBP reacted with 3-mcpd or glycidol (75) was transferred into a cuvette, with the absorbance measured at around 530-560 nm using a spectrophotometer.

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 concentration of 3-mcpd, glycidol and their esters (Fig. 1 a).

Example 2. Detecting the amount of 3-monochloropropane diol (3-mcpd) and/or 3-mcpd ester

The detection of the amount of 3-monochloropropane diol (3-mcpd) and/or 3-mcpd ester in the samples was carried out using the apparatus and reagents listed above. Similar to Example 1 , the detection process is divided into two parts: sample preparation (6) and detection (8), as shown in Fig. 5. This method is substantially the same as the method in Example 1 , but it includes an additional step at the sample preparation phase to remove epoxide functional groups in glycidols and glycidol esters that might be present in the samples. This effectively deactivates the glycidols and glycidol esters, so that they will not react with NBP in the detection step to give a colour change. This detection method has been carried out on foodstuff samples which include RBDPO (refined, bleached and deodorised palm oil), palm olein, interesterified palm oil, sunflower oil, canola oil, solid stearin, cream margarine for cakes, confectionary fats for chocolate products and rice bran oil.

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

Firstly, 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. The addition of Reagent 1 is an additional step (compared to Example 1 ) and is important for the removal of epoxide functional groups in glycidols (24) and glycidol esters (14) that might be present in the samples (shown in the reaction scheme in Fig. 6).

4 mL of Reagent 2 (20) was then added to each sample (10), mixed and incubated at room temperature for 10-15 min. The addition of Reagent 2 is to covert 3-mcpd esters and other esters into 3-mcpd and the respective alcohols (as shown in the reaction scheme in Fig. 2).

Thereafter, sufficient amount (~3.4 mL) of Reagent 3 (30) was added to each samples, mixed and vortexed to neutralise the alkaline conditions (Fig. 5). The samples were then centrifuged at ref of 2,000 or higher for at least 1 min to give two layers. The organic layer was discarded and the aqueous layer was transferred into a new tube. Care was taken to ensure that the organic layer did not contaminate the aqueous layer sample. This was then followed by the addition of 15 pL of Reagent 4 (40) to the sample to ensure that the sample was neutralised (Fig. 5). This gave the as-prepared samples 55 which were used directly in the subsequent detection step.

Sample detection (8) To detect the amount of 3-mcpd in the as-prepared samples 55 (from previous preparation step), 1 ml. of Reagent A (60) was added to each tube and vortexed thoroughly. The tubes were then incubated in boiling water bath for 15-40 min to allow the reaction of the NBP with 3-mcpd and glycidol to proceed completely (as shown in the reaction scheme in Fig. 3). The tubes were then removed from the water bath and cooled rapidly in ice. Thereafter, 0.5 ml. of Reagent B (70) and 1 ml. of Reagent C (80) were added to each tube and vortex thoroughly (Fig. 5). The tubes were then centrifuged to give two layers. The organic layer (95) containing the NBP reacted with 3-mcpd (75) was transferred into a cuvette, with the absorbance measured at around 530-560 nm using a spectrophotometer.

The concentration of 3-mcpd and/or 3-mcpd esters can be determined from a calibration curve generated using standard samples containing various concentration of 3-mcpd and its esters (Fig. 1 b). A comparison of the concentrations of 3-mcpd and/or 3-mcpd esters (in the various samples) determined by the current method to the concentrations as determined by GC/MS shows that the current method is able to achieve an accuracy and sensitivity comparable to that of the conventional GC/MS method (Table 1 ). Table 1. Comparing the various concentrations of 3-mcpd and/or 3-mcpd esters determined by the current method to the concentrations as determined by GC/MS

Example 3. Detecting the amount of glycidol and/or glycidol ester

The amount of glycidol and/or glycidol ester in the samples 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 glycidol ester (in Example 1 ) as shown below:

Concentration of = Total concentration of 3-mcpd, 3-mcpd esters, glycidol and/or glycidol glycidol and/or ester (as determined in Example 1 )

glycidol ester

Concentration of 3-mcpd and/or 3-mcpd ester (as determined in Example 2) A comparison of the concentrations of glycidol and/or glycidol ester (in the various samples) determined by the current method to that determined by GC/MS shows that the current method is able to achieve an accuracy and sensitivity comparable to that of the conventional GC/MS method (Table 2). Table 2. Comparing the various concentrations of glycidol and/or glycidol ester determined by the current method to the concentrations as determined by GC/MS

Example 4. Comparison of the current detection method with official methods by the American Oil Chemists’ Society

The currently claimed method of quantifying 3-mcpd and glycidol was compared with American Oil Chemists’ Society (AOCS) official methods Cd-29a-13, Cd-29b-13 and Cd-29c- 13 (referred as Method A, B and C respectively) and the advantages are summarised in Table 3 below.

Table 3. Summary of the advantages of the current method in comparison with the official methods by the AOCS.