A method for simultaneously detecting limonene and linalool in a solution

Abstract

The application discloses a method for simultaneously detecting limonene and linalool in a solution, and relates to the technical field of detection methods. The method comprises the following steps: providing a sample solution to be detected; mixing limonene standard samples, linalool standard samples and a solvent to obtain a mixed standard solution; diluting the mixed standard solution into a plurality of mixed standard solution diluents with different concentration gradients by using the solvent; detecting the mixed standard solution diluents by using GC-FID, collecting the retention time and the chromatographic peak area of limonene and linalool; establishing a standard curve of limonene and linalool; detecting the sample solution to be detected by using the same method and collecting data; and determining the content of limonene and linalool in the sample solution to be detected according to the standard curve, the retention time of the standard samples and the data collected from the sample to be detected. The GC-FID is easy to operate and has a good separation effect. The mixed standard solution can be used to simultaneously detect limonene and linalool, and the analysis efficiency is high. The establishment of the standard curve improves the detection stability and the accuracy.

Description

Technical Field

[0001] This invention relates to the field of detection methods, and in particular to a method for simultaneously detecting limonene and linalool in a solution. Background Technology

[0002] In the food and flavor industries, citrus essential oils are an important class of natural flavorings, with limonene and linalool being among their main flavor components. To obtain high-quality, water-soluble citrus essential oils in industrial production, eutectic solvents are typically used for extraction to achieve a certain degree of terpene removal, thereby improving the flavor and quality of the citrus essential oils. After terpene removal, the contents of linalool and limonene in the eutectic solvent (extract phase) and the treated citrus essential oil (raffinate phase) will change. Therefore, it is necessary to accurately determine the extent of terpene removal in the raffinate phase and the extraction effect of the extract phase by measuring the precise contents of limonene and linalool in both phases.

[0003] Existing methods for detecting linalool and limonene include high performance liquid chromatography, gas chromatography, and gas chromatography-mass spectrometry.

[0004] However, existing analytical methods have the following drawbacks: 1) poor separation effect; 2) low analytical efficiency; 3) poor detection stability and low accuracy; 4) mainly applicable to systems such as aromatherapy, and cannot be used for complex systems, such as systems containing eutectic solvents; 5) mainly semi-quantitative, without accurate quantification. Summary of the Invention

[0005] The main objective of this invention is to propose a method for the simultaneous detection of limonene and linalool in solution, aiming to solve the problems of poor separation effect, low analytical efficiency, unstable detection, low accuracy, inability to be used in complex systems, and inability to accurately quantify in existing technologies.

[0006] To achieve the above objectives, this invention proposes a method for simultaneously detecting limonene and linalool in solution, comprising the following steps:

[0007] S10. Provide the sample solution to be tested;

[0008] S20. Mix limonene standard, linalool standard and solvent to obtain mixed standard solution;

[0009] S30. Dilute the mixed standard solution with solvent to prepare multiple mixed standard solution dilution solutions with different concentration gradients;

[0010] S40. The GC-FID detection method was used to detect the mixed standard solution dilutions with multiple concentration gradients, and the retention time and chromatographic peak area of ​​limonene and linalool in the mixed standard solution dilutions with multiple concentration gradients were collected.

[0011] S50. Establish standard curves for limonene and linalool based on the retention times and chromatographic peak areas of mixed standard solutions with multiple concentration gradients.

[0012] S60. The GC-FID detection method was used to detect the sample solution to be tested, and the retention time and chromatographic peak area of ​​limonene and linalool in the sample solution to be tested were collected.

[0013] S70. Based on the standard curves of limonene and linalool, and the retention times and peak areas of limonene and linalool in the sample solution to be tested, determine the content of limonene and linalool in the sample solution to be tested.

[0014] Optionally, in step S20, the solvent includes one of methanol and isopropanol; and / or,

[0015] In step S30, the solvent includes either methanol or isopropanol.

[0016] Optionally, in step S20, the mass concentration ratio of limonene to linalool in the mixed standard solution is 1:(1-2).

[0017] Optionally, in step S30, the number of the multiple mixed standard solution diluents with different concentration gradients is n, and the value of n is a positive integer greater than or equal to 5.

[0018] Optionally, in step S30, the concentrations of the mixed standard solution diluents with different concentration gradients are 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

[0019] Optionally, in step S40, the material of the capillary column of the GC-FID chromatographic column includes (5% phenyl)-methylpolysiloxane; and / or,

[0020] In step S60, the material of the capillary column of the GC-FID chromatographic column includes (5% phenyl)-methylpolysiloxane.

[0021] Optionally, in step S40, the chromatographic conditions for the GC-FID include:

[0022] The injection port temperature is 250℃~280℃; the carrier gas is N2; the flow rate is 1.0~1.2mL / min; the injection volume is 1.0μL; and the split ratio is (30~40):1.

[0023] Temperature program: Initial temperature 80℃, hold for 0 min, increase to 150℃ at 10℃ / min, hold for 3 min, then increase to 200℃ at 10℃ / min, hold for 0 min; and / or,

[0024] In step S60, the chromatographic conditions for GC-FID include:

[0025] The injection port temperature is 250℃~280℃; the carrier gas is N2; the flow rate is 1.0~1.2mL / min; the injection volume is 1.0μL; and the split ratio is (30~40):1.

[0026] Temperature rise program: Initial temperature 80℃, hold for 0 min, rise to 150℃ at 10℃ / min, hold for 3 min, then rise to 200℃ at 10℃ / min, hold for 0 min.

[0027] Optionally, in step S40, the FDI detector conditions of the GC-FID include:

[0028] Detector temperature: 280℃~300℃; air flow rate: 350~400mL / min; hydrogen flow rate: 30~35mL / min; make-up gas flow rate: 25~30mL / min; and / or,

[0029] In step S60, the FDI detector conditions of the GC-FID include:

[0030] Detector temperature: 280℃~300℃; air flow rate: 350~400mL / min; hydrogen flow rate: 30~35mL / min; tail gas flow rate: 25~30mL / min.

[0031] Optionally, in step S50: the detection limit of the limonene standard curve is 6 mg / kg; and / or,

[0032] The limit of quantitation for the limonene standard curve is 20 mg / kg.

[0033] Optionally, in step S50: the detection limit of the linalool standard curve is 5 mg / kg; and / or,

[0034] The limit of quantitation for the linalool standard curve is 20 mg / kg.

[0035] This invention proposes a method for simultaneously detecting limonene and linalool in solution. Limonene and linalool are mixed with a solvent to obtain a mixed standard solution, meaning the mixed standard solution contains both limonene and linalool standards, which can be detected simultaneously during analysis, improving analytical efficiency. The mixed standard solution is then diluted with a solvent to create multiple diluted mixed standard solution solutions with different concentration gradients. These diluted solutions contain both limonene and linalool standards, allowing for separate detection of the limonene and linalool content in each diluted solution during analysis, resulting in high analytical efficiency. The method utilizes GC-FID detection, which is convenient to operate and can effectively detect the mixed standard solution. Limonene and linalool are completely separated in the solution, meaning their chromatographic peaks do not overlap and are independent, improving the separation efficiency. Standard curves for limonene and linalool are established based on the retention times and peak areas of diluted mixed standard solutions at multiple concentration gradients. These standard curves are applicable to samples with limonene and linalool content across the entire concentration gradient, improving analytical efficiency and detection stability. Based on the standard curves and the retention times and peak areas of limonene and linalool in the sample solution, the absolute content of limonene and linalool in the sample solution can be quickly determined, improving detection accuracy. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0037] Figure 1 The results show the peak areas of linalool and limonene at different time points provided in Example 1 of this invention.

[0038] Figure 2 The chromatogram of the mixed standard solution of linalool and limonene with a mass concentration of 50 mg / mL provided in Example 1 of the present invention;

[0039] Figure 3 The chromatogram of sweet orange essential oil provided in Example 1 of this invention;

[0040] Figure 4 This is a chromatogram of the extract phase provided in Example 1 of the present invention;

[0041] Figure 5 This is a chromatogram of the raffinate phase provided in Example 1 of the present invention.

[0042] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] In the food and flavor industry, citrus essential oils are an important class of natural flavorings, with limonene and linalool being among their main flavor components. To obtain high-quality, water-soluble citrus essential oils in industrial production, eutectic solvents are typically used for extraction to remove terpenes, thereby improving the flavor and quality of the citrus essential oils. After terpene removal, the content of linalool and limonene in the eutectic solvent (extract phase) and the treated citrus essential oil (raffinate phase) will change. Existing methods for detecting linalool and limonene include high-performance liquid chromatography (HPLC), gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS). However, existing analytical methods have the following drawbacks: 1) poor separation effect; 2) low analytical efficiency; 3) poor detection stability and low accuracy; 4) mainly applicable to aromatherapy systems and not suitable for complex systems, such as those containing eutectic solvents; 5) primarily semi-quantitative, lacking accurate quantification. In view of this, the present invention provides a method for simultaneously detecting limonene and linalool in solution, aiming to solve the problems of poor separation effect, low analytical efficiency, unstable detection, low accuracy, inability to be used in complex systems, and inability to accurately quantify in the prior art.

[0045] In this invention, the method for simultaneously detecting limonene and linalool in solution includes the following steps:

[0046] S10. Provide the sample solution to be tested;

[0047] S20. Mix limonene standard, linalool standard and solvent to obtain mixed standard solution;

[0048] S30. Dilute the mixed standard solution with solvent to prepare multiple mixed standard solution dilution solutions with different concentration gradients;

[0049] S40. The GC-FID detection method was used to detect the mixed standard solution dilutions with multiple concentration gradients, and the retention time and chromatographic peak area of ​​limonene and linalool in the mixed standard solution dilutions with multiple concentration gradients were collected.

[0050] S50. Establish standard curves for limonene and linalool based on the retention times and chromatographic peak areas of mixed standard solutions with multiple concentration gradients.

[0051] S60. The GC-FID detection method was used to detect the sample solution to be tested, and the retention time and chromatographic peak area of ​​limonene and linalool in the sample solution to be tested were collected.

[0052] S70. Based on the standard curves of limonene and linalool, and the retention times and peak areas of limonene and linalool in the sample solution to be tested, determine the content of limonene and linalool in the sample solution to be tested.

[0053] Existing methods for detecting linalool and limonene include high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). However, these methods are mainly used for simple systems such as aromatherapy and fragrance products. In practical industrial applications, many complex solution systems require the simultaneous detection of the absolute content of both linalool and limonene. For example, citrus essential oils generally require "terpene removal" treatment (i.e., extraction) using eutectic solvents. Both the treated citrus essential oil (raffinate phase) and the extracted eutectic solvent (extract phase) contain limonene and linalool. When using existing methods to detect the extract and raffinate phases, current HPLC analysis is inaccurate, mainly due to the presence of a large number of salt ions in solutions such as eutectic solvents, which causes background interference, i.e., matrix effect. This has a significant impact on liquid chromatography analysis, resulting in poor separation, poor detection stability, and low accuracy. Compared to HPLC, this invention eliminates the need for mobile phase preparation, and reduces detection time, reagent types, and reagent quantities. Furthermore, the eutectic solvent composition is complex, and the salt compounds and inorganic substances (metal ions) contained therein can damage the mass spectrometer in a gas chromatography-mass spectrometry (GC-MS) system and easily cause background interference or increased matrix effects. At the same time, the maintenance cost of GC-MS is relatively higher than that of GC-FID. In contrast to the above two methods, the GC-FID method of this invention has better stability and higher accuracy.

[0054] In the technical solution of this invention, limonene and linalool are mixed with a solvent to obtain a mixed standard solution, meaning the mixed standard solution contains both limonene and linalool standards simultaneously, which can be detected concurrently during analysis, improving analytical efficiency. The mixed standard solution is then diluted with a solvent to create multiple diluted mixed standard solution solutions with different concentration gradients, meaning each of these diluted solutions contains both limonene and linalool standards. During analysis, the content of limonene and linalool in these diluted solutions with different concentration gradients can be detected separately, resulting in high analytical efficiency. GC-FID detection is convenient to operate and can detect both limonene and linalool in the mixed standard solution. Linalool and limonene are completely separated, meaning their chromatographic peaks do not overlap and are independent, improving separation efficiency. Standard curves for limonene and linalool are established based on the retention times and peak areas of diluted mixed standard solutions at multiple concentration gradients. These standard curves are applicable to samples with limonene and linalool content across the entire concentration gradient, improving analytical efficiency and detection stability. The absolute content of limonene and linalool in the sample solution can be rapidly determined based on the standard curves and the retention times and peak areas of limonene and linalool in the sample solution, improving detection accuracy.

[0055] The solvents for dissolving the sample, dissolving limonene and linalool standards, and diluting the mixed standard solution can be methanol, propylene glycol, ethanol, or isopropanol. These solvents can effectively disperse complex systems, such as sweet orange essential oil treated with a eutectic solvent and a eutectic solvent system treated with sweet orange essential oil.

[0056] Further, in step S20, the solvent includes either methanol or isopropanol; and / or, in step S30, the solvent includes either methanol or isopropanol. Propylene glycol has poor fluidity, making it difficult to mix the extract / raffinate phase evenly, resulting in longer sample processing time and reduced efficiency. Because eutectic solvents contain propylene glycol and glycerol matrices (hydrogen bond acceptors), citrus essential oils, such as sweet orange essential oil, contain many terpenes and oxygenated terpenes. When diluted with ethanol in an HP-5 column, limonene and glycerol cannot be completely separated in sweet orange essential oil treated with eutectic solvents. Compared to other solvents, methanol and isopropanol have relatively good solubility and can completely separate limonene and glycerol in the extract phase. This invention uses only one solvent, isopropanol or methanol, for dilution and filtration before analysis, simplifying the pretreatment process, reducing the types of solvents used, indirectly reducing pollution to human health and the environment, and eliminating the need for complex experimental steps and professional skills, thus reducing operational difficulty.

[0057] Further, in step S20, the mass concentration ratio of limonene to linalool in the mixed standard solution is 1:(1-2). The mass concentration ratio of limonene to linalool in the mixed standard solution can be 1:1, 1:1.5, or 1:2, and a clearer peak shape can be obtained within this range.

[0058] Furthermore, in step S30, the number of mixed standard solution diluents with different concentration gradients is n, and the value of n is a positive integer greater than or equal to 5. The number of mixed standard solution diluents with different concentration gradients, n, can be 5, 6, 7, 8, or 9. Within this range, n can make the established standard curves for limonene and linalool have better linearity, and ultimately make the detection of the content of limonene and linalool in the test solution more accurate.

[0059] Furthermore, in step S30, the concentrations of the mixed standard solution diluents with different concentration gradients are 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL, respectively. Selecting these six concentrations of mixed standard solution diluents ensures a good linear regression coefficient for the standard curve, making the standard curve more reflective of the true situation and the measurement values ​​more accurate.

[0060] Further, in step S40, the capillary column material of the GC-FID chromatographic column includes (5% phenyl)-methylpolysiloxane; and / or, in step S60, the capillary column material of the GC-FID chromatographic column includes (5% phenyl)-methylpolysiloxane. Other capillary column packing materials can also be polyethylene glycol, modified polyethylene glycol columns, and methylsiloxane. Taking standards as an example, the diluted mixed standard solution of limonene and linalool has a relatively long retention time on medium polarity columns (such as DB-35), which is not conducive to improving analysis time and efficiency; polar capillary columns (such as HP-INNOWAX) are themselves greatly affected by temperature (Agilent's maximum temperature is 270°C), and continuous high temperatures lead to severe column bleed and are not durable. Compared with medium polarity columns and polar capillary columns, weakly polar chromatographic columns packed with (5% phenyl)-methyl polysiloxane (such as HP-5: 30m × 0.32mm × 0.25μm) can rapidly adsorb and desorb linalool and limonene standards, shortening the analysis time and improving the analysis efficiency. At the same time, the weakly polar chromatographic columns packed with (5% phenyl)-methyl polysiloxane have different adsorption and desorption rates for linalool and limonene, which can completely separate the chromatographic peaks of the two in a short time (6min), thus improving the separation effect.

[0061] Further, in step S40, the chromatographic conditions for the GC-FID include:

[0062] The injection port temperature is 250℃~280℃; the carrier gas is N2; the flow rate is 1.0~1.2mL / min; the injection volume is 1.0μL; and the split ratio is (30~40):1.

[0063] Temperature program: Initial temperature 80℃, hold for 0 min, increase to 150℃ at 10℃ / min, hold for 3 min, then increase to 200℃ at 10℃ / min, hold for 0 min; and / or,

[0064] In step S60, the chromatographic conditions for GC-FID include:

[0065] The injection port temperature is 250℃~280℃; the carrier gas is N2; the flow rate is 1.0~1.2mL / min; the injection volume is 1.0μL; and the split ratio is (30~40):1.

[0066] Temperature rise program: Initial temperature 80℃, hold for 0 min, rise to 150℃ at 10℃ / min, hold for 3 min, then rise to 200℃ at 10℃ / min, hold for 0 min.

[0067] The injection port temperature can be 250℃, 260℃, 270℃, or 280℃. If the injection port temperature is too low, incomplete sample atomization and insufficient injection will result in poor repeatability of the target analyte. An appropriate injection port temperature range ensures good detection stability and repeatability. The carrier gas flow rate can be 1.0 mL / min, 1.1 mL / min, or 1.2 mL / min. An appropriate carrier gas flow rate range ensures a split ratio of (30–40):1, i.e., a split ratio of 30:1, 35:1, or 40:1. A suitable split ratio range ensures the integrity of the target peak shape, resulting in high detection sensitivity. A suitable temperature program ensures good peak separation of linalool and limonene in the sample.

[0068] Further, in step S40, the FDI detector conditions of the GC-FID include:

[0069] Detector temperature: 280℃~300℃; air flow rate: 350~400mL / min; hydrogen flow rate: 30~35mL / min; tail gas flow rate: 25~30mL / min; and / or,

[0070] In step S60, the FDI detector conditions of the GC-FID include:

[0071] Detector temperature: 280℃~300℃; air flow rate: 350~400mL / min; hydrogen flow rate: 30~35mL / min; tail gas flow rate: 25~30mL / min.

[0072] The detector temperature can be 280℃, 290℃, or 300℃. A suitable detector temperature range can improve separation efficiency and make the detection results more accurate. The air flow rate can be 350mL / min, 360mL / min, 370mL / min, 380mL / min, or 400mL / min. A suitable air flow rate range can improve separation efficiency and make the detection results more accurate. The hydrogen flow rate can be 30mL / min, 32mL / min, 34mL / min, or 35mL / min. A suitable hydrogen flow rate range can improve separation efficiency and make the detection results more accurate. The tail gas flow rate can be 25mL / min, 27mL / min, 29mL / min, or 30mL / min. A suitable tail gas flow rate range can improve separation efficiency and make the detection results more accurate.

[0073] Further, in step S50: the limit of detection (LOD) of the limonene standard curve is 6 mg / kg; and / or, the limit of quantitation (LOQ) of the limonene standard curve is 20 mg / kg. A LOD of 6 mg / kg for the limonene standard curve ensures greater accuracy of the method while also providing more accurate qualitative and quantitative analysis of limonene and linalool in solutions such as eutectic solvents and sweet orange essential oil. A LOQ of 20 mg / kg for the limonene standard curve ensures greater accuracy of the method while also providing more accurate qualitative and quantitative analysis of limonene and linalool in solutions such as eutectic solvents and sweet orange essential oil.

[0074] Further, in step S50: the limit of detection (LOD) of the linalool standard curve is 5 mg / kg; and / or, the limit of quantitation (LOQ) of the linalool standard curve is 20 mg / kg. A LOD of 6 mg / kg for the limonene standard curve ensures greater accuracy of the method while also providing more accurate qualitative and quantitative analysis of limonene and linalool in solutions such as eutectic solvents and sweet orange essential oil. A LOQ of 20 mg / kg for the limonene standard curve ensures greater accuracy of the method while also providing more accurate qualitative and quantitative analysis of limonene and linalool in solutions such as eutectic solvents and sweet orange essential oil.

[0075] The method for simultaneously detecting limonene and linalool in solution can be applied to special application scenarios including but not limited to cosmetics, food, cleaning, or fragrance.

[0076] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0077] Example 1

[0078] A method for simultaneously detecting limonene and linalool in solution includes the following steps:

[0079] S10. Accurately weigh 0.1g (accurate to 0.0001g) of the sample to be tested, dilute to 10mL with isopropanol, vortex and shake well, let stand for 1min, filter through a 0.45μm needle filter to obtain the sample solution to be tested, take 1mL of the sample solution to be tested into the injection vial, and wait for GC-FID analysis.

[0080] S20. Accurately transfer 1 mL each of linalool and limonene into a 10 mL volumetric flask, and dilute to the mark with isopropanol to obtain a mixed standard solution with a mass concentration of 100 μg / mL. The mass concentrations of linalool and limonene in the mixed standard solution are 100 μg / mL and 100 μg / mL, respectively.

[0081] S30. The mixed standard solution was serially diluted with isopropanol to obtain mixed standard solution dilutions with concentrations of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL and 50 μg / mL.

[0082] S40. The GC-FID method was used to detect the diluted mixed standard solutions of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL. The retention times and chromatographic peak areas of limonene and linalool in the diluted mixed standard solutions of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL were collected.

[0083] The chromatographic conditions for the GC-FID included: an Agilent J&W HP-5 column with a (5% phenyl)-methylpolysiloxane stationary phase (30m × 0.32mm × 0.25μm); an injection port temperature of 250℃; a carrier gas of N2 (purity: 99.999%); a flow rate of 1.2mL / min; an injection volume of 1.0μL; a split ratio of 40:1; and a temperature program of initial temperature 80℃, holding for 0 min, increasing to 150℃ at 10℃ / min, holding for 3 min, then increasing to 200℃ at 10℃ / min, and holding for 0 min.

[0084] The FID detector conditions for the GC-FID are: detector temperature 300℃, air flow rate: 400mL / min, hydrogen flow rate: 35mL / min, and tail gas flow rate: 30mL / min.

[0085] S50. Standard curves for limonene and linalool were established based on the retention times and peak areas of diluted mixed standard solutions at concentrations of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL. Linear regression analysis was performed with the peak area Y of limonene in the diluted mixed standard solution as the ordinate and the corresponding mass concentration X (μg / mL) of limonene in the diluted mixed standard solution as the abscissa to obtain the regression equations and correlation coefficients for limonene and linalool. The regression equation and correlation coefficient for linalool were obtained using the same method. Simultaneously, 12 parallel determinations were performed on the lowest concentration standard solution (1.0 μg / mL), and the standard deviation (S) was calculated. The limit of detection (LOD) was set at 3 times the LOD, and the limit of quantitation (LOQ) was set at 10 times the LOD. The detection limits for linalool and limonene were 5 mg / kg and 6 mg / kg, respectively, and the LOQ for both was 20 mg / kg.

[0086] S60. The GC-FID detection method was used to detect the sample solution to be tested, and the retention time and chromatographic peak area of ​​limonene and linalool in the sample solution to be tested were collected.

[0087] The chromatographic conditions for the GC-FID included: an Agilent J&W HP-5 column with a (5% phenyl)-methylpolysiloxane stationary phase (30m × 0.32mm × 0.25μm); an injection port temperature of 250℃; a carrier gas of N2 (purity: 99.999%); a flow rate of 1.2mL / min; an injection volume of 1.0μL; a split ratio of 40:1; and a temperature program of initial temperature 80℃, holding for 0 min, increasing to 150℃ at 10℃ / min, holding for 3 min, then increasing to 200℃ at 10℃ / min, and holding for 0 min.

[0088] The FID detector conditions for the GC-FID are: detector temperature 300℃, air flow rate: 400mL / min, hydrogen flow rate: 35mL / min, and tail gas flow rate: 30mL / min.

[0089] S70. Based on the standard curves of limonene and linalool, and the retention times and peak areas of limonene and linalool in the sample solution to be tested, determine the content of limonene and linalool in the sample solution to be tested.

[0090] Comparative Example 1

[0091] Comparative Example 1: Determination of limonene and linalool content in the test solution by high performance liquid chromatography (HPLC):

[0092] High-performance liquid chromatography (HPLC) pretreatment method: Weigh 0.1 g (accurate to 0.1 mg) of sample into a 10 mL volumetric flask, dilute to the mark with a mixed solvent (acetonitrile / ultrapure water: 4:6, v / v), filter through a 0.45 μm syringe filter into a vial, and wait for HPLC analysis;

[0093] Preparation of mixed standard solution: Accurately transfer 1 mL each of linalool and limonene into a 10 mL volumetric flask, and dilute to the mark with acetonitrile:ultrapure water = 4:6 (v / v) to obtain a mixed standard solution with a mass concentration of 100 μg / mL. The mass concentrations of linalool and limonene in the mixed standard solution are 100 μg / mL and 100 μg / mL, respectively.

[0094] Preparation of mixed standard solution dilution buffer: The mixed standard solution was serially diluted with acetonitrile:ulpure water = 4:6 (v / v) to obtain mixed standard solution dilution buffers with concentrations of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL and 50 μg / mL respectively;

[0095] The HPLC method was used to detect the dilutions of mixed standard solutions at concentrations of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL. The retention times and peak areas of limonene and linalool in the mixed standard solution dilutions at these concentrations were collected.

[0096] The HPLC chromatographic conditions included: an Agilent 1100 high-performance liquid chromatograph; an Agilent ZORBAX Eclipse Plus C18 column (3.5 μm, 4.6 mm × 100 mm); mobile phase A was acetonitrile; mobile phase B was ultrapure water; the detector was a DAD; the injection volume was 5.0 μL; the wavelength was 200 nm; the flow rate was 1 mL / min; the column temperature was 30 °C; and the elution method was gradient elution, as shown in Table 1.

[0097] Table 1. HPLC gradient elution

[0098]

[0099]

[0100] Standard curves for limonene and linalool were established based on the retention times and peak areas of diluted mixed standard solutions at concentrations of 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL. Linear regression analysis was performed with the peak area Y of limonene in the diluted mixed standard solution as the ordinate and the corresponding mass concentration X (μg / mL) of limonene in the diluted mixed standard solution as the abscissa to obtain the regression equations and correlation coefficients for limonene and linalool. The regression equation and correlation coefficient for linalool were obtained using the same method.

[0101] The GC-FID detection method was used to detect the sample solution and the retention time and chromatographic peak area of ​​limonene and linalool in the sample solution were collected.

[0102] The HPLC chromatographic conditions included: an Agilent 1100 high-performance liquid chromatograph; an Agilent ZORBAX Eclipse Plus C18 column (3.5 μm, 4.6 mm × 100 mm); mobile phase A was acetonitrile; mobile phase B was ultrapure water; the detector was a DAD; the injection volume was 5.0 μL; the wavelength was 200 nm; the flow rate was 1 mL / min; the column temperature was 30 °C; and the elution method was gradient elution, as shown in Table 1.

[0103] The contents of limonene and linalool in the test sample solution were determined based on the standard curves of limonene and linalool, the retention times of limonene and linalool in the test sample solution, and the chromatographic peak areas.

[0104] Since Comparative Example 1 did not use GC-FID and external standard methods in combination, the pretreatment of Comparative Example 1 required the preparation of mixed solvents, which was time-consuming, had baseline interference, and resulted in relatively long analysis time, relatively poor detection stability, and relatively low detection accuracy.

[0105] Performance testing

[0106] The stability of the methods for detecting limonene and linalool in solutions in Example 1 and Comparative Example 1 was studied: The prepared mixed standard solutions of linalool and limonene (both with a mass concentration of 100 μg / mL) were measured at different time points (0 h, 5 h, 10 h, 12 h, 24 h) under the same GC-FID / HPLC conditions. Peak area data of linalool and limonene in the mixed standard solutions at different time points were collected. The results of Example 1 are as follows: Figure 1 As shown in Table 2, the results of Comparative Example 1 are shown in Table 2.

[0107] Table 2. Stability test results of the methods for detecting limonene and linalool in solutions in Example 1 and Comparative Example 1.

[0108]

[0109]

[0110] Depend on Figure 1 As shown in Table 2, the relative standard deviations (RSDs) of the peak area measurements of linalool and limonene at the five time points in the detection method of Example 1 were 1.1% and 1.0%, respectively, indicating that linalool and limonene in isopropanol were relatively stable within 24 hours. As shown in Table 2, the relative standard deviations (RSDs) of the peak area measurements of linalool and limonene at the five time points in the detection method of Comparative Example 1 were 2.3% and 2.9%, respectively, indicating that the detection method of Comparative Example 1 had poorer stability compared to Example 1.

[0111] The methods of Example 1 and Comparative Example 1 were used to detect limonene and linalool in sweet orange essential oil, the extract phase (hereinafter referred to as the extract phase) obtained from sweet orange essential oil treated with a eutectic solvent, and the raffinate phase (hereinafter referred to as the raffinate phase) obtained from sweet orange essential oil treated with a eutectic solvent, respectively; the linear range of the standard curve in this method was also analyzed. Example 1: The chromatogram of a mixed standard solution of linalool and limonene with a mass concentration of 50 mg / mL is shown below. Figure 2 As shown, the chromatogram of sweet orange essential oil is as follows: Figure 3 As shown, the chromatogram of the extract phase is as follows: Figure 4 As shown, the chromatogram of the raffinate phase is as follows: Figure 5 As shown in Table 3, the linear range results are shown in Table 4, and the determination results for limonene and linalool are shown in Table 5. Comparative Example 1: The linear range results are shown in Table 3.

[0112] Table 3. Linear equations for the standard curves of limonene and linalool in the detection method of Example 1.

[0113]

[0114] As shown in Table 3, in Example 1, the standard curve equation for limonene was Y = 0.4604X - 2.7532, and the standard curve equation for linalool was Y = 0.4846X - 4.3566, with a correlation coefficient of 0.9999 for both. This indicates that the standard curves prepared by this method have good linearity and high detection accuracy. Table 3 also shows that in Comparative Example 1, the standard curve equation for limonene was Y = 69.8264X + 0.2285, with a correlation coefficient of 0.99981; the standard curve equation for linalool was Y = 32.1468X + 2.9979, with a correlation coefficient of 0.99966. Compared with the correlation coefficients of the standard curves in Example 1, the correlation coefficients of the standard curves in the comparative examples are lower, indicating that the standard curves prepared in Example 1 have poor linearity, low detection accuracy, and do not closely approximate the true detection values.

[0115] Table 4 shows the results of detecting limonene and linalool in sweet orange essential oil, the extract phase, and the raffinate phase using the detection method of Example 1.

[0116]

[0117] Table 4 shows that, using the detection method of Example 1, the content ratios of linalool and limonene in sweet orange essential oil were 2.16% and 96.18%, respectively. Using the linalool and limonene content in sweet orange essential oil as control data, the contents of linalool and limonene in the extract phase after eutectic solvent extraction were 1.27% and 7.95%, respectively, and the contents of linalool and limonene in the raffinate phase were 0.75% and 87.60%, respectively. This indicates that the eutectic solvent has a good extraction effect on linalool (an oxygenated terpene alcohol compound) in sweet orange essential oil.

[0118] The recovery rates of limonene and linalool in the solutions of Example 1 and Comparative Example 1 were studied by spiked analysis. The extract phase (hereinafter referred to as the extract phase) and the raffinate phase (hereinafter referred to as the raffinate phase) of sweet orange essential oil treated with a eutectic solvent were used as the research objects. Linalool and limonene standard solutions (the mass concentration of linalool and limonene was 100 μg / mL) were added to a 10 mL volumetric flask containing 0.1 g of the analyte. The specific steps were as follows: Low concentration: 30 μL (3 μg) was added to 10 mL. In volumetric flasks, 50 μL (5 μg) of medium-sized spiked sample was added to a 10 mL volumetric flask and diluted to the mark with solvent (same as above). In volumetric flasks, 100 μL (10 μg) of high-sized spiked sample was added to a 10 mL volumetric flask and diluted to the mark with solvent (same as above). Each spiked sample was measured 5 times. The spiked recovery rate was calculated. The spiked recovery results of the extract phase are shown in Table 5, and the spiked recovery results of the raffinate phase are shown in Table 6.

[0119] Table 5. Spike recovery results of the extract phases in Example 1 and Comparative Example 1 (n=5)

[0120]

[0121]

[0122] Table 5 shows that in Example 1, the average recovery rate of linalool in the extract phase was 99.19%, with an average relative standard deviation of 5.51%, and the recovery rate of limonene was 93.69%, with an average relative standard deviation of 5.18%. This indicates that the method has good specificity, anti-interference ability, and accuracy. In Comparative Example 1, the average recovery rate of linalool in the extract phase was 77.73%, with an average relative standard deviation of 11.9%, and the recovery rate of limonene was 83.55%, with an average relative standard deviation of 10.9%. Compared with Example 1, the method in Comparative Example 1 had a lower recovery rate and a higher relative standard deviation, indicating that the detection results were inaccurate. This shows that the detection method in Comparative Example 1 had poor specificity, anti-interference ability, and accuracy.

[0123] Table 6. Spike recovery results of the raffinate phase in Example 1 and Comparative Example 1 (n=5)

[0124]

[0125]

[0126] Table 6 shows that in Example 1, the spiked recovery rate of linalool in the raffinate phase was 99.21%, with an average relative standard deviation of 5.23%, and the spiked recovery rate of limonene was 99.27%, with an average relative standard deviation of 6.92%. This indicates that the method has good specificity, anti-interference ability, and accuracy. The accuracy of the target analyte determination results was over 95% through spiked recovery rate verification and relative standard deviation calculation for limonene and linalool. In Comparative Example 1, the spiked recovery rate of linalool in the raffinate phase was 80.99%, with an average relative standard deviation of 11.58%, and the spiked recovery rate of limonene was 83.61%, with an average relative standard deviation of 8.75%. Compared with Example 1, the method in Comparative Example 1 had a lower spiked recovery rate and a higher relative standard deviation, indicating that the detection results were inaccurate. This shows that the detection method in Comparative Example 1 had poor specificity, anti-interference ability, and accuracy.

[0127] In summary, the method for simultaneous detection of limonene and linalool in solution according to the present invention is an easy-to-operate method with good separation effect, high analytical efficiency, and high detection stability and accuracy.

[0128] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. A method for simultaneously detecting limonene and linalool in solution, characterized in that, Includes the following steps: S10. Provide a sample solution to be tested; the sample solution to be tested is the raffinate phase and the extract phase obtained after treating citrus essential oils with a eutectic solvent to remove terpenes. S20. Mix limonene standard, linalool standard and solvent to obtain mixed standard solution; S30. Dilute the mixed standard solution with solvent to prepare multiple mixed standard solution dilution solutions with different concentration gradients; S40. The GC-FID detection method was used to detect the mixed standard solution dilutions with multiple concentration gradients, and the retention time and chromatographic peak area of ​​limonene and linalool in the mixed standard solution dilutions with multiple concentration gradients were collected. S50. Establish standard curves for limonene and linalool based on the retention times and chromatographic peak areas of mixed standard solutions with multiple concentration gradients. S60. The GC-FID detection method was used to detect the sample solution to be tested, and the retention time and chromatographic peak area of ​​limonene and linalool in the sample solution to be tested were collected. S70. Based on the standard curves of limonene and linalool, and the retention times and chromatographic peak areas of limonene and linalool in the sample solution to be tested, determine the content of limonene and linalool in the sample solution to be tested. In steps S20 and S30, the solvent is methanol or isopropanol; In steps S40 and S60, the chromatographic conditions for GC-FID include: The injection port temperature was 250℃~280℃; the carrier gas was N2; the flow rate was 1.0~1.2 mL / min; the injection volume was 1.0 μL; and the split ratio was (30~40):

1. Temperature program: Initial temperature 80℃, hold for 0 min, increase to 150℃ at 10℃ / min, hold for 3 min, then increase to 200℃ at 10℃ / min, hold for 0 min; The capillary column material of the GC-FID chromatographic column includes (5%-phenyl)-methylpolysiloxane; The FID detector conditions for GC-FID include: Detector temperature: 280℃~300℃; air flow rate: 350~400 mL / min; hydrogen flow rate: 30~35 mL / min; tail gas flow rate: 25~30 mL / min.

2. The method for simultaneously detecting limonene and linalool in solution as described in claim 1, characterized in that, In step S20, the mass concentration ratio of limonene to linalool in the mixed standard solution is 1:(1~2).

3. The method for simultaneous detection of limonene and linalool in solution as described in claim 1, characterized in that, In step S30, the number of mixed standard solution diluents with different concentration gradients is n, and the value of n is a positive integer greater than or equal to 5.

4. The method for simultaneously detecting limonene and linalool in solution as described in claim 1, characterized in that, In step S30, the concentrations of the mixed standard solution diluents with different concentration gradients are 1.0 μg / mL, 2.5 μg / mL, 5.0 μg / mL, 10 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

5. The method for simultaneous detection of limonene and linalool in solution as described in claim 1, characterized in that, In step S50: the detection limit of the limonene standard curve is 6 mg / kg; the quantitation limit of the limonene standard curve is 20 mg / kg.

6. The method for simultaneous detection of limonene and linalool in solution as described in claim 1, characterized in that, In step S50: the detection limit of the linalool standard curve is 5 mg / kg; The limit of quantitation for the linalool standard curve is 20 mg / kg.

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