Identification and quantification of astaxanthin esters in shrimp (Pandalus borealis) and in a microalga (Haematococcus pluvialis) by liquid chromatography mass spectrometry using negative ion atmospheric pressure chemical ionization

Negative ion liquid chromatography-atmospheric pressure chemical ionization mass spectrometry [negative ion LC-(APCI)MS] was used for the identification of astaxanthin esters in extracts of commercial shrimp (Pandalus borealis) and dried microalga (Haematococcus pluvialis) samples. A cleanup step using a normal phase solid phase extraction (SPE) cartridge was applied prior to analysis. Recovery experiments with astaxanthin oleate as model compound proved the applicability of this step (98.5 +/- 7.6%; n = 4). The assignment of astaxanthin esters in negative ion LC-(APCI)MS was based on the detection of the molecular ion (M.-) and the formation of characteristic fragment ions, resulting from the loss of one or two fatty acids. Quantification of individual astaxanthin esters was performed using an astaxanthin calibration curve, which was found to be linear over the required range (1-51 mumol/L; r(2) = 0.9996). Detection limits, based on the intensity of M.-, a signal-to-noise ratio of 3: 1, and an injection volume of 20 muL, were estimated to be 0.05 mug/mL (free astaxanthin), 0.28 mug/mL (astaxanthin-C16:0), and 0.78 mug/mL (astaxanthin-C16:0/C16:0), respectively. This LC(APCI)MS method allows for the first time the characterization of native astaxanthin esters in P. borealis and H. pluvialis without using time-consuming isolation steps with subsequent gas chromatographic analyses of fatty acid methyl esters. The results suggest that the pattern of astaxanthin-bound polyunsaturated fatty acids of P. borealis does not reflect the respective fatty acid pattern found in triacylglycerides. Application of the presented LC-(APCI)MS technique in common astaxanthin ester analysis will forestall erroneous xanthophyll ester assignment in natural sources.