UPLC-UV-MSE analysis for quantification and identification of major carotenoid and chlorophyll species in algae

Abstract

A fast method for quantification and identification of carotenoid and chlorophyll species utilizing liquid chromatography coupled with UV detection and mass spectrometry has been demonstrated and validated for the analysis of algae samples. This method allows quantification of targeted pigments and identification of unexpected compounds, providing isomers separation, UV detection, accurate mass measurements, and study of fragment ions for structural elucidation in a single run. This is possible using parallel alternating low- and high-energy collision spectral acquisition modes, which provide accurate mass full scan chromatograms and accurate mass high-energy chromatograms. Here, it is shown how this approach can be used to confirm carotenoid and chlorophyll species by identification of key diagnostic fragmentations during high-energy mode. The developed method was successfully applied for the analysis of Dunaliella salina samples during defined red LED lighting growth conditions, identifying 37 pigments including 19 carotenoid species and 18 chlorophyll species, and providing quantification of 7 targeted compounds. Limit of detections for targeted pigments ranged from 0.01 ng/mL for lutein to 0.24 ng/mL for chlorophyll a. Inter-run precision ranged for of 3 to 24 (RSD%) while inter-run inaccuracy ranged from -17 to 11.

Introductory Chapter: Chlorophyll Molecules and Their Technological Relevance

“…These microorganisms demonstrate considerable biocatalytic potential in biotechnological processes due to their robustness and require nutritional simplicity [18]. Chlorophyll-a is present in all organisms that perform photosynthesis and chlorophyll-b oxygen (Figure 1), which are the most abundant pigment in green algae, is found in higher plants in nature, and other similar structures are used as accessory pigments in photosynthetic process, called chlorophyll-c, chlorophyll-d, chlorophyll-e, and chlorophyll-f [6][7][8]13]. These speciic pigments present in the cells of the photosynthetic organisms inluence eiciency of dispersion of light, because each species has its own distinct characteristic pigment [9].…”

“…Basically, chlorophyll molecules are conjugated tetrapyrroles to which a cyclopentanone ring jointly with the third ring linked together by bridges methylene, with the central atom, magnesium (Figure 1). This structure also contains at the C-17, a propionic acid chain esteriied with the phytol, diterpene alcohol [3,[6][7][8].…”

Rapid and highly sensitive analysis of chlorophylls and carotenoids from marine phytoplankton using ultra-high performance liquid chromatography (UHPLC) with the first derivative spectrum chromatogram (FDSC) technique

“…However, as far as we know, no methodological paper has been published on the UHPLC analysis for chlorophylls and carotenoids from phytoplankton assemblages in the ocean. Recently, Fu et al (2012) andPacini et al (2015) introduced powerful techniques for analyzing chlorophylls and carotenoids in phytoplankton cultures using UHPLC combined with UV-VIS detection and mass spectrometry. The run-time of their UHPLC systems was 20 min, but the resolutions of polar pigments such as the Chl c group and peridinin (Peri) were not reported in Fu et al (2012) and Pacini et al (2015).…”

“…Recently, Fu et al (2012) andPacini et al (2015) introduced powerful techniques for analyzing chlorophylls and carotenoids in phytoplankton cultures using UHPLC combined with UV-VIS detection and mass spectrometry. The run-time of their UHPLC systems was 20 min, but the resolutions of polar pigments such as the Chl c group and peridinin (Peri) were not reported in Fu et al (2012) and Pacini et al (2015). Although UHPLC has the potential to achieve faster and higher resolution analyses, the maximum sample injection volume of UHPLC is generally lower than that of HPLC to minimize the extra-column band spreading.…”

Induction of carotenoid and phytosterol accumulation in microalgae

“…The extracts (20 µL) were injected onto a YMC C30 Carotenoid Column, 3 µm, 4.6 x 250 mm (Waters, Milford, MA, USA). Using the HPLC conditions described above, an MS scan was undertaken between 530 and 610 mass units in the APCI+ mode (Fu et al, 2012), using the following system and conditions. An Acquity UPLC H-Class system connected to a Quattro The carotenoids were identified by comparison of retention times, UV/Vis spectra and mass spectra against authentic standards (Lu et al, 2009), and the concentrations of the identified carotenoids were determined using individual calibration curves.…”