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Development and Validation of a Liquid ChromatographyTandem Mass Spectrometry Method for the Quantitation of Microcystins in Blue-Green Algal Dietary Supplements Christine H. Parker, Whitney L. Stutts, and Stacey L DeGrasse J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04292 • Publication Date (Web): 15 Oct 2015 Downloaded from http://pubs.acs.org on October 22, 2015

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Development and Validation of a Liquid

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Chromatography-Tandem Mass Spectrometry

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Method for the Quantitation of Microcystins in

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Blue-Green Algal Dietary Supplements

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Christine H. Parker,* Whitney L. Stutts, and Stacey L. DeGrasse

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U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition,

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5100 Paint Branch Parkway, College Park, MD 20740

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*To whom correspondence should be addressed:

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Phone: (240) 402-2019

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Fax: (301) 436-1052

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Email: [email protected]

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ACS Paragon Plus Environment

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ABSTRACT A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was

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developed for the simultaneous detection and quantitation of seven microcystin congeners (1–7)

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and nodularin-R (8) in blue-green algal dietary supplements. Single-laboratory method validation

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data were collected in four supplement matrices (capsule, liquid, powder, and tablet) fortified at

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toxin concentrations from 0.25–2.00 µg/g (ppm). Average recoveries and relative standard

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deviations (RSD) using matrix-corrected solvent calibration curves were 101% (6% RSD) for all

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congeners and supplements investigated. Limits of detection (0.006–0.028 µg/g) and quantitation

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(0.018–0.084 µg/g) were sufficient to confirm the presence of microcystin contamination at the

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Oregon-mandated guidance concentration of 1.0 µg microcystin-LReq/g. Quantitated

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concentrations of microcystin contamination in market-available Aphanizomenon flos-aquae

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blue-green algal supplements ranged from 0.18–1.87 µg microcystin-LReq/g for detected

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congeners microcystin-LR, microcystin-LA, and microcystin-LY (3–5). Microcystin-RR, -YR, -

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LW, -LF, and nodularin-R (1–2, 6–8) were not detected in the supplements examined.

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KEYWORDS: microcystin, cyanotoxin, blue-green algae, dietary supplements, A. flos-aquae,

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tandem mass spectrometry


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INTRODUCTION Cyanobacteria (previously classified as blue-green algae) are a diverse group of photo-

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autotrophic organisms which are found in terrestrial and aquatic environments. While serving as

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a nutrient dense resource in many ecosystems, under euphotic conditions massive blooms of

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toxic cyanobacteria occur in both freshwater and marine environments. Cyanobacterial toxins are

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structurally classified as cyclic peptides (microcystin and nodularin), alkaloids (anatoxin,

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saxitoxin, and cylindrospermopsin), and lipopolysaccharides.1,2 Of the cyanobacterial toxins,

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microcystins are the most abundant known toxins of bloom-forming cyanobacteria.

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Microcystins pose a major threat to drinking and irrigation water supplies, vegetation,

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aquatic wildlife, terrestrial animals, and humans that have been in contact with or consume

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products harvested from contaminated waters. The most severe human exposure to microcystins

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was reported in 1996, when contaminated water was inadvertently distributed to 126 renal

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dialysis patients in Brazil, resulting in 60 deaths.3,4 While acute and short-term hepatotoxic

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effects have been associated with human exposure to microcystin contaminated water, the

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potential exposure to cyanotoxins through organic cyanobacteria- and algae-derived dietary

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supplements pose a risk to consumers of blue-green algal supplements.

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Cyanobacteria- and algae-derived dietary supplements can be divided into three main

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categories: Aphanizomenon flos-aquae, Spirulina (Arthrospira platensis and Arthrospira

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maxima), and Chlorella pyrenoidosa products.5,6 Marketed to children and adults, blue-green

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algal supplements are advertised for treatment of fatigue, anxiety, depression, attention deficit-

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hyperactivity disorder (ADHD), diabetes, and high cholesterol while supporting weight loss,

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stimulating immune function, and elevating energy.6–9 Whereas Spirulina was initially

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commercialized as a non-toxic dietary supplement sourced from constructed ponds, A. flos-


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aquae entered the market as an organic cyanobacterial source harvested from natural lakes. The

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remarkably stable abundance and highly available biomass of A. flos-aquae in Upper Klamath

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Lake, Klamath Falls, Oregon renders this natural lake one of the largest viable commercial

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sources for harvesting of cyanobacterial-derived dietary supplements.

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Microcystins are characterized by a common chemical structure containing three D-amino

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acids [D-glutamic acid, D-alanine, and D-MeAsp (D-erythro-β-methylaspartic acid)], two non-

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proteinogenic amino acids [Mdha (N-methyldehydroalanine) and Adda (2S,3S,8S,9S-3-amino-9-

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methoxy-2,6,8-trimethyl-10-phenyldeca-4E,6E-dienoic acid)], and two variable L-amino acids.10

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A generalized chemical structure is shown in Figure 1A where structural variants are named

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according to the one-letter abbreviation of the two variable amino acids at positions R1 and R2.

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Among the 94 variants of microcystins that have been reported,11 microcystin-RR, microcystin-

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YR, and microcystin-LR (1–3) are the most common in cyanobacterial blooms worldwide.12,13

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While extensive toxicological data are available for microcystin-LR (3), the relative potencies of

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cyanotoxin congeners are primarily dependent on L-amino acid substituted chemical variants.14

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Similar in structure to microcystin, the cyclic pentapeptide nodularin-R is commonly isolated

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from the cyanobacterium Nodularia spumigena15,16 and most abundant in cyanobacterial blooms

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from brackish water bodies in the Baltic Sea and estuaries of Australia.17,18 Nodularin-R is

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characterized by a chemical structure of D-glutamic acid, N-methyldehydrobutyrine (Mdhb), D-

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MeAsp, Adda, and L-arginine (R2) (Figure 1B).

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To date, all reported tests of algal dietary supplements produced from Klamath Lake have

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failed to detect the presence of anatoxin, cylindrospermopsin, nodularin-R, and saxitoxin

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cyanotoxins.19–23 The risk of chronic exposure to microcystins, however, led the Oregon Health

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Division and the Oregon Department of Agriculture to establish a regulatory limit of 1 µg


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microcystin-LReq/g for microcystins in blue-green algal products.24 Health Canada followed in

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1999, releasing a precautionary advisory statement recommending the discontinued consumption

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of blue-green algal supplements for children. Currently, the levels of algal toxins in food

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supplements are unregulated at the federal level in the United States. The U.S. Environmental

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Protection Agency (EPA-820R15102) and Harmful Algal Bloom and Hypoxia Research and

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Control Amendments Act of 2014, however, have emphasized a need for research-driven action

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strategies to mitigate stakeholder response to freshwater harmful algal blooms and safeguard

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consumers from potential exposure to toxin contaminated sources.

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Current methods for microcystin detection can be characterized into approaches for

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screening and quantitation including: enzyme-linked immunosorbent assays (ELISA),25–26

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protein phosphatase inhibition assays,27,28 liquid chromatography (LC) combined with ultraviolet

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(UV)29,30 or mass spectrometric (MS) detection,31–33 polymerase chain reaction (PCR)

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taxonomic-based assays,34–35and surface plasmon resonance (SPR) biosensors.36–37 Although

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biochemical and physiochemical methods for microcystin detection are suitable for general

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monitoring purposes, limitations in sensitivity and specificity constrain quantitation of individual

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structural variants. LC-MS-based approaches provide a powerful technology capable of meeting

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required needs for sensitivity while allowing simultaneous quantitation and structural

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characterization of multiple microcystin analogs. The contamination of A. flos-aquae-based blue-

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green algal dietary supplements with microcystins, primarily microcystin-LR (3) and

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microcystin-LA (4), has been confirmed for products surveyed worldwide with concentrations

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ranging from 95% by high performance

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liquid chromatography (HPLC). Leucine enkephalin (Protea Biosciences, Morgantown, WV) and

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angiotensin I (Sigma Aldrich, St. Louis, MO) were reconstituted to stock concentrations of

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9 fmol/µL and 100 pmol/µL, respectively, and added to each sample prior to injection as a

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quality control standard and background matrix surrogate, respectively. Optima grade solvents

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for sample reconstitution, sample pretreatment, and LC analysis were purchased from Fisher

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Scientific (Pittsburg, PA). All sample preparation was performed using Eppendorf Protein

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LoBind microcentrifuge tubes (Fisher Scientific). LC-MS certified clear glass total recovery


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vials with presplit PTFE/silicone septa (Waters Corporation, Manchester, UK) were utilized as

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sample injection vials for all samples.

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Calibration Standard Preparation

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Lyophilized microcystin and nodularin-R standards were reconstituted to a stock

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concentration of 10 µg/mL in methanol. Sample dilutions were made with calibrated pipettes in

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80% methanol, 20% water, 9 fmol/µL leucine enkephalin, and 1 pmol/µL angiotensin I. To

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prevent solvent evaporation and sample concentration, exposure of the solutions to air was

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minimized. Standard solutions were stored in the dark at −20 oC or colder (up to six months).

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Neat calibration solutions were analyzed in a randomized order of injection over the range of

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1.5–197 pg/µL (1.5, 3.1, 6.2, 12.3, 24.6, 49.3, 98.5, and 197 pg/µL). The 12.3 pg/µL (0.19 µg/g)

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standard was evaluated in 3 h intervals for quality control performance.

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Preparation of Extracts from Blue-Green Algal Dietary Supplements

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Five commercially purchased blue-green algal dietary supplements were investigated for

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total microcystin content. Supplements, with corresponding daily serving quantities and

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excipients in parenthesis, included: A. flos-aquae capsule (1 g; plant cellulose and water), A. flos-

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aquae liquid (1 Tbsp), A. flos-aquae powder (1 g), A. flos-aquae Lot A and Lot B tablet (1 g;

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microcrystalline cellulose), and Spirulina powder (7 g). Each homogenized powdered

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supplement (0.100 + 0.004 g) was weighed into a 2.0 mL microcentrifuge tube. Twelve tablets

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(500 mg each) from each sample lot (60 count) were ground into a fine homogenized powder in

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a 50 mL Nalgene high-speed centrifuge tube with polypropylene screw cap (Fisher Scientific)

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using a 3/8” stainless steel grinding ball (SPEX, Metuchen, NJ) and SPEX Sample Prep 2010

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Geno/Grinder (1400 rpm × 1 min × 5 cycles and 1500 rpm × 1 min × 1 cycle). Similarly,

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powdered contents from twelve capsules (500 mg each) were emptied and homogenized into a


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uniform mixture (500 count). Aliquots (1 mL) of the mixed thawed liquid supplement (16 oz)

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were dried by vacuum centrifugation for 12–16 h to an equivalent dry weight of 0.050 + 0.010 g.

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The liquid supplement was stored at 2–8 oC for 7–10 d according to manufacturer

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recommendations.

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Weighed powdered supplements were extracted in 1.0 mL of 80% methanol, 20% water

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[1:10 supplement to solvent (w/v) extraction]. Samples were vortex mixed (1400 rpm) using an

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Eppendorf ThermoMixer for 15 min at room temperature (23 oC), rotated end-over-end (0.23 ×

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g) at room temperature, and centrifuged at 14,000 × g for 10 min at 20 oC. The supernatant was

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transferred to a new 1.5 mL microcentrifuge tube. Liquid supplement sample extractions were

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performed in a total volume of 500 µL to maintain a 1:10 (w/v) ratio.

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Five independent sample extractions were performed for each matrix source. Fortified matrix

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samples were prepared at 0.25, 0.50, 1.00, and 2.00 µg/g concentrations for each analyte by

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spiking equivalent volumes of a neat cyanotoxin standard solution into each matrix prior to

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extraction. Fortified concentrations were based upon the Oregon established regulatory limit of

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1 µg microcystin-LReq/g for products containing blue-green algae.24

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Sample Pretreatment of Blue-Green Algal Extracts

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Sample extract pretreatment procedures were evaluated to optimize sample recovery

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without analyte bias and to limit matrix interference. The following sorbents were assessed in

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method development: Phenomenex (Torrance, CA) Strata C18-E SPE (1 mL; 50 mg sorbent

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mass), Phenomenex Strata-X Polymeric SPE (1 mL; 30 mg sorbent mass), and Pierce (Thermo

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Fisher Scientific, Waltham, MA) Graphite Spin Columns (500 µL; 10 mg sorbent mass).

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Preparatory columns were used according to manufacturer recommendations with slight

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modifications to accommodate sample extract conditions and optimize sample recovery.


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Phenomenex SPE cartridges were activated in 100% methanol (2 × 1 mL) and equilibrated in

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94.5% water, 5% methanol, and 0.5% trifluoroacetic acid (TFA) (2 × 1 mL). Sample extracts

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(400 µL) were combined 1:4 (v/v) with 100% water (1600 µL) to facilitate analyte binding in

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SPE. Diluted sample extracts were added to the SPE column in two aliquots (1000 µL each) and

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passed through the sorbent a second time to enhance binding capacity. The column was washed

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with 94.5 % water, 0.5%TFA (2 × 1 mL) and sample eluted in 80% methanol, 19.9% water, and

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0.1% formic acid (2 × 100 µL). Pierce graphite spin columns were utilized as filters for pigment

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and auxiliary contaminant removal. Columns were activated and equilibrated according to

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manufacturer recommendations. Sample extracts (100 µL) were applied to the prepared resin bed

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and centrifuged at 1000 × g for 3 min without incubation or vortex mixing. The graphite resin

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was washed with a 50 µL aliquot of 80% methanol, 20% water and centrifuged for an additional

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3 min at 1000 × g.

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Cyanotoxin Infusion and Fragmentation Identification

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Infusions of cyanotoxin standards were performed on a hybrid LTQ-Orbitrap Elite

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(Thermo Scientific, San Jose, CA) with a Digital PicoView (New Objective, Woburn, MA)

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nanospray source operated at a flow rate of 300 nL/min. Individual analyte solutions were

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prepared in 50% methanol, 49.9% water, and 0.1% formic acid. The purity of individual

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microcystin standards was evaluated by full scan MS over the mass range m/z 400–1250. Higher-

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energy collisional dissociation (HCD) spectra were acquired for each microcystin congener and

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nodularin-R at 10 microscans/spectrum with a maximum injection time of 100 ms. Each high-

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resolution Fourier transform (FT) MS/MS spectrum was collected over a scan range m/z 100–

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1250 for a specified precursor mass (isolation width m/z 2.0) at a resolving power of 120,000.

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Normalized collision energies (activation time 0.100 ms) were optimized for each analyte and


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empirically adjusted based upon precursor charge so as to assign primary structural

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fragmentation while limiting pathways of secondary fragmentation. An automatic gain control

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(AGC) target allowed accumulation of up to 5 × 104 ions for FT MS/MS scans.

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In FT MS3 scans, precursor masses were first isolated for ion trap collision-induced

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dissociation (CID) at a precursor ion isolation width of m/z 2.0, using an AGC target of 1 × 104,

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10 microscans per spectrum, and a maximum ion accumulation time of 100 ms. Fragmentation

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was performed with experimentally determined normalized collision energies and an activation

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time of 10 ms (Qact = 0.250). Directly following each MS/MS experiment, a user-specified

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product ion was isolated and fragmented by HCD (isolation width m/z 2.0; activation time

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0.100 ms) to obtain a MS3 spectrum over the scan range m/z 100–1000.

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Instrumental Analysis for Quantitation

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An ACQUITY UPLC system with 150 mm × 1 mm i.d., 1.7 µm (130 Å), ACQUITY

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UPLC C18 BEH analytical column (Waters Corporation) was used for reverse phase separation

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at 40 oC. Column flow rate was maintained at 50 µL/min. Mobile phase A was prepared with

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0.1% (v/v) formic acid in water and mobile phase B with 0.1% (v/v) formic acid in acetonitrile.

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Weak needle wash and strong needle wash solvent compositions matched that of mobile phase A

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and B, respectively. Sample injections were made in partial loop mode at 2 µL volumes (10 µL

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sample loop) and the autosampler temperature was thermostated to 8 oC.

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Cyanotoxin congeners were eluted with a step gradient of 35–45% B in 5 min and 45–

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75% B from 5 to 6 min. The gradient was ramped to 90% B and re-equilibrated at initial starting

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conditions for a total run time of 14 min. Mass spectrometric analyses were accomplished using

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a 5500 QTRAP (AB Sciex, Framingham, MA) operated in positive ionization mode. Turbo V

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ion source parameters (ion spray voltage, source temperature, gas flows) were optimized


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collectively for all analytes under the chromatographic conditions specified above. The curtain

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gas was set at 20 au, CAD gas at High (12 au), ion spray voltage at 5000 V, source temperature

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at 400 oC, gas 1 pressure at 40 au, gas 2 pressure at 30 au, and entrance potential at 10 V. Using a

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syringe pump for infusion, compound dependent parameters (declustering potential, collision

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energy, and collision cell exit potential) were optimized for solvent standards of each analyte at a

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flow rate of 7 µL/min. Consensus values for declustering potential (80 V), entrance potential

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(10 V), and collision cell exit potential (18 V) were determined for each analyte. Individual

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compound transitions are shown in Table 1 with corresponding retention times, optimized

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collision energy voltages, and approximate analyte relative abundances. Retention times for the

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target compounds were determined by analyzing a mixed solution of cyanotoxin standards, under

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the conditions described above, without scheduling. The MS/MS data for all validation samples

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were collected in scheduled multiple reaction monitoring (MRM) mode with low resolution for

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Q1, unit resolution for Q3, a 5 ms pause between mass ranges, a MRM detection window of

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120 s and a targeted scan time of 1 s. Quantitative data analysis was performed using Skyline v

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2.6.39

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RESULTS AND DISCUSSION

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Optimization of Sample Pretreatment

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Dietary supplement extractions were based upon previous literature reports to optimize

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conditions for microcystin recovery.19,40,41 Representative A. flos-aquae-based (capsule, liquid,

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powder, and tablet) and Spirulina-based (powder) blue-green algal dietary supplements were

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selected for method development. Spirulina, commercialized as a non-toxic algal supplement,

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was evaluated in the context of this work as a potential matrix reference blank for quantitation.


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Extraction efficiencies were evaluated for various supplement-solvent ratios, methanol-water

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ratios, acidities, mechanical agitations, and sequential re-extractions. A 1:10 dietary supplement

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to solvent (w/v) extraction with 80% methanol, 20% water using gentle agitation (vortex and

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rotation) was selected based upon method efficacy and recovery. To minimize sample-surface

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contact and facilitate the provisioning of numerous fortified samples, a 1.0 mL sample extraction

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volume was prepared for each 100 mg sample of homogenized supplement.

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The stability of sample extracts was evaluated at varying temperatures (−20 oC and 4 oC)

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and methanol-water ratios (8% methanol and 80% methanol) over the course of five consecutive

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days. Sample stability was maximized for samples stored in sealed, light-shielded vials at −20 oC

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and freshly aliquoted prior to LC-MS analysis. The effect of methanol concentration was

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demonstrated to have a noteworthy influence on analyte recovery with an average increase in

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peak area of 77 + 3% for samples prepared and injected in 80% methanol.

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Solid-phase sorbents were investigated for contaminant removal from supplement

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extracts. Analyte recoveries were compared between pre- and post-fortified sample extracts for a

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C18 silica-based SPE cartridge, a polymeric SPE cartridge, and a graphitized carbon spin

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column. C18 sorbents are traditionally employed in the literature for the concentration of

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microcystins from various aqueous matrices of contaminated water, fish, and blue-green algal

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dietary supplements.19,31,42,43 The C18 silica- and polymeric-based SPE sorbents, however,

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yielded reduced recoveries (

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