Development and Validation of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Quantitation of Microcystins in Blue-Green Algal Dietary Supplements

Abstract

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous detection and quantitation of seven microcystin congeners (1-7) and nodularin-R (8) in blue-green algal dietary supplements. Single-laboratory method validation data were collected in four supplement matrices (capsule, liquid, powder, and tablet) fortified at toxin concentrations from 0.25-2.00 μg/g (ppm). Average recoveries and relative standard deviations (RSD) using matrix-corrected solvent calibration curves were 101% (6% RSD) for all congeners and supplements investigated. Limits of detection (0.006-0.028 μg/g) and quantitation (0.018-0.084 μg/g) were sufficient to confirm the presence of microcystin contamination at the Oregon-mandated guidance concentration of 1.0 μg of microcystin-LReq/g. Quantitated concentrations of microcystin contamination in market-available Aphanizomenon flos-aquae blue-green algal supplements ranged from 0.18-1.87 μg of microcystin-LReq/g for detected congeners microcystin-LR, microcystin-LA, and microcystin-LY (3-5). Microcystin-RR, -YR, -LW, and -LF and nodularin-R (1, 2, and 6-8) were not detected in the supplements examined.

Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit

“…To assess the accuracy of MC measurements in BGA-containing dietary supplements, MC-LA, LF, LR, LW, LY, RR, YR, and nodularin were spiked individually into a representative powdered BGA supplement containing 100% Spirulina (species not identified by manufacturer). AFA-based BGA products were not used as a blank matrix because preliminary analysis of representative products in powdered, capsule, and tablet form did not identify any products below the limit of detection for MC contamination as determined by LC-MS/MS ( Parker et al., 2015 , unpublished data). Following the supplemental procedure for the analysis of dried algae as dietary supplements, as described above in the Sample Preparation section, 100 mg of Spirulina powder was weighed into a 15 mL disposable polypropylene centrifuge tube followed by the addition of 5 mL of extraction solvent.…”

“…These products represented a mixture of powdered (3), tablet (2), and capsule (2) forms. Full internet searches were not performed for Spirulina or Chlorella products because previous studies found that products containing only these ingredients were less likely to contain MC contamination ( Lawrence et al., 2001 ; Heussner et al., 2012 ; Vichi et al., 2012 ; Parker et al., 2015 ; Yakes et al., 2015 ). Altogether, 67 algal dietary supplement products were tested for MC contamination using the Microcystin/Nodularin PP2A microtiter plate test kit following the supplemental instructions for dried algal dietary supplements as described above.…”

“…Numerous methods have been employed for screening or confirmation of MC contamination in BGA-containing dietary supplement products including enzyme-linked immunosorbent assays (ELISAs) typically targeting the conserved Adda group, antibody-based surface plasmon resonance (SPR) biosensors, DNA-based polymerase chain reaction (PCR) assays screening for the presence of MC producing genes (i.e., screening for the potential for MC production), and chemical confirmatory methods such as Liquid Chromatography with UV detection (LC-UV) and Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) ( Lawrence et al., 2001 ; Heussner et al., 2012 ; Vichi et al., 2012 ; Parker et al., 2015 ; Yakes et al., 2015 ). The mechanism of hepatotoxicity of MC to mammals involves the potent inhibition of protein phosphatases comprising PP1, PP2A, and related enzymes, which dephosphorylate serine, threonine, and histidine residues in proteins and are critical to the control of several biological processes ( MacKintosh et al., 1995 ).…”

“…To accomplish the primary objective of this study we first evaluated the performance of the MicroCystest for use on BGA-containing dietary supplements, and then used this test to assess the prevalence of MC contamination above the Oregon 1 μg/g limit in BGA dietary supplements currently available to U.S. consumers. For confirmation, LC-MS/MS was performed following the previously validated method of Parker et al. (2015) on any samples equal to or exceeding 0.5 μg/g MC.…”

Dietary supplements: food safety issues and detection strategies

“…已有的分析方法大多属于已知目标物的筛查或测 定. 其中, LC-MS/MS 技术将 HPLC 的高效分离与高灵 敏度、强专属性的串联质谱如三重四极杆相结合, 增 强了复杂体系中多成分的准确定量和结构确证能力, 已成为检测食品添加物最常用的手段 [8,10,11] .…”