Method for simultaneously detecting various carotenoids in egg yolk

Abstract

The invention belongs to the technical field of food detection, and discloses a method for simultaneously detecting various carotenoids in egg yolk. According to the method, the C30 reversed-phase chromatographic column is creatively adopted, and an optimized binary mobile phase gradient elution procedure is combined, so that the problem of co-elution of structural analogues (especially lutein and zeaxanthin) is successfully solved. The method provided by the invention realizes simultaneous baseline separation of five main carotenoids, namely lutein, zeaxanthin, cantharidin yellow, beta-cryptoxanthin and beta-carotene, peak shapes are sharp and symmetrical, the separation degree is obviously superior to that of a traditional C18 chromatographic column method, and a solid foundation is laid for accurate quantification.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, specifically to a method for the simultaneous detection of multiple carotenoids in egg yolks. Background Technology

[0002] Carotenoids are an important class of natural pigments widely found in nature. They not only give egg yolks their distinctive golden color but also play a crucial role in antioxidation, immune regulation, and visual health. Egg yolks are rich in various carotenoids, mainly including lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene. Their composition and content are among the core indicators for evaluating the nutritional and sensory quality of eggs.

[0003] Currently, the main methods for detecting carotenoids include spectrophotometry, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). Among them, HPLC has become the mainstream analytical technique in this field due to its high sensitivity and good applicability. However, existing technologies have significant shortcomings: First, in the complex matrix of egg yolk, most existing HPLC methods only detect one or two pigments such as lutein and zeaxanthin, resulting in a single analytical dimension and failing to provide comprehensive carotenoid spectral information. Second, many methods (such as AOAC-based ultraviolet spectrophotometry) can only determine the total carotenoid content and estimate it using β-carotene equivalents, failing to achieve accurate quantification and differentiation of specific components and thus failing to reflect the true composition. Furthermore, structurally similar carotenoids (such as lutein and zeaxanthin) have poor separation on conventional C18 columns, making effective separation difficult and placing higher demands on chromatographic conditions (especially the selection of mobile phase and column).

[0004] Furthermore, existing sample pretreatment and detection methods primarily target raw or fully cooked egg yolks, neglecting the common state of soft-boiled egg yolks, where the degree of heat processing falls between the two. Soft-boiled egg yolks possess a unique semi-solid physical structure, and their lipid binding state and carotenoid extraction efficiency may differ significantly from those of raw and cooked egg yolks. Currently, there is a lack of a systematic detection method that can simultaneously cover raw, soft-boiled, and cooked egg yolks, and can efficiently separate and accurately quantify various carotenoids.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to solve at least one of the above technical problems and provides a method for simultaneous detection of multiple carotenoids in egg yolks.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Methods for simultaneous detection of multiple carotenoids in egg yolks include: Preparation of test solutions for egg yolk samples; The test solution was analyzed using high performance liquid chromatography. The high-performance liquid chromatography (HPLC) analysis used a C30 reversed-phase column and a gradient elution program. Mobile phase A was a mixture of methanol / tert-butyl methyl ether / water at a volume ratio of (80~86):(14.5~15.5):(1.9~2.1), and mobile phase B was a mixture of tert-butyl methyl ether / methanol / water at a volume ratio of (87~93):(6.8~7.2):(2.9~3.1). The gradient elution program is as follows: at 0 min, mobile phase A is 99% and mobile phase B is 1%; at 50 min, mobile phase A is 50% and mobile phase B is 50%; at 52 min, mobile phase A is 99% and mobile phase B is 1%.

[0008] Preferably, the multiple carotenoids include lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene.

[0009] Preferably, the C30 reversed-phase chromatographic column has a size of 250 mm × 4.6 mm and a diameter of 5 μm. The flow rate for high-performance liquid chromatography analysis is 1.0–1.2 mL / min, the injection volume is 10–30 μL, the detection wavelength is 450 nm, and the column temperature is 20–30 ℃.

[0010] Preferably, the test solution for preparing the egg yolk sample includes: extracting carotenoids from the egg yolk sample, saponifying, washing and dehydrating, and concentrating and adjusting the volume, wherein the egg yolk sample is selected from raw egg yolk, soft-boiled egg yolk, or cooked egg yolk.

[0011] Preferably, the carotenoid extraction is performed using a mixed solvent consisting of methanol, ethyl acetate, and petroleum ether in a volume ratio of 1:1:1, and the mixed solvent contains 0.08-0.012% by weight / volume of the antioxidant di-tert-butyl-p-cresol.

[0012] Preferably, the extraction includes ultrasound-assisted extraction, with ultrasound conditions of 40 kHz frequency, 20~35 ℃ temperature, and 10~20 min extraction time.

[0013] Preferably, the saponification is performed using a 30% methanol-KOH solution at room temperature for 2-4 hours under light-protected conditions.

[0014] Preferably, the washing and dehydration include washing with brine using a 5% NaCl aqueous solution and dehydrating with anhydrous sodium sulfate.

[0015] Preferably, the concentration and volume adjustment are performed using a solution containing 0.8–1.2% di-tert-butyl-p-cresol, which is a mixture of methanol, tert-butyl methyl ether, and water in a volume ratio of (0.8–1.2):(0.8–1.2):(0.8–1.2).

[0016] Preferably, the method uses a standard calibration curve to perform qualitative and quantitative analysis of lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention, through the innovative use of a C30 reversed-phase column combined with an optimized binary mobile phase gradient elution program, successfully solves the co-elution problem of structurally similar compounds (especially lutein and zeaxanthin). It achieves simultaneous baseline separation of five major carotenoids—lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene—with sharp and symmetrical peaks, demonstrating significantly superior resolution compared to traditional C18 column methods, thus laying a solid foundation for accurate quantification.

[0018] This invention systematically incorporates three different processing states of egg yolks—raw egg yolks, soft-boiled egg yolks, and hard-boiled egg yolks—into the same detection system for the first time. The optimized pretreatment process effectively adapts to the differences in physical properties of egg yolks from liquid to semi-solid to fully solid states, ensuring the efficiency and stability of carotenoid extraction under different conditions. This fills the gap in existing methods for detecting carotenoids in soft-boiled eggs and enables comparable analysis across sample states.

[0019] This invention minimizes the oxidative degradation and loss of target analytes during analysis through comprehensive measures, including operating in the dark throughout the process, adding the antioxidant di-tert-butyl-p-cresol (BHT) in key steps, and using saponification to remove lipid interference. Combined with optimized chromatographic conditions, the established standard curves for each component exhibit good linearity (R² ≥ 0.9952), demonstrating high sensitivity and excellent repeatability over a wide concentration range, resulting in accurate and reliable quantitative results. Attached Figure Description

[0020] Figure 1 The HPLC chromatograms are of five standard carotenoids, where 1: lutein; 2: zeaxanthin; 3: canthaxanthin; 4: β-cryptoxanthin; and 5: β-carotene. Figure 2 The HPLC chromatogram of carotenoids in the raw egg yolk in Example 1 is shown below. Figure 3 The HPLC chromatogram of carotenoids in the soft-boiled egg yolk in Example 1; Figure 4 The HPLC chromatogram of carotenoids in the yolk of a cooked egg in Example 1; Figure 5 The HPLC chromatogram of carotenoids in raw eggs obtained from the elution degree of Comparative Example 1 is shown. Figure 6 The HPLC chromatogram of carotenoids in raw eggs obtained from the elution degree of Comparative Example 2 is shown. Figure 7 The HPLC chromatogram of carotenoids in raw eggs from Example 2 is shown below. Figure 8 This is the HPLC chromatogram of carotenoids in raw eggs from Example 3. Detailed Implementation

[0021] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0022] This invention provides a method for the simultaneous detection of multiple carotenoids in egg yolks, including: Preparation of test solutions for egg yolk samples; The test solution was analyzed using high performance liquid chromatography. The high performance liquid chromatography analysis used a C30 reversed-phase column and a gradient elution program. Mobile phase A was a mixture of methanol / tert-butyl methyl ether / water at a volume ratio of 83:15:2, and mobile phase B was a mixture of tert-butyl methyl ether / methanol / water at a volume ratio of 90:7:3. The gradient elution program is as follows: at 0 min, mobile phase A is 99% and mobile phase B is 1%; at 50 min, mobile phase A is 50% and mobile phase B is 50%; at 52 min, mobile phase A is 99% and mobile phase B is 1%.

[0023] Unlike the commonly used C18 chromatographic column, this invention creatively employs a C30 reversed-phase chromatographic column. C30 exhibits stronger hydrophobic interactions and shape selectivity: the C30 bonded phase possesses longer alkyl chains, providing stronger hydrophobic forces compared to C18, resulting in better retention of highly hydrophobic carotenoids (such as β-carotene). More importantly, the longer carbon chains form a higher-density and more rigid "solid-phase alkyl layer," enabling better identification and differentiation of subtle stereostructural differences between carotenoid molecules (such as functional group positions, cis-trans isomerism, etc.). This plays a decisive role in separating structurally similar isomers, such as lutein and zeaxanthin (which differ only in the position of a single hydroxyl group in their molecules). While these two are readily co-eluted on a C18 column, baseline separation can be achieved on a C30 column thanks to its superior shape selectivity. In addition, carotenoids are prone to producing cis isomers during extraction and processing. The C30 column is far superior to the C18 column in separating these isomers, thus providing purer chromatographic peaks and more accurate principal component quantification results.

[0024] The gradient elution procedure provided in this invention is a key kinetic process for achieving efficient separation of five target analytes. Initial high polarity conditions (99% A): Mobile phase A (methanol / tert-butyl methyl ether / water = 83:15:2) has high polarity, which initially allows for the gentle retention of relatively polar lutein and zeaxanthin at the column head, initiating separation, while simultaneously ensuring rapid elution of strongly polar matrix interferences. Linear reduction of polarity (50% of phase A): Within 50 minutes, the polarity of the entire mobile phase system is systematically reduced by linearly increasing the proportion of the moderately polar mobile phase B (tert-butyl methyl ether / methanol / water = 90:7:3). This slow and linear gradient change provides optimal conditions for the sequential elution of carotenoids of different polarities. The elution sequence generally follows a polarity from highest to lowest: lutein / zeaxanthin → canthaxanthin → β-cryptoxanthin → β-carotene. The introduction of tert-butyl methyl ether is crucial; as a moderately polar solvent, it effectively adjusts the elution intensity, ensuring sufficient separation while reasonably shortening the elution time of the most hydrophobic component. Column equilibration recovery (rapid return to initial conditions): Gradient elution is terminated at 50 min, and the gradient is rapidly restored to initial conditions within 50–52 min and maintained for a period to ensure the column is fully reequilibrated before the next injection, guaranteeing excellent reproducibility and stability of the analytical method.

[0025] In some preferred embodiments, the C30 reversed-phase chromatographic column has a size of 250 mm × 4.6 mm and a diameter of 5 μm. The flow rate for high-performance liquid chromatography analysis is 1.0–1.2 mL / min, the injection volume is 10–30 μL, the detection wavelength is 450 nm, and the column temperature is 20–30 °C.

[0026] To make the method of this invention applicable to the detection of egg yolks in three different physical states—raw egg yolk, soft-boiled egg yolk, and hard-boiled egg yolk—a ternary extraction solvent (methanol, ethyl acetate, and petroleum ether mixed in a volume ratio of (0.8–1.2):(0.8–1.2):(0.8–1.2)) was used to extract carotenoids from egg yolks during sample preparation. This ternary extraction solvent possesses both polar and nonpolar properties; methanol can disrupt the lipoprotein structure of egg yolks, while ethyl acetate and petroleum ether can effectively dissolve and release carotenoids bound to nonpolar lipids. This composite solvent system exhibits good penetration and dissolution capabilities for egg yolks in different coagulation states, overcoming extraction resistance caused by protein denaturation and lipid solidification (such as in fully cooked egg yolks). To prevent oxidative degradation of carotenoids during extraction, 0.08%–0.012% (w / v) of di-tert-butyl-p-cresol (BHT) was added to the mixed solvent as an antioxidant. In addition, ultrasonic-assisted extraction is used during the extraction process. The intense vibration and local high temperature and pressure generated by ultrasonic cavitation can effectively break the egg yolk particles, increase the contact area between the solvent and the target, and significantly improve the extraction efficiency from semi-solid (soft-boiled egg) and solid (cooked egg) matrices, ensuring the completeness and consistency of carotenoid extraction.

[0027] There are no special limitations on the parameters for ultrasound-assisted extraction; those skilled in the art can choose them adaptably based on the extraction effect. For example, the ultrasound frequency can be 40 kHz, the temperature 25–35°C, and the extraction time 10–20 min.

[0028] Because carotenoids are highly sensitive to light, oxygen, and heat, any degradation will lead to significantly lower quantitative results. Therefore, this method involves operating in the dark throughout the process and using brown glassware to cut off the photo-induced degradation pathway. BHT is added to the extraction and volume-adjusting solvents as a chain-terminating antioxidant, effectively capturing and eliminating peroxide free radicals generated during extraction and concentration, preventing the chain oxidation reaction of carotenoids. Simultaneously, the saponification step (using KOH-methanol solution) effectively removes neutral lipids such as triglycerides from the sample through ester bond hydrolysis. This "liberates" the carotenoids bound to lipids, improving the extraction rate; furthermore, it removes lipid interferences that may co-elute on the chromatogram, reducing background noise and improving detection sensitivity and accuracy.

[0029] This invention utilizes standard calibration curves to quantitatively analyze lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene. Details are as follows: (1) Qualitative analysis: The structure of carotenoids was identified based on the retention time of each peak in the high performance liquid chromatogram of carotenoid standards.

[0030] (2) Quantitative analysis: Record the peak area corresponding to each concentration of various carotenoid standards, plot the standard curve of various standards with concentration as the abscissa and peak area as the ordinate, and obtain the corresponding linear regression equation; then substitute the peak area of ​​the identified carotenoid into the regression equation of the corresponding standard to calculate the content of the corresponding substance in the test solution.

[0031] The following provides several specific examples illustrating the methods and effects of simultaneous detection of multiple carotenoids in egg yolks.

[0032] Example 1: Method for simultaneous detection of multiple carotenoids in egg yolks at different cooking levels Sample selection and preparation: Fresh eggs were taken and divided into three categories according to the degree of cooking: raw egg yolk, soft-boiled egg yolk (heating time was controlled to make the yolk semi-solid), and hard-boiled egg yolk (heated until the yolk was completely solidified); 3 eggs of each type of yolk were taken, thoroughly mixed and homogenized, and 1.000 g of sample was weighed and stored in a brown glass bottle for later use. Avoid light exposure to prevent degradation of carotenoids. The entire process was carried out under light-protected conditions. Carotenoid extraction: The extraction solvent is a mixture of methanol, ethyl acetate, and petroleum ether in a volume ratio of 1:1:1, with 0.01% (w / v) of BHT antioxidant added and mixed thoroughly. First extraction: Add 15 mL of extraction solvent to the sample, sonicate in a water bath (40 kHz, 25 ℃) for 15 min, centrifuge at 5000 rpm for 6 min, and collect the supernatant into a clean centrifuge tube A. Repeat extraction 1-2 times: Add 10 mL of extraction solvent to the residue, repeat the extraction and combine the extracts into tube A, until the color of the extract becomes significantly lighter or almost colorless. Saponification: Add 10 mL of 30% methanol-KOH to the combined organic phases and react at room temperature in the dark for 2 h; dilute with an equal volume of 5% NaCl aqueous solution and transfer to a separatory funnel; Salt washing and dehydration: Add 5 mL of 5% NaCl, shake for 1 min, let stand for 30 min and discard the lower aqueous phase; add an appropriate amount of anhydrous sodium sulfate to dehydrate for 10 min, and filter to remove sodium sulfate; Concentration and volume adjustment: Rotary evaporate to near dryness, leaving a small amount of oily residue; transfer to a 5 mL brown volumetric flask, redissolve and adjust to volume with 4 mL of methanol / tert-butyl methyl ether / water (83:15:2, v / v / v) containing 1% BHT; HPLC detection: Sample was filtered (0.45 μm) and then injected. Column: YMC C30 reversed-phase column (250 × 4.6 mm, 5 μm); Mobile phase: A (methanol / tert-butyl methyl ether / water = 83:15:2), B (tert-butyl methyl ether / methanol / water = 90:7:3); Gradient: 0 min 99%A / 1%B → 50 min 50%A / 50%B → 52 min 99%A / 1%B; Flow rate 1.0 mL / min, injection volume 30 μL, detection wavelength 450 nm, column temperature 25 ℃.

[0033] Example 2: Method for simultaneous detection of multiple carotenoids in raw egg yolks Sample selection and preparation: Take the yolks of 3 fresh eggs, mix them thoroughly and homogenize them, then weigh 1.000 g of the sample into a brown glass bottle for later use. Avoid light exposure to prevent degradation of carotenoids. The entire process should be carried out under light-protected conditions.

[0034] Carotenoid extraction: The extraction solvent is a mixture of methanol, ethyl acetate, and petroleum ether in a volume ratio of 1:1.2:0.8, with 0.008% (w / v) of BHT antioxidant added and mixed thoroughly. First extraction: Add 15 mL of extraction solvent to the sample, sonicate in a water bath (40 kHz, 20 ℃) ​​for 10 min, centrifuge at 5000 rpm for 5 min, and collect the supernatant into a clean centrifuge tube A. Repeat extraction 1-2 times: Add 10 mL of extraction solvent to the residue, repeat the extraction and combine the extracts into tube A until the color of the extract becomes significantly lighter or almost colorless.

[0035] Saponification: Add 10 mL of 30% methanol-KOH to the combined organic phases and react at room temperature in the dark for 3 h; dilute with an equal volume of 5% NaCl aqueous solution and transfer to a separatory funnel.

[0036] Salt washing and dehydration: Add 5 mL of 5% NaCl, shake for 1 min, let stand for 30 min and discard the lower aqueous phase; add an appropriate amount of anhydrous sodium sulfate to dehydrate for 10 min, and filter to remove sodium sulfate.

[0037] Concentration and volume adjustment: Rotary evaporate to near dryness, leaving a small amount of oily residue; transfer to a 5 mL brown volumetric flask, redissolve and volume up with 4 mL of methanol / tert-butyl methyl ether / water (83:15:2, v / v / v) containing 0.8% BHT.

[0038] HPLC detection: Sample was filtered (0.45 μm) and then injected. Column: YMC C30 reversed-phase column (250 × 4.6 mm, 5 μm); Mobile phase: A (methanol / tert-butyl methyl ether / water = 83:15:2), B (tert-butyl methyl ether / methanol / water = 90:7:3); Gradient: 0 min 99%A / 1%B → 50 min 50%A / 50%B → 52 min 99%A / 1%B; Flow rate 1.0 mL / min, injection volume 10 μL, detection wavelength 450 nm, column temperature 20 ℃.

[0039] Example 3: Method for simultaneous detection of multiple carotenoids in raw egg yolks Sample selection and preparation: Take the yolks of 3 fresh eggs, mix them thoroughly and homogenize them, then weigh 1.000 g of the sample into a brown glass bottle for later use. Avoid light exposure to prevent degradation of carotenoids. The entire process should be carried out under light-protected conditions.

[0040] Carotenoid extraction: The extraction solvent is a mixture of methanol, ethyl acetate, and petroleum ether in a volume ratio of 1:0.8:1.2, with 0.012% (w / v) of BHT antioxidant added and mixed thoroughly. First extraction: Add 15 mL of extraction solvent to the sample, sonicate in a water bath (40 kHz, 30 ℃) for 20 min, centrifuge at 5000 rpm for 8 min, and collect the supernatant into a clean centrifuge tube A. Repeat extraction 1-2 times: Add 10 mL of extraction solvent to the residue, repeat the extraction and combine the extracts into tube A until the color of the extract becomes significantly lighter or almost colorless.

[0041] Saponification: Add 10 mL of 30% methanol-KOH to the combined organic phases and react at room temperature in the dark for 4 h; dilute with an equal volume of 5% NaCl aqueous solution and transfer to a separatory funnel.

[0042] Salt washing and dehydration: Add 5 mL of 5% NaCl, shake for 1 min, let stand for 30 min and discard the lower aqueous phase; add an appropriate amount of anhydrous sodium sulfate to dehydrate for 10 min, and filter to remove sodium sulfate.

[0043] Concentration and volume adjustment: Rotary evaporate to near dryness, leaving a small amount of oily residue; transfer to a 5 mL brown volumetric flask, redissolve and adjust to volume with 4 mL of methanol / tert-butyl methyl ether / water (83:15:2, v / v / v) containing 1% BHT.

[0044] HPLC detection: Sample was filtered (0.45 μm) and then injected. Column: YMC C30 reversed-phase column (250 × 4.6 mm, 5 μm); Mobile phase: A (methanol / tert-butyl methyl ether / water = 83:15:2), B (tert-butyl methyl ether / methanol / water = 90:7:3); Gradient: 0 min 99%A / 1%B → 50 min 50%A / 50%B → 52 min 99%A / 1%B; Flow rate 1.2 mL / min, injection volume 20 μL, detection wavelength 450 nm, column temperature 30 ℃.

[0045] Comparative Example 1 Fresh egg samples were taken, and after separating the yolks, carotenoid extraction and detection were performed directly according to the method in Example 1 without heat treatment. Compared with Example 1, the elution program of this comparative example was as follows: 0 min, 99% mobile phase A and 1% mobile phase B; 0–30 min, 80% mobile phase A and 20% mobile phase B; 30–40 min, 65% mobile phase A and 35% mobile phase B; 40–50 min, 55% mobile phase A and 45% mobile phase B; 51–52 min, 99% mobile phase A and 1% mobile phase B. The remaining detection steps were the same as in Example 1.

[0046] Comparative Example 2 Fresh egg samples were taken, and after separating the yolks, carotenoid extraction and detection were performed directly according to the method in Example 1 without heat treatment. Compared with Example 1, the elution program of this comparative example was as follows: 0 min, 99% mobile phase A and 1% mobile phase B; 0–20 min, 40% mobile phase A and 60% mobile phase B; 20–35 min, 50% mobile phase A and 50% mobile phase B; 35–50 min, 45% mobile phase A and 55% mobile phase B; 51–52 min, 99% mobile phase A and 1% mobile phase B. The remaining detection steps were the same as in Example 1.

[0047] Based on the above examples and comparative examples, standard curves were established using reference standards. Peak areas were then used for quantitative analysis of lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene. The standard curves are as follows: The standard curve for lutein is: y = 71.161x - 26.004, R0 2 =0.998, showing a good linear relationship in the range of 6-30 μg / mL; The standard curve for maize xanthine is: y = 76.967x + 53.11, R0 2 =0.9965, showing a good linear relationship in the range of 3-20 μg / mL; The standard curve for cantharidin is: y = 50.36x + 12.059, R0 2=0.9952, showing a good linear relationship in the range of 1-8 μg / mL; The standard curve for β-cryptoxanthin is: y = 260.27 - 34.313, R0 2 =0.9991, showing a good linear relationship in the range of 0.2-5 μg / mL; The standard curve for β-carotene is: y = 19.819x - 2.3197, R0 2 =0.9998, showing a good linear relationship in the range of 0.2-4 μg / mL.

[0048] HPLC chromatograms of five standard carotenoids, and HPLC chromatograms of various samples in Example 1 are shown below. Figure 1-4 As shown, the quantitative detection results are presented in Table 1. In Table 1, "UV method" refers to the results obtained from the UV spectrophotometric AOAC method for detecting total carotenoid content, estimated using β-carotene equivalents, as described in "Fletcher D L. An evaluation of the AOAC method of yolk color analysis[J]. Poultry Science, 1980, 59(5):1059-1066." The results show that the target carotenoids of this invention eluted completely within 0–40 minutes in chromatographic analysis, and the spectrum only truncated within this range to display all peaks.

[0049] according to Figure 1-4 By comparing the peak shape and retention time of egg yolks from raw eggs, soft-boiled eggs, and hard-boiled eggs, it was found that the peak shapes of all types of egg yolks were complete and good, and the retention time was consistent. The peak area of ​​carotenoids in soft-boiled eggs and hard-boiled eggs was slightly lower than that in raw eggs, but the relative proportions were consistent. The method of this invention can stably and accurately determine the carotenoids in egg yolks under different cooking conditions.

[0050] Table 1 .

[0051] The contents of lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin and β-carotene in the raw eggs measured in Example 2 were 24.901 μg / g, 12.357 μg / g, 0.760 μg / g, 0.405 μg / g and 0.700 μg / g, respectively.

[0052] The contents of lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin and β-carotene in the raw eggs measured in Example 3 were 18.767 μg / g, 8.655 μg / g, 0.672 μg / g, 0.378 μg / g and 0.688 μg / g, respectively.

[0053] Figure 5-6Chromatograms of raw eggs were obtained using different elution procedures for Comparative Examples 1 and 2, combined with... Figure 2 It can be seen that under the low gradient conditions of Comparative Example 1, the mobile phase polarity changes slowly, resulting in weak elution capacity. The main carotenoids in the sample only gradually eluted after 9–15 min, with close peak spacing, insufficient separation, and some pigments exhibiting co-elution. In Comparative Example 2, the gradient rises too quickly, causing weakly polar pigment components to elute prematurely, resulting in significant peak overlap. Some major peaks could not be effectively separated, baseline fluctuations increased, and tailing and delayed elution of impurity peaks occurred between 15–30 min, leading to poor separation selectivity. In contrast, the embodiments of this invention, by optimizing the B-phase rise rate, achieved a stable mobile phase polarity change, resulting in good separation of early-eluting peaks and components with medium to high retention times. The resulting chromatograms showed sharp peaks, clear inter-peak separation, and stable baselines, with overall separation performance significantly superior to Comparative Example 1 and Comparative Example 2.

[0054] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A method for simultaneous detection of multiple carotenoids in egg yolk, characterized in that, include: Preparation of test solutions for egg yolk samples; The test solution was analyzed using high performance liquid chromatography. The high-performance liquid chromatography (HPLC) analysis used a C30 reversed-phase column and a gradient elution program. Mobile phase A was a mixture of methanol / tert-butyl methyl ether / water at a volume ratio of (80~86):(14.5~15.5):(1.9~2.1), and mobile phase B was a mixture of tert-butyl methyl ether / methanol / water at a volume ratio of (87~93):(6.8~7.2):(2.9~3.1). The gradient elution program is as follows: at 0 min, mobile phase A is 99% and mobile phase B is 1%; at 50 min, mobile phase A is 50% and mobile phase B is 50%; at 52 min, mobile phase A is 99% and mobile phase B is 1%.

2. The method as described in claim 1, characterized in that, The various carotenoids include lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene.

3. The method as described in claim 1, characterized in that, The C30 reversed-phase column has dimensions of 250 mm × 4.6 mm and a diameter of 5 μm. The flow rate for high-performance liquid chromatography (HPLC) analysis is 1.0 ~ 1.2 mL / min, the injection volume is 10 ~ 30 μL, the detection wavelength is 450 nm, and the column temperature is 20 ~ 30 °C.

4. The method as described in claim 1, characterized in that, The preparation of the test solution for the egg yolk sample includes: extracting carotenoids from the egg yolk sample, saponifying, washing and dehydrating, and concentrating and adjusting the volume. The egg yolk sample is selected from raw egg yolk, soft-boiled egg yolk, or cooked egg yolk.

5. The method as described in claim 4, characterized in that, The carotenoids were extracted using a mixed solvent consisting of methanol, ethyl acetate, and petroleum ether in a volume ratio of (0.8–1.2):(0.8–1.2):(0.8–1.2), and the mixed solvent contained 0.008% to 0.012% by weight / volume of the antioxidant di-tert-butyl-p-cresol.

6. The method as described in claim 5, characterized in that, The extraction includes ultrasound-assisted extraction, with ultrasound conditions of 40 kHz frequency, 25~35 ℃ temperature, and 10~20 min extraction time.

7. The method as described in claim 4, characterized in that, The saponification was performed using a 30% methanol-KOH solution at room temperature for 2-4 hours under light-protected conditions.

8. The method as described in claim 4, characterized in that, The washing and dehydration process includes washing with brine using a 5% NaCl aqueous solution and dehydrating with anhydrous sodium sulfate.

9. The method as described in claim 4, characterized in that, The concentration and volume adjustment are performed using a solution containing 0.8% to 1.2% di-tert-butyl-p-cresol, which is a mixture of methanol, tert-butyl methyl ether, and water in a volume ratio of 83:15:

2.

10. The method according to any one of claims 1-9, characterized in that, The method describes the qualitative and quantitative analysis of lutein, zeaxanthin, canthaxanthin, β-cryptoxanthin, and β-carotene using a standard calibration curve.

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