Method and portable test kit for detecting 3-monochloropropane-1,2-DIOL (3-MCPD) esters and glycidyl esters (GE) in refined, bleached, and deodorized palm oil (RBDPO)

Abstract

A method for detecting 3-Monochloropropane-1,2-diol (3-MCPD) esters and Glycidyl esters (GE) in a sample containing refined, bleached, and deodorized palm oil (RBDPO) comprising: adding a predetermined amount of a first reagent containing Dibutyltin dilaurate to the sample and forming a mixture; heating the mixture containing the first reagent to a temperature between 55°C and 65°C for a predetermined period to initiate a reaction between the first reagent and the 3-MCPD esters and GE, forming reaction products containing target analytes; adding a second reagent comprising an ethanol-based solvent to selectively dissolve the reaction products and separating them from the RBDPO; wherein a separate ethanol layer is formed, the layer containing the target analytes; adding a third reagent comprising a dye solution to bind with the separated target analytes; wherein adding the third reagent to the separated target analytes, enables detection of the 3-MCPD esters and GE in the sample. A portable test kit is also provided herein.

Description

METHOD AND PORTABLE TEST KIT FOR DETECTING 3- MONOCHLOROPROPANE-1,2-DIOL (3-MCPD) ESTERS AND GLYCIDYL ESTERS (GE) IN REFINED, BLEACHED, AND DEODORIZED PALM OIL (RBDPO)FIELD OF INVENTION

[0001] The present invention generally relates to a method and a portable test kit for detecting the presence of 3 -Monochloropropane- 1,2-diol (3-MCPD) esters and Glycidyl esters (GE) in refined, bleached, and deodorized palm oil (RBDPO).BACKGROUND

[0002] 3 -monochloropropane- 1,2-diol (3-MCPD) and glycidyl esters (GE) are two known byproducts formed during the refining process of palm oil, which includes bleaching and deodorizing steps. These contaminants have raised concerns due to their potential toxicity and possible health risks when consumed in significant amounts. For instance, 3-MCPD esters are of particular concern for their potential carcinogenicity and impact on renal health, while GE is recognized as a precursor to glycidol, a compound linked to mutagenic and genotoxic risks.

[0003] Existing detection methods for 3-MCPD esters and GE rely heavily on advanced analytical technologies, including gas chromatography (GC), high-performance liquid chromatography (HPLC), and mass spectrometry (MS). While these techniques are highly sensitive and accurate, they are also associated with significant drawbacks. The equipment involved is often prohibitively expensive and requires not only substantial capital investment but also ongoing maintenance and operational costs. Furthermore, these methods demand highly skilled personnel to conduct the analysis and interpret results accurately, limiting their practicality to well -equipped laboratories. Additionally, the time-intensive nature of sample preparation, analysis, and data processing restricts the feasibility of these methods for routine or large-scale screening.

[0004] This reliance on complex, centralized testing infrastructure creates significant barriers to widespread monitoring of 3-MCPD esters and GE, particularly for smaller-scale producers or regulatory bodies operating in regions with limited access to laboratory facilities.SUMMARY

[0005] In one aspect, the present invention provides a method for detecting 3-monochloropropane-l,2-diol (3-MCPD) esters and Glycidyl esters (GE) in a sample containing refined, bleached, and deodorized palm oil (RBDPO) comprising: adding a predetermined amount of a first reagent containing Dibutyltin dilaurate to the sample and forming a mixture; heating the mixture containing the first reagent to a temperature between 55°C and 65°C for a predetermined period to initiate a reaction between the first reagent and the 3-MCPD esters and GE and forming reaction products containing target analytes; adding a second reagent comprising an ethanol-based solvent to selectively dissolve the reaction products and separating them from the RBDPO; wherein a separate ethanol layer is formed, the layer containing the target analytes; adding a third reagent comprising a dye solution to bind with the separated target analytes; wherein adding the third reagent to the separated target analytes initiates a reaction that forms dye-bound analytes, enabling detection of the 3-MCPD esters and GE in the sample.

[0006] In an embodiment, the method further includes vortexing the mixture containing the third reagent and subsequently allowing the mixture to settle to form the separate ethanol layer with dye-bound target analytes.

[0007] In an embodiment, the reaction products comprise target analytes defined as compounds derived from 3-MCPD esters and Glycidyl esters through interaction with the first reagent.

[0008] In another embodiment, the mixture is heated to a temperature of 60°C. Typically, the mixture is heated for 20 minutes.

[0009] Advantageously, heating the first reagent with the mixture enables the chemical conversion of MCPD esters and GE thereby forming detectable reaction products.

[0010] In another embodiment, the amount of the first reagent is the amount of the first reagent is lOOpL.

[0011] In yet a further embodiment, the method further includes allowing the mixture to settle for 10 minutes upon adding the second reagent comprising ethanol-based solvent to allow phase separation to occur.

[0012] In an embodiment, wherein the amount of the first reagent is added to the sample in avolume range of 3 to 3.5 %v / v relative to the total volume of the sample. Typically, the volume of the sample ranges from 1ml to 3ml.

[0013] In another embodiment, the amount of the second reagent added to the sample in a volume range of 100% v / v to 135% v / v relative to the volume of the sample.

[0014] In yet another embodiment, the amount of the third reagent is added within the range of 0.5 to 1 % v / v relative to the volume of the sample.

[0015] In yet a further embodiment, the visual detection includes UV-VIS spectrophotometer.

[0016] In an embodiment, the method includes the UV-VIS spectrophotometer generating an absorbance spectrum that correlates directly with the concentration of MCPD esters and GE, providing a quantitative measure of these contaminants.

[0017] Advantageously, the reaction between the third reagent and the RhB-bound analytes results in the formation RhB-MCPD ester and RhB-GE complexes, enabling the concentration of MCPD esters and GE to be quantified.

[0018] In a second aspect, the present invention provides a portable test kit for detecting 3-Monochloropropane-l,2-diol (3-MCPD) esters and Glycidyl esters (GE) in a sample of refined, bleached, and deodorized palm oil (RBDPO), comprising: a first reagent containing Dibutyltin dilaurate Dibutyltin dilaurate to react with 3-MCPD esters and GE when mixed with the RBDPO sample and heated; a heating device capable of maintaining a temperature between 55 °C and 65 °C for a predetermined period to facilitate the reaction between the first reagent and the 3-MCPD esters and GE; a second reagent comprising an ethanol-based solvent for dissolving the reaction products after heating and for separating the ethanol layer containing the dissolved reaction products; a third reagent comprising a dye solution containing acetonitrile for binding with the dissolved reaction products, enabling visual detection of the 3-MCPD esters and GE; and a portable UV-VIS for quantifying the concentration of the 3-MCPD esters and GE in the sample based on the detected 3-MCPD esters and GE.

[0019] In an embodiment, the UV-VIS spectrophotometer is designed for portability, enabling on-site testing and real-time analysis.BRIEF DESCRIPTION OF DRAWINGS

[0020] The invention will be more understood by reference to the description below taken in conjunction with the accompanying drawings herein:

[0021] FIG. 1 provides a flowchart for the method in accordance with an embodiment of the present invention.DETAILED DESCRIPTION

[0022] In line with the above summary, the following description of a number of specific and alternative embodiments is provided to understand the inventive features of the present invention. It shall be apparent to one skilled in the art, however that this invention may be practiced without such specific details. Some of the details may not be described at length so as not to obscure the invention. For ease of reference, common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures.

[0023] Embodiments of the invention are described by way of illustration. As will be realized, the invention is capable of other and different embodiments and its several details are capable of modifications in various respects, all without departing from the scope of the present invention. It should be noted that the drawings include schematic description of how the process in accordance with the preferred embodiments can be carried out. The necessary pumps, valves, and other standard equipment or components may have not been illustrated since they are known in the art.

[0024] With reference to the flowchart in FIG. 1, the present invention provides a method for detecting 3-MCPD esters and GE in refined, bleached, and deodorized palm oil (RBDPO) using a combination of specific reagents that react with these contaminants. The method involves adding a first reagent to the RBDPO sample (S 101) and forming a mixture, followed by heating the mixture to atemperature between 55°C and 65°C for a specific duration (S102). This heating step induces a reaction between the first reagent and the 3-MCPD esters and GE, converting them into detectable reaction products. Following the reaction, the method proceeds with the addition of a second reagent (SI 03), to dissolve the reaction products. Phase separation occurs, allowing the ethanol layer, containing the dissolved reaction products, to be isolated (S104). A thirdreagent, a dye solution containing acetonitrile is then introduced to bind with the reaction products, forming detectable complexes that can be observed visually or analyzed using spectrophotometric techniques (S105). The presence of the complexes indicates the presence of 3-MCPD esters and GE in the sample, and the concentration can be measured using a UV-VIS spectrophotometer (SI 06).

[0025] In addition to the method, the present invention further provides a portable test kit that includes two reagents and equipment required to perform the detection. This test kit is adapted for on-site testing.

[0026] The first reagent comprises Dibutyltin dilaurate that functions as a catalyst or derivatizing agent, facilitating the chemical conversion of MCPD esters and GE within the RBDPO sample into forms detectable by UV-VIS spectrophotometry. The mixture, comprising the RBDPO oil matrix and the first reagent, may be heated for a duration of 15 to 25 minutes. This heating facilitates optimal conditions for a chemical reaction between the MCPD esters, GE, and the first reagent, leading to the formation of reaction products that can subsequently be detected using UV-VIS spectrophotometry.

[0027] The second reagent comprises ethanol, or ethanol-based solvent to act as an extraction solvent. Ethanol, due to its polarity, making it effective at dissolving polar reaction products such as ester or intermediates while leaving non-polar components such as the oil matrix, largely unaffected. The selective solubility ensures that the reaction products containing the target analytes or MCPD ester and GE derivatives are separated from the oil matrix. In this method, the mixture is thoroughly shaken to enable separation and subsequently allowed to settle for the phase separation to occur, in which ethanol layer containing the dissolved reaction products rises to the top.

[0028] The third reagent is a dye solution comprising Rhodamine B (RhB) dissolved in acetonitrile at a concentration of 5 mg per 10 mb. RhB functions as a chromophore, forming complexes with the extracted analytes to enhance their optical properties for detection. The ethanol layer from the previous step is combined with the third reagent, allowing RhB to interact with the target analytes. This interaction facilitates the detection and quantification of the analytes via UV-VIS spectrophotometry.

[0029] In typical applications, the sample volume generally ranges from 1 mL to 3 mL. The first reagent is added to the sample in a volume range of 3% v / v to 3.5% v / v, relative to the total volume of the sample. This corresponds to a volume of 100 pL for a 3 mL sample. The second reagent is introduced within a volume range of 100% v / v to 135% v / v relative to the sample volume, equating to a volume of 4 mL for a 3 mL sample. The third reagent is added within a volume range of 0.5% v / v to 1% v / v, which is 100 pL for a 3 mL sample. These specific volumes and ranges ensure that the method remains adaptable to varying sample sizes while maintaining the desired efficiency, accuracy, and reproducibility of the process, providing consistent and reliable results across a range of uses.

[0030] To prepare a sample for analysis, a 3 mL sample of RBDPO, which serves as the oil matrix for detecting 3-MCPD esters and GE is prepared. The first reagent, comprising 100 pL of Dibutyltin dilaurate, is added to this sample. Dibutyltin dilaurate functions as a catalyst or derivatizing agent, facilitating the chemical conversion of MCPD esters and GE within the RBDPO sample into forms detectable by UV-VIS spectrophotometry. Next, the mixture is heated to 60°C for 20 minutes to allow the reaction between MCPD esters, GE, and the reagent, resulting in the formation of detectable intermediates or reaction products. Following this heating step, the mixture is allowed to cool for 5 minutes to stabilize the reaction products, preparing the sample for further processing. 4 mL of ethanol is added as the second reagent for dissolving the reaction products and separating them from the oil matrix. The mixture is thoroughly shaken to enable extraction of the MCPD ester and GE derivatives, then allowed to settle for 10 minutes (S104). During this period, phase separation occurs, with the ethanol layer, now containing the target analytes, rising to the top. The third reagent, comprising 100 pL of Rhodamine B (RhB) in acetonitrile at a concentration of 5 mg per 10 mL, is then added to the mixture. RhB serves as a chromophoric agent, that binds to the extracted analytes to enhance their visibility and thereby forming complexes that enables their detection under UV-VIS spectrophotometry. The mixture is vortexed for 1 minute to ensure thorough interaction between the RhB dye and the extracted analytes in the ethanol layer. After vortexing, the mixture is allowed to settle for an additional 5 minutes to ensure proper phase separation and concentration of the RhB-bound analytes in the ethanol layer. The ethanol layer containing the RhB-bound reaction products (complexes formed by interaction between the dye solution and the target analytes) is then extracted carefully for spectrophotometric analysis. Using a UV-VIS spectrophotometer, the absorbance of the RhB-bound reaction products is measured at specific wavelengths corresponding to the RhB-MCPD ester and RhB-GE complexes. The resulting absorbance data allows for quantifying theconcentration of MCPD esters and GE in the original RBDPO sample, thus providing a reliable measure of contamination levels.

[0031] The method of the present invention provides significant advantages over traditional methods for detecting 3-MCPD esters and GE in RBDPO, primarily through simplified, cost-effective, and user-friendly sample preparation. Unlike existing methods that involve complex, multi-stage processes with high costs, this method uses straightforward, reagent-based reactions that reduce handling and accelerate the detection process. Its portability, enabled by a compact UV-VIS spectrophotometer, supports on-site testing at processing facilities and remote areas, which allows immediate feedback and decision-making.

[0032] The test kit makes it viable for use in smaller labs and developing regions. The procedure is quick and efficient, possibly yielding results in minutes. This ease of use extends to the operability of the kit itself, whereby anyone with minimal training can perform tests and interpret results accurately.

[0033] The present invention makes contaminant detection in edible oils more accessible, practical, and affordable, offering a reliable tool for routine testing in quality control and regulatory compliance within the palm oil industry by combining reagent-based chemistry with portable UV-VIS spectrophotometry. Further, environmental impact is minimized using small volumes of less hazardous chemicals, making the method more sustainable than traditional techniques. The method could improve analytical performance by providing enhanced sensitivity and specificity, with direct, single-step reactions that reduce the risk of errors and contamination, while real-time monitoring allows for timely interventions in production environments.

[0034] While the invention has been described as required in terms of preferred embodiments and specific operating ranges and conditions, those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described.

Claims

CLAIMS1. A method for detecting 3 -Monochloropropane- 1,2-diol (3-MCPD) esters and Glycidyl esters (GE) in a sample comprising:- adding a predetermined amount of a first reagent containing Dibutyltin dilaurate to the sample and forming a mixture (S 101);- heating the mixture (S102) to a temperature between 55°C and 65°C for a predetermined period to initiate a reaction between the first reagent, and the 3-MCPD esters and / or GE, thereby forming reaction products containing 3-MCPD esters and / or GE target analytes;adding a second reagent (S 103) comprising an ethanol-based solvent to the mixture, wherein the second reagent selectively dissolves the reaction products to form a separate ethanol layer containing the target analytes is formed;- adding a third reagent (SI 05) comprising a dye solution to bind with the separated target analytes;wherein adding the third reagent to the separated target analytes initiates a reaction that forms dye-bound analytes, enabling detection of the 3-MCPD esters and / or GE in the sample (S106).

2. The method according to claim 1, wherein the sample contains refined, bleached, and deodorized palm oil (RBDPO).

3. The method according to claim 1, wherein the target analytes comprise compounds derived from 3-MCPD esters and Glycidyl esters.

4. The method according to claim 1, wherein the mixture containing the first reagent is heated to a temperature of 60°C.

5. The method according to claim 1, wherein the mixture containing the first reagent is heated for 20 minutes.

6. The method according to claim 1, wherein the method further includes allowing the mixture containing the second reagent to settle for 10 minutes to allow phase separation to occur.

7. The method according to claim 1, wherein the dye solution of the third reagent is Rhodamine B (RhB).

8. The method according to claim 1, wherein the third reagent comprises acetonitrile.

9. The method according to claim 7, wherein the method further includes vortexing the mixture containing the third reagent and subsequently allowing the mixture to settle to form the separate ethanol layer with RhB-bound target analytes.

10. The method according to claim 9, wherein the reaction between the third reagent and the RhB-bound analytes results in the formation RhB-MCPD ester and RhB-GE complexes, enabling the concentration of MCPD esters and GE to be quantified.

11. The method according to claim 1, wherein the volume of the sample ranges from 1 to 3ml.

12. The method according to claim 1, wherein the amount of the first reagent is added to the sample in a volume range of 3 to 3.5 %v / v relative to the volume of the sample.

13. The method according to claim 1, wherein the amount of the second reagent added to the sample in a volume range of 100% v / v to 135% v / v relative to the volume of the sample.

14. The method according to claim 1, wherein the amount of the third reagent is added within the range of 0.5 to 1 % v / v relative to the volume of the sample.

15. The method according to claim 1, wherein the detection includes by means of UV-VIS spectrophotometer.

16. The method according to claim 15, wherein the UV-VIS spectrophotometer generates an absorbance spectrum that correlates directly with the concentration of MCPD esters and GE, providing a quantitative measure of these contaminants.

17. A portable test kit for detecting 3 -Monochloropropane- 1,2-diol (3-MCPD) esters and Glycidyl esters (GE) in a sample of refined, bleached, and deodorized palm oil (RBDPO), comprising:a first reagent containing Dibutyltin dilaurate to react with 3-MCPD esters and GE when mixed with the RBDPO sample and heated;a heating device capable of maintaining a temperature between 55 °C and 65 °C for a predetermined period to facilitate the reaction between the first reagent and the 3-MCPD esters and GE;a second reagent comprising an ethanol-based solvent for dissolving the reaction products after heating and for separating the ethanol layer containing the dissolved reaction products;a third reagent comprising a dye solution for binding with the dissolved reaction products, enabling detection of the 3-MCPD esters and GE;a portable UV-VIS for quantifying the concentration of the 3-MCPD esters and GE in the sample based on the detected 3-MCPD esters and GE.

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