Determination of Carotenoids in Human Serum and Breast Milk Using High Performance Liquid Chromatography Coupled with a Diode Array Detector (HPLC-DAD)

Abstract

High performance liquid chromatography (HPLC) coupled with a diode array detector (HPLC-DAD) for the identification and quantification of carotenoids, namely all-trans lutein, zeaxanthin, β-cryptoxanthin, α-carotene, and β-carotene, in biological samples such as human serum and breast milk, has been developed and validated. Good chromatography separation was achieved using a binary mobile phase system on a YMC C30 column (150 × 2.1 mm, 3 µm) at 30 °C. Owing to the smaller column particle size and diameter of the column, the separation was achieved in 18 min, which is significantly reduced from the typical 30–40 min of other methods. The diode array detector (DAD) acquisition was set at a wavelength of 445 nm; 3D spectra ranging from wavelengths of 240–600 nm were also recorded. Peaks were identified by matching their retention time and spectra with those of standards. Quantification was achieved by internal standard calibration using echinenone as the internal standard. Good linearity was obtained for each compound (R2 > 0.9999). The method quantification limits (MQLs) for serum and breast milk were 10 ng/mL and 5 ng/mL, in matrix, respectively. A spike recovery study and standard reference material (SRM) from the National Institute of Standards and Technology (NIST) 968e analysis has proven that the method has a high degree of accuracy, precision, and robustness. The stability study showed that the carotenoid standard and sample extracts could be stored in a chilled autosampler at 8 °C up to 48 h without being comprised, which provides guidance on re-test time frames. The freeze/thaw process was found to be detrimental to carotenoids, and should always be avoided. Most importantly, UV standardization of the stock standard is to be performed prior to each assay, and simply taking the values on Certificate of Analysis (CoA) for calculation of the standard concentration is not recommended.