TWI828935B - Testing methods for free lutein and free zeaxanthin - Google Patents
Testing methods for free lutein and free zeaxanthin Download PDFInfo
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Abstract
本發明係揭示一種游離葉黃素、游離玉米黃質之檢驗方法，包含製備標準樣品及待測物樣品，並利用高效液相層析儀搭配X橋式分離管柱，以水、乙腈調製出不同溶劑濃度梯度的沖提液以進行沖提，藉以檢測標準樣品中游離葉黃素、游離玉米黃質的停留時間以及波峰面積，並據以建立標準曲線，進而在量測待測物樣品時，可供對照而獲得待測物樣品中葉黃素、玉米黃質的濃度。尤其，本發明方法的靈敏度及再現性相當高，所以非常適用於家禽蛋黃、食品中游離葉黃素與玉米黃質的含量分析。The invention discloses a method for testing free lutein and free zeaxanthin, which includes preparing a standard sample and a sample of a substance to be tested, and using a high-performance liquid chromatograph with an X-bridge separation column to prepare the sample with water and acetonitrile. Elution with different solvent concentration gradients is used to detect the residence time and peak area of free lutein and free zeaxanthin in the standard sample, and establish a standard curve based on this, so as to measure the analyte sample. , which can be used for comparison to obtain the concentration of lutein and zeaxanthin in the test object sample. In particular, the method of the present invention has very high sensitivity and reproducibility, so it is very suitable for analyzing the content of free lutein and zeaxanthin in poultry egg yolks and foods.
Description
本發明係有關於一種游離葉黃素、游離玉米黃質之檢驗方法，尤其是利用前處理及萃取技術，藉高效液相層析儀，搭載X橋式管柱，而以水、乙腈作為移動相，並分別使用不同的溶劑濃度梯度進行沖提，最後分別得到游離型的葉黃素、玉米黃質的標準曲線，並藉備製樣品而製成測試液，再利用高效液相層析儀檢測樣品中葉黃素、玉米黃質的停留時間以及波峰面積，進而參考標準曲線以獲得葉黃素、玉米黃質的濃度，很適用於家禽產業蛋黃、食品及保健食品中游離葉黃素、玉米黃質的含量分析。The present invention relates to a method for testing free lutein and free zeaxanthin, especially by utilizing pre-treatment and extraction technology, using a high-performance liquid chromatograph equipped with an X-bridge column, and using water and acetonitrile as mobile phase, and used different solvent concentration gradients for elution, and finally obtained the standard curves of free lutein and zeaxanthin respectively, and prepared the samples to make test solutions, and then used high-performance liquid chromatography Detect the residence time and peak area of lutein and zeaxanthin in the sample, and then refer to the standard curve to obtain the concentration of lutein and zeaxanthin. It is very suitable for free lutein and corn in egg yolks, food and health foods in the poultry industry. Xanthin content analysis.
眾所周知，蛋黃中的葉黃質（xanthophylls）和胡蘿蔔素(carotene)是蛋黃紅色跟黃色色素的良好來源，其中葉黃素（lutein）、玉米黃質(Zeaxanthin)皆屬於葉黃質一種，不僅能夠提供蛋黃色素且具有抗氧化、增強免疫功能及預防人類視網膜病變等多種功能。As we all know, xanthophylls and carotene in egg yolks are good sources of red and yellow pigments in egg yolks. Among them, lutein and zeaxanthin are both a type of xanthophylls, which not only can It provides egg yolk pigment and has many functions such as antioxidant, enhancing immune function and preventing human retinopathy.
再者，人類對葉黃素和玉米黃質的生物可利用率會依來源而產生差異，目前已知食用富含葉黃素的雞蛋較其他補充品或菠菜的利用率較高，換言之，雞蛋是最好提供葉黃素的天然補充食品。Furthermore, the bioavailability of lutein and zeaxanthin to humans varies depending on the source. It is currently known that consumption of eggs rich in lutein is more efficient than other supplements or spinach. In other words, eggs are the most It is good to provide natural supplementary food of lutein.
由於天然類葉黃素含有羥基(hydroxyl group)，常與脂肪酸鍵結形成酯鍵因而，將葉黃素及玉米黃素型態分成二種：游離型(free form)及酯化型(ester form)。而酯化的葉黃質或胡蘿蔔素在被吸收前必須經過皂化後轉化為，才得以被吸收。然而，市售雞蛋所含之葉黃素型態標示不明確，在這一情況下，會造成消費者花錢購買宣告富含高含量之葉黃素，卻未能有效吸收而感到困惑，因而限制高品質雞蛋的市場機制，若能建立有效且快速地將游離型的葉黃素、玉米黃質定性與定量，並標示明確是非常必須的。Since natural lutein contains a hydroxyl group, it often bonds with fatty acids to form ester bonds. Therefore, the forms of lutein and zeaxanthin are divided into two types: free form and ester form. ). The esterified lutein or carotene must be saponified and converted into lutein before it can be absorbed. However, the type of lutein contained in commercially available eggs is not clearly labeled. In this case, consumers will spend money to buy products that are declared to be rich in high levels of lutein, but they will not be able to effectively absorb them and become confused. Therefore, In order to restrict the market mechanism of high-quality eggs, it is very necessary to establish an effective and rapid identification and quantification of free lutein and zeaxanthin, and to clearly label them.
在習知技術中，中國畜牧學會在中國畜牧學會會誌 45(1)：1~12, 2016的標題為” 蛋雞飼料添加金盞花萃取物對蛋黃顏色和葉黃素濃度之影響”之研究報告中指出，利用高效液相層析儀(high performance liquid chromatography，HPLC)以搭配管柱(Phenomenex C18 250*4.6 mm)，並在柱溫30℃下以檢測葉黃素、玉米黃質。In the known technology, the Chinese Animal Husbandry Society published a research report titled "The Effect of Adding Calendula Extract to Laying Hen Feed on Egg Yolk Color and Lutein Concentration" in Journal of the Chinese Animal Husbandry Society 45(1): 1~12, 2016 It is pointed out that high performance liquid chromatography (HPLC) is used with a column (Phenomenex C18 250*4.6 mm), and the column temperature is 30°C to detect lutein and zeaxanthin.
此外，在食品藥物研究年報中，5 : 17-25 2014 Ann. Rept. Food Drug Res. 5 : 17-25 2014，標題為：利用高效液相層析法檢測膠囊錠狀食品中葉黃素及玉米黃素之含量，是使用HPLC搭配分離管柱Ascentis RP-Amide 150*4.6 mm，而檢測葉黃素、玉米黃質的RT，分別為16.5及18.1。In addition, in the Annual Report of Food and Drug Research, 5: 17-25 2014 Ann. Rept. Food Drug Res. 5: 17-25 2014, the title is: Detection of lutein and corn in capsule and tablet foods using high-performance liquid chromatography The content of flavin was measured using HPLC with a separation column Ascentis RP-Amide 150*4.6 mm. The RTs for detecting lutein and zeaxanthin were 16.5 and 18.1 respectively.
再者，大陸國標的GB5009.248以及GB5009.83藉示出檢測葉黃素、玉米黃質的方式，且使用分離管柱C30 150*4.6 mm，並利用由甲醇(MeOH)、水(H2 O)、乙腈(Acetonitrile, ACN)、甲基第三丁基醚(methyl tert-butyl ether，MTBE)所調製的沖提液，葉黃素、玉米黃質的量測結果為RT分別是13.3、16.1，不過未能揭示出線性回歸分析的線性度R2 值，而且甲基第三丁基醚(MTBE)具有一定的毒性，易於與水融合，可滲入土壤，破壞地下水質，是一種污染物。Furthermore, the mainland national standards GB5009.248 and GB5009.83 also show the method of detecting lutein and zeaxanthin, and use a separation column C30 150*4.6 mm, and use methanol (MeOH), water (H 2 O), acetonitrile (ACN), and methyl tert-butyl ether (MTBE). The measurement results of lutein and zeaxanthin were RT 13.3 and MTBE respectively. 16.1, however, the linearity R 2 value of the linear regression analysis cannot be revealed, and methyl tert-butyl ether (MTBE) has a certain toxicity, easily merges with water, can penetrate into the soil, and destroys groundwater quality. It is a pollutant .
此外，MTBE主要可經由呼吸道而被人體吸收，或經皮膚和消化道吸收，動物在高濃度的MTBE中可能會致癌，而對小鼠的麻醉濃度為1.0mmol/L，致死濃度為1.6mmol/L，對人體的影響主要表現在上呼吸道、眼睛黏膜的刺激反應，長期接觸可使皮膚乾燥。In addition, MTBE can be mainly absorbed by the human body through the respiratory tract, or through the skin and digestive tract. Animals may be carcinogenic in high concentrations of MTBE. The anesthetic concentration for mice is 1.0mmol/L, and the lethal concentration is 1.6mmol/L. L, the impact on the human body is mainly manifested in the irritation of the upper respiratory tract and eye mucosa. Long-term exposure can make the skin dry.
因此，非常需要一種新穎設計的檢驗方法，利用前處理及萃取技術，藉高效液相層析儀，搭載X橋式管柱，而以水、乙腈作為移動相而調製出具不同溶劑濃度梯度的沖提液以供進行沖提，因而分別得到游離型的葉黃素、玉米黃質的標準曲線，再進行備製樣品而製成量測用的測試液，且利用高效液相層析儀檢測其中葉黃素、玉米黃質的停留時間以及波峰面積，進而參考標準曲線以獲得葉黃素、玉米黃質的濃度，藉以克服習知技術的問題。Therefore, there is a great need for a novel testing method that uses pre-treatment and extraction technology, high-performance liquid chromatography, equipped with an X-bridge column, and uses water and acetonitrile as mobile phases to prepare flushing solutions with different solvent concentration gradients. The liquid is extracted for flushing, so as to obtain the standard curves of free lutein and zeaxanthin respectively, and then prepare samples to make test solutions for measurement, and use high-performance liquid chromatography to detect them. The residence time and peak area of lutein and zeaxanthin are then determined by referring to the standard curve to obtain the concentrations of lutein and zeaxanthin, thereby overcoming the problems of conventional techniques.
本發明之主要目的在於提供一種游離葉黃素、游離玉米黃質之檢驗方法，是利用包含步驟S10、S20、S30、S32、S34、S40、S50、S60以及S70，用以實現前處理及萃取技術而獲得靈敏度及再現性相當高的量測數據。The main purpose of the present invention is to provide a method for testing free lutein and free zeaxanthin, which uses steps S10, S20, S30, S32, S34, S40, S50, S60 and S70 to achieve pre-treatment and extraction. technology to obtain measurement data with high sensitivity and reproducibility.
具體而言，步驟S10是利用乙醇溶解葉黃素而備製具不同葉黃素濃度的多個葉黃素標準測試品，且利用乙醇溶解玉米黃質而備製具不同玉米黃質濃度的多個玉米黃質標準測試品，該不同葉黃素濃度以及該不同玉米黃質濃度是在一預設濃度範圍內。Specifically, step S10 is to use ethanol to dissolve lutein to prepare multiple lutein standard test samples with different lutein concentrations, and to use ethanol to dissolve zeaxanthin to prepare multiple samples with different zeaxanthin concentrations. A zeaxanthin standard test product, the different lutein concentrations and the different zeaxanthin concentrations are within a preset concentration range.
在步驟S20中，利用水以及乙腈以備製具不同的溶劑濃度梯度的沖提液。In step S20, water and acetonitrile are used to prepare eluents with different solvent concentration gradients.
在步驟S30中，利用沖提液，搭載高效液相層析儀以及分離管柱，用以量測每個葉黃素標準測試品的停留時間以及波峰面積，再利用該等波峰面積對應該等不同葉黃素濃度以進行回歸分析而計算並繪製葉黃素標準曲線。 In step S30, the elution liquid is used, equipped with a high-performance liquid chromatograph and a separation column, to measure the residence time and peak area of each lutein standard test product, and then the peak areas are used to correspond to the corresponding levels. Different lutein concentrations were calculated using regression analysis and a lutein standard curve was drawn.
在步驟S32，利用沖提液，搭載高效液相層析儀以及分離管柱，用以量測每個玉米黃質標準測試品的停留時間以及波峰面積，再利用該等波峰面積對應該等不同玉米黃質濃度以進行回歸分析而計算並繪製玉米黃質標準曲線。 In step S32, the elution liquid is used to carry a high-performance liquid chromatograph and a separation column to measure the residence time and peak area of each zeaxanthin standard test product, and then use the peak areas to correspond to the different levels. Zeaxanthin concentration was calculated using regression analysis and a zeaxanthin standard curve was drawn.
然後進入步驟S34，備製樣品，且樣品是由鳥禽類蛋中取出蛋黃部分而形成，或是包含食品或保健食品，之後執行步驟S40，搭配丙酮、乙醇、正己烷進行超音波萃取，而後移入分液漏斗進行分離、純化、濃縮，而由樣品備製回溶過濾液，並安置於褐色玻璃瓶瓶中，並在步驟S50，保存包含回溶過濾液的褐色玻璃瓶在低於0℃的低溫保存溫度。 Then enter step S34 to prepare a sample, and the sample is formed by removing the yolk part from a bird egg, or contains food or health food, and then performs step S40 to perform ultrasonic extraction with acetone, ethanol, and n-hexane, and then moves in The separatory funnel is used for separation, purification, and concentration, and the re-dissolved filtrate is prepared from the sample and placed in a brown glass bottle. In step S50, the brown glass bottle containing the re-dissolved filtrate is stored below 0°C. Low temperature storage.
在步驟S60中，由褐色玻璃瓶，取出部分的回溶過濾液，並添加甲醇而稀釋至預設濃度範圍內，當作葉黃素、玉米黃質測試樣品液。在步驟S70中，利用沖提液，搭載高效液相層析儀以及分離管柱，用以量測測試樣品液的葉黃素波峰面積以及玉米黃質波峰面積，並藉葉黃素標準曲線以及玉米黃質標準曲線，而獲得測試樣品液的葉黃素濃度以及玉米黃質濃度。 In step S60, part of the redissolved filtrate is taken out from the brown glass bottle, and methanol is added to dilute it to a preset concentration range to serve as a lutein and zeaxanthin test sample solution. In step S70, the elution liquid is used, equipped with a high-performance liquid chromatograph and a separation column, to measure the lutein peak area and zeaxanthin peak area of the test sample liquid, and the lutein standard curve and Zeaxanthin standard curve, and obtain the lutein concentration and zeaxanthin concentration of the test sample liquid.
要注意的是，上述步驟S30、S32以及S34的執行次序可為任意逐一排列。 It should be noted that the execution order of the above-mentioned steps S30, S32 and S34 can be any one by one.
因此，本發明利用利用前處理及萃取技術，利用高效液相層析-光電二極體陣列(HPLC-PDA)，搭載管柱(分離管柱X橋式C18 250*4.6mm)，並並以水、乙腈作為移動相，配置成具不同溶劑濃度梯度的沖提液以供進行沖提，進而得到游離型的葉黃素、玉米黃質的標準曲線，當作定量的檢測數據，而且針對待測樣品進行沖提而量測其中葉黃素、玉米黃質的停留時間及波峰面積，並利用標準曲線而對應出葉黃素、玉米黃質的濃度，而且，由於靈敏度與再現性較高，所以非常適用於家禽產業蛋黃中游離葉黃素、玉米黃質的含量分析，以及食品及保健食品中游離葉黃素、玉米黃質的含量分析。Therefore, the present invention utilizes pre-treatment and extraction technology, high-performance liquid chromatography-photodiode array (HPLC-PDA), equipped with a column (separation column Water and acetonitrile are used as mobile phases, and eluents with different solvent concentration gradients are configured for elution, and then the standard curves of free lutein and zeaxanthin are obtained, which can be used as quantitative detection data, and for the target The test sample is washed to measure the residence time and peak area of lutein and zeaxanthin in it, and the standard curve is used to correspond to the concentration of lutein and zeaxanthin. Moreover, due to high sensitivity and reproducibility, Therefore, it is very suitable for the analysis of the content of free lutein and zeaxanthin in egg yolks in the poultry industry, as well as the analysis of the content of free lutein and zeaxanthin in food and health foods.
以下配合圖示及元件符號對本發明之實施方式做更詳細的說明，俾使熟習該項技藝者在研讀本說明書後能據以實施。The following is a more detailed description of the embodiments of the present invention with reference to diagrams and component symbols, so that those skilled in the art can implement them after reading this specification.
請參閱第一圖，本發明實施例游離葉黃素、游離玉米黃質之檢驗方法的操作流程示意圖。如第一圖所示，本發明實施例的游離葉黃素、游離玉米黃質之檢驗方法是包含步驟S10、S20、S30、S32、S34、S40、S50、S60以及S70，用以實現前處理及萃取技術而獲得靈敏度及再現性相當高的量測數據。Please refer to the first figure, which is a schematic diagram of the operation flow of the testing method for free lutein and free zeaxanthin according to the embodiment of the present invention. As shown in the first figure, the testing method for free lutein and free zeaxanthin according to the embodiment of the present invention includes steps S10, S20, S30, S32, S34, S40, S50, S60 and S70 to achieve pre-processing and extraction technology to obtain measurement data with high sensitivity and reproducibility.
首先，本發明實施例的檢驗方法是由步驟S10開始，主要是備製具不同葉黃素濃度的多個葉黃素標準測試品、具不同玉米黃質濃度的多個玉米黃質標準測試品。進一步而言，是利用乙醇( Ethanol，EtOH )分別溶解葉黃素、玉米黃質，尤其，不同葉黃素濃度、不同玉米黃質濃度是在預設濃度範圍內，且葉黃素預設濃度範圍是較佳的為5ppm至100ppm，玉米黃質預設濃度範圍是較佳的為2ppm至50ppm，不過可依據實際需要而縮小範圍或放大範圍。First, the testing method of the embodiment of the present invention starts from step S10, which mainly involves preparing multiple lutein standard test products with different lutein concentrations and multiple zeaxanthin standard test products with different zeaxanthin concentrations. . Furthermore, ethanol (EtOH) is used to dissolve lutein and zeaxanthin respectively. In particular, different lutein concentrations and different zeaxanthin concentrations are within the preset concentration range, and the preset lutein concentration The preferred range is 5 ppm to 100 ppm, and the preferred preset concentration range of zeaxanthin is 2 ppm to 50 ppm, but the range can be narrowed or enlarged according to actual needs.
在步驟S20中，是利用水以及乙腈以備製具不同的溶劑濃度梯度的沖提液。舉例而言，沖提液中水、乙腈的相對體積比例為20:80至0:100，因而達成二種不同的溶劑濃度梯度。In step S20, water and acetonitrile are used to prepare eluents with different solvent concentration gradients. For example, the relative volume ratio of water and acetonitrile in the eluent is 20:80 to 0:100, thus achieving two different solvent concentration gradients.
接著要注意的是，本發明中步驟S30、步驟S32的執行次序為任意逐一排列，不過為方便說明，下文中是依序執行步驟S30、步驟S32。Next, it should be noted that the execution order of steps S30 and S32 in the present invention is arbitrary. However, for convenience of explanation, steps S30 and S32 are executed sequentially in the following.
在步驟S30中，利用沖提液，並搭載高效液相層析儀(high performance liquid chromatography，HPLC)以及分離管柱，用以量測每個葉黃素標準測試品的停留時間(Retention Time，RT)以及波峰面積，如第二圖的HPLC色譜圖所示，再利用該等波峰面積對應該等不同葉黃素濃度，藉以進行回歸分析而計算並繪製供後續比對用的葉黃素標準曲線，如第五圖所示。In step S30, the elution liquid is used and equipped with a high performance liquid chromatography (HPLC) and a separation column to measure the retention time (Retention Time) of each lutein standard test product. RT) and the peak area, as shown in the HPLC chromatogram in the second picture, and then use the peak areas to correspond to the different lutein concentrations to perform regression analysis to calculate and draw the lutein standard for subsequent comparison. Curve, as shown in Figure 5.
在步驟S32中，同樣是利用沖提液，並搭載高效液相層析儀以及分離管柱，用以量測每個玉米黃質標準測試品的停留時間以及波峰面積，如第三圖的HPLC色譜圖所示，再利用該等波峰面積對應該等不同玉米黃質濃度，藉以進行回歸分析而計算並繪製供後續比對用的玉米黃質標準曲線，如第六圖所示。In step S32, the eluent is also used and equipped with a high-performance liquid chromatograph and a separation column to measure the residence time and peak area of each zeaxanthin standard test product, as shown in the HPLC in the third figure As shown in the chromatogram, the peak areas are then used to correspond to the different zeaxanthin concentrations to perform regression analysis to calculate and draw a zeaxanthin standard curve for subsequent comparison, as shown in the sixth figure.
再者，第四圖的HPLC色譜圖係顯示本發明方法可同時量測相互混合的游離的葉黃素及玉米黃質。Furthermore, the HPLC chromatogram in Figure 4 shows that the method of the present invention can simultaneously measure free lutein and zeaxanthin mixed with each other.
上述的高效液相層析儀可包含高效液相層析-光電二極體陣列(high performance liquid chromatography-Photo Diode Array Detector，HPLC-PDA)，而分離管柱可包含X橋式分離管柱(XBridge)，且較佳的，X橋式分離管柱的尺寸大小可為250*4.6mm。由於高效液相層析儀在習知技術中是很常用的量測置，所以下文中並不對相關的操作細節做詳細的描述。 The above-mentioned high performance liquid chromatography may include a high performance liquid chromatography-Photo Diode Array Detector (HPLC-PDA), and the separation column may include an X-bridge separation column ( XBridge), and preferably, the size of the X-bridge separation column can be 250*4.6mm. Since high performance liquid chromatography is a very commonly used measuring device in the prior art, the relevant operating details will not be described in detail below.
然後進入步驟S34，備製待測的樣品，其中樣品可由一般的鳥禽類蛋中取出蛋黃部分而形成，或者，樣品可包含食品或保健食品。 Then step S34 is entered to prepare a sample to be tested, where the sample can be formed by taking out the yolk part from ordinary bird eggs, or the sample can include food or health food.
在步驟S40中，搭配丙酮、乙醇、正己烷進行超音波萃取，而後移入分液漏斗進行分離、純化、濃縮，而由步驟S34所備製的樣品中，進一步備製回溶過濾液，並安置於褐色玻璃瓶瓶中而保存，防止被外界光線照射而破壞其中的成分。 In step S40, acetone, ethanol, and n-hexane are used for ultrasonic extraction, and then moved into a separatory funnel for separation, purification, and concentration. From the sample prepared in step S34, a filtrate is further prepared and placed. Store in brown glass bottles to prevent damage to the ingredients by external light.
更加具體而言，步驟S40可包含以下步驟。 More specifically, step S40 may include the following steps.
首先，藉添加丙酮至樣品，並利用超音波萃取機而由樣品中萃取出萃取液；接著，移入分液漏斗，利用水與正己烷進行樣品分離、純化處理，進而取得上清液；將澄清液置入樣品瓶中，並進行減壓濃縮處理以去除正己烷；之後，添加甲醇回溶樣品；最後，過濾回溶樣品而獲得回溶過濾液，並安置於褐色玻璃瓶瓶中。 First, acetone is added to the sample, and an ultrasonic extraction machine is used to extract the extract from the sample; then, it is moved into a separatory funnel, and water and n-hexane are used to separate and purify the sample to obtain the supernatant; the clarified The solution was placed into a sample bottle and concentrated under reduced pressure to remove n-hexane; then, methanol was added to redissolve the sample; finally, the redissolved sample was filtered to obtain the redissolved filtrate, which was placed in a brown glass bottle.
此外，上述的減壓濃縮處理可包含利用抽真空機，抽出樣品瓶中的氣體，進而降低壓力，移除正己烷。 In addition, the above-mentioned reduced pressure concentration process may include using a vacuum machine to extract the gas in the sample bottle, thereby reducing the pressure and removing n-hexane.
在步驟S40之後，進入步驟S50，主要是將包含回溶過濾液的褐色玻璃瓶保存在低於0℃的低溫保存溫度，比如低溫保存溫度是在-10℃至-25℃之間。較佳的，可利用冰箱以保存回溶過濾液在低溫保存溫度。After step S40, step S50 is entered, which mainly involves storing the brown glass bottle containing the redissolved filtrate at a low-temperature storage temperature lower than 0°C, for example, the low-temperature storage temperature is between -10°C and -25°C. Preferably, a refrigerator can be used to store the re-dissolved filtrate at a low temperature.
在步驟S60中，由褐色玻璃瓶，取出部分的回溶過濾液，並添加適量的對應溶劑，如:葉黃素、玉米黃質應以甲醇稀釋至預設濃度範圍內，當作測試樣品液，較佳的，葉黃素預設濃度範圍為5ppm至100ppm，玉米黃質預設濃度範圍為2ppm至50ppm。In step S60, take out part of the redissolved filtrate from the brown glass bottle, and add an appropriate amount of the corresponding solvent, such as: lutein and zeaxanthin should be diluted with methanol to a preset concentration range as the test sample solution , preferably, the preset concentration range of lutein is 5ppm to 100ppm, and the preset concentration range of zeaxanthin is 2ppm to 50ppm.
然後進入步驟S70，利用沖提液，並搭載高效液相層析儀以及分離管柱，用以量測測試樣品液的葉黃素波峰面積以及玉米黃質波峰面積，並分別藉第五圖的葉黃素標準曲線以及第六圖的玉米黃質標準曲線，而獲得測試樣品液的葉黃素濃度以及玉米黃質濃度。Then enter step S70, use the elution liquid, and install a high-performance liquid chromatograph and a separation column to measure the lutein peak area and zeaxanthin peak area of the test sample liquid, and use the values of the fifth figure respectively. The lutein standard curve and the zeaxanthin standard curve in the sixth figure are used to obtain the lutein concentration and zeaxanthin concentration of the test sample liquid.
進一步舉例而言，在本發明的方法中，沖提液在步驟S30、S32以及S70中的流速可為1ml/min，且高效液相層析儀在步驟S70的檢測波長為450nm，此外，分離管柱的柱溫為35℃。不過要注意的是，上述的操作參數都只是示範性實例而已，並非用以限定發明的範圍，亦即，操作參數可依據實際樣品中葉黃素、玉米黃質的含量高低而適度調整，藉以達到最佳操作效率。For further example, in the method of the present invention, the flow rate of the eluate in steps S30, S32 and S70 can be 1 ml/min, and the detection wavelength of the high-performance liquid chromatograph in step S70 is 450 nm. In addition, the separation The column temperature of the column is 35°C. However, it should be noted that the above operating parameters are only exemplary examples and are not used to limit the scope of the invention. That is, the operating parameters can be appropriately adjusted according to the content of lutein and zeaxanthin in the actual sample, so as to achieve Optimum operating efficiency.
以蛋黃為例，實際檢測出的游離型玉米黃質、葉黃素之RT分別為: 14.5、15.5分鐘，其中回歸分析(主要是透過線性回歸分析)後所得的回歸係數R2 值高達0.9995，非接近於1，優於一般習知技術，而且其靈敏度與再現性較高，所以非常適用於家禽產業蛋黃中游離葉黃素、玉米黃質的含量分析，以及食品及保健食品中游離葉黃素、玉米黃質的含量分析。Taking egg yolk as an example, the actual detected RTs of free zeaxanthin and lutein are: 14.5 and 15.5 minutes respectively. The regression coefficient R2 value obtained after regression analysis (mainly through linear regression analysis) is as high as 0.9995, which is not close. In 1, it is better than the general conventional technology, and its sensitivity and reproducibility are high, so it is very suitable for the analysis of free lutein and zeaxanthin in egg yolks in the poultry industry, as well as the analysis of free lutein and zeaxanthin in food and health foods. Analysis of zeaxanthin content.
再進一步舉例而言，在備製樣品時，可取出蛋黃1 g精秤至0.01克而置入樣品瓶中，再加入10ml濃度百分之五十之丙酮，然後進行超音波萃取，歷時5 分鐘，從而獲得萃取液；接著加入30ml含百分之十濃度之氫氧化鉀的乙醇，混合30秒，並利用50℃進行水浴加熱30分鐘，待冷卻後移入分液漏斗，並利用些許水與40ml正己烷進行分離、純化處理取得澄清液，再放入25ml的褐色樣品瓶中以進行減壓濃縮，濃縮到完全無正己烷為止；再添加10ml甲醇以回溶；回溶後，以0.45μm的濾網過濾到1.5ml的vail瓶中；最後，放入-20℃冰箱進行保存。 For a further example, when preparing a sample, 1 g of egg yolk can be taken out and weighed to 0.01 g accurately and placed in a sample bottle, then 10 ml of acetone with a concentration of 50% can be added, and then ultrasonic extraction can be performed for 5 minutes. , to obtain the extract; then add 30 ml of ethanol containing 10% potassium hydroxide, mix for 30 seconds, and heat in a water bath at 50°C for 30 minutes. After cooling, transfer to a separatory funnel, and use a little water and 40 ml Separate and purify the n-hexane to obtain the clear liquid, then put it into a 25 ml brown sample bottle for concentration under reduced pressure until there is no n-hexane at all; then add 10 ml of methanol to dissolve it back; after dissolving it, dissolve it with 0.45 μm Filter into a 1.5ml vail bottle; finally, store in a -20°C refrigerator.
接著，進行HPLC分析，主要是利用分離管柱(X橋式C18 250*4.6mm)，並以溶劑乙腈、水配製成具二種不同濃度梯度的沖提液，且ACN(乙腈)：H2O=80：20、100：0，其中柱溫為35℃，總流速為1ml/min，檢測波長為450nm。結果，葉黃素(Lutein)的RT為15.5分鐘，玉米黃質(Zeaxanthin)的RT為14.5分鐘。 Then, perform HPLC analysis, mainly using a separation column (X-bridge C18 250*4.6mm), and using the solvent acetonitrile and water to prepare two eluents with different concentration gradients, and ACN (acetonitrile): H 2 O=80:20, 100:0, where the column temperature is 35°C, the total flow rate is 1ml/min, and the detection wavelength is 450nm. As a result, the RT of Lutein was 15.5 minutes and the RT of Zeaxanthin was 14.5 minutes.
換言之，玉米黃質、葉黃素的RT分別為14.5分鐘、15.5分鐘，如第二圖、第三圖、第四圖的HPLC色譜圖所示。 In other words, the RTs of zeaxanthin and lutein are 14.5 minutes and 15.5 minutes respectively, as shown in the HPLC chromatograms of the second, third and fourth figures.
再者，備製葉黃素標準品時，是取出1mg葉黃素，溶於10ml乙醇中，而配置為濃度100ppm，且備製玉米黃質標準品時，是取出1mg玉米黃質，溶於10ml乙醇中，而配置為濃度100ppm。 Furthermore, when preparing the lutein standard, 1 mg of lutein is taken out and dissolved in 10 ml of ethanol, and the concentration is 100 ppm. When preparing the zeaxanthin standard, 1 mg of zeaxanthin is taken out and dissolved in 10 ml of ethanol. In 10ml of ethanol, the concentration is 100ppm.
接著進行標曲製備，取葉黃素標準品的母液，配置濃度為5、10、20、50ppm，並精確量取0.5、1、2、5mL分別定溶至10ml；取玉米黃質標準品的母液，配置濃度為2、5、10、20、50ppm，並精確量取0.2、0.5、1、2、5mL分別定溶至10ml。 Then prepare the standard song, take the mother solution of the lutein standard, and configure the concentrations to be 5, 10, 20, and 50 ppm, and accurately measure 0.5, 1, 2, and 5 mL respectively and dissolve to 10 ml; take the zeaxanthin standard The mother liquor is prepared at concentrations of 2, 5, 10, 20, and 50ppm, and accurately measure 0.2, 0.5, 1, 2, and 5mL and dissolve to 10ml respectively.
量測結果如第五圖、第六圖所示，分別顯示游離葉黃素、玉米黃質的線性回歸分析圖，其中游離葉黃素、玉米黃質的R2為0.9952、0.9965。 The measurement results are shown in the fifth and sixth figures, which show the linear regression analysis plots of free lutein and zeaxanthin respectively, in which the R 2 of free lutein and zeaxanthin are 0.9952 and 0.9965.
綜合而言，本發明的特點主要在於利用高效液相層析-光電二極體陣列(HPLC-PDA)，搭載管柱(分離管柱X橋式C18 250*4.6mm)，並以水、乙腈作為移動相，配置成具不同溶劑濃度梯度的沖提液以供進行沖提，進而得到游離型的葉黃素、玉米黃質的標準曲線，當作定量的檢測數據，而且針對待測樣品進行沖提而量測其中葉黃素、玉米黃質的停留時間及波峰面積，並利用標準曲線而對應出葉黃素、玉米黃質的濃度。In summary, the main feature of this invention is the use of high-performance liquid chromatography-photodiode array (HPLC-PDA), equipped with a column (separation column As a mobile phase, eluents with different solvent concentration gradients are configured for elution, and then a standard curve of free lutein and zeaxanthin is obtained, which is used as quantitative detection data and is carried out on the sample to be tested. After dilution, the residence time and peak area of lutein and zeaxanthin were measured, and the concentration of lutein and zeaxanthin was determined using the standard curve.
由於本發明的檢驗方法很容易實施，且操作成本低廉，能快速檢測一般市售雞蛋所包含之游離型的葉黃素、玉米黃質的定性與定量數據，當作客觀的簡測數據以供一般民眾檢視，藉以建立對產品的正確認知，避免不肖業利用廣告宣傳迷惑不了解相關知識的廣大群眾而牟求不當得利益，同時可保障民眾的健康。Since the test method of the present invention is easy to implement and has low operating cost, it can quickly detect the qualitative and quantitative data of free lutein and zeaxanthin contained in general commercial eggs, and serve as objective simple test data for Inspection by the general public can help establish a correct understanding of the product, prevent unscrupulous industries from using advertising to confuse the masses who do not know the relevant knowledge and seek undue benefits, and at the same time protect the health of the public.
以上所述者僅為用以解釋本發明之較佳實施例，並非企圖據以對本發明做任何形式上之限制，是以，凡有在相同之發明精神下所作有關本發明之任何修飾或變更，皆仍應包括在本發明意圖保護之範疇。The above are only used to explain the preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Therefore, any modifications or changes related to the present invention are made under the same spirit of the invention. , should still be included in the scope of protection intended by the present invention.
S10、S20、S30、S32、S34、S40:步驟 S50、S60、S70:步驟S10, S20, S30, S32, S34, S40: steps S50, S60, S70: steps
第一圖顯示本發明實施例游離葉黃素、游離玉米黃質之檢驗方法的操作流程示意圖。 第二圖顯示游離的葉黃素的HPLC色譜圖。 第三圖顯示游離的玉米黃質的HPLC色譜圖。 第四圖顯示游離的葉黃素及玉米黃質的HPLC色譜圖。 第五圖顯示游離的葉黃素標準曲線圖。 第六圖顯示游離的玉米黃質標準曲線圖。The first figure shows a schematic diagram of the operation flow of the testing method for free lutein and free zeaxanthin according to the embodiment of the present invention. The second figure shows the HPLC chromatogram of free lutein. The third panel shows the HPLC chromatogram of free zeaxanthin. The fourth figure shows the HPLC chromatogram of free lutein and zeaxanthin. The fifth graph shows the free lutein standard curve. Figure 6 shows the free zeaxanthin standard curve plot.
S10、S20、S30、S32、S34、S40:步驟S10, S20, S30, S32, S34, S40: steps
S50、S60、S70:步驟S50, S60, S70: steps
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