- 期刊
利用HPLC/UV建立β-胡蘿蔔素於保健食品中分析方法
Development of a High Performance Liquid Chromatography with Ultraviolet Detection for Quantification of β-carotene in Health Food
摘要
本研究利用簡易快速兩段式固-液萃取操作，及高效液相層析紫外光檢測器(high performance liquid chromatography/ultraviolet detector, HPLC/UV)，開發保健食品固態劑型中β-胡蘿蔔素之分析方法。傳統分析方法利用液－液萃取，可能存在回收率較差、前處理時間較久等缺點。本研究步驟為取0.1 g檢體，加入含1.0% 2,6-二丁基羥基甲苯(BHT)之乙醇溶液及超純水，進行分散及萃取，接著以四氫呋喃(THF)進行第二段萃取。上清液以氮氣吹至近乾，再以0.025% BHT之乙醇:甲基第三丁基醚(MTBE)(1:1，V/V)回溶，並以YMC Carotenoid C30 (5 m, 4.6×250 mm)管柱搭配甲醇、乙腈及MTBE做梯度流洗，最後以紫外光偵測器分析定量。結果顯示，標準曲線範圍0.5~20 g/mL，其相關係數R^2 = 0.9998。於空白樣品中分別添加0.2、0.4及0.8%濃度之β-胡蘿蔔素標準品，平均回收率各別為104.86%、97.41%與95.37%，變異係數各別為1.77%、1.53%與2.11%。方法確效結果之準確度及精確度均佳，符合AOAC方法規範。檢測市售保健產品固態劑型中β-胡蘿蔔素含量，已知添加量為4.16 mg/g，利用此方法結果為4.09 mg/g，符合包裝的標示含量3.13 mg/g，證實此方法可應用於保健食品固態劑型中β-胡蘿蔔素含量檢測。
並列摘要
Preparation for β-carotene quantification is traditionally achieved by liquid-liquid extraction, which is time-consuming and suffers from poor yields. In this study, an easy, fast, and two-stage extraction method coupled with high performance liquid chromatography/ultraviolet detector (HPLC/UV) was developed and validated for determination of β-carotene in solid dosage forms of health food. To achieve this, the homogenized samples (0.1 g each) were extracted with 1% butylated hydroxytoluene (BHT) in ethanol and water, followed by a second extraction with tetrahydrofuran (THF). The supernatants were collected and evaporated under a stream of nitrogen, and the residue was reconstituted with 0.025% BHT in ethanol: methyl tertiarybutyl ether (MTBE) (1:1, V/V). The samples were analyzed using HPLC/UV on YMC carotenoid C30 (5 m, 4.6×250 mm) and eluted with mobile phase composed of methanol, acetonitrile, and MTBE. For validation of the method, a linear range of calibration curve (R^2 = 0.9998) was generated with 0.5-20 g/mL of β-carotene. Standards for evaluation of the method were spiked with 0.2, 0.4 and 0.8% of β-carotene. The average extraction yields from these standards were 104.86%, 97.41% and 95.37%, and the coefficients of variation were 1.77%, 1.53% and 2.11%, respectively. The sensitivity and accuracy of the method were found to conform to the requirements of AOAC (Association of Official Analytical Chemists). The method was then applied to a commercial product purchased from the drugstore. Results showed that the amount (4.09 mg/g) of β-carotene determined by this method was very close to the declared content (3.13 mg/g) on package label, indicating that this method can be used to quantify β-carotene in solid dosage form of health food.