Development and Validation of a Liquid Chromatography-Tandem

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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

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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 (

Development and Validation of a Liquid Chromatography-Tandem

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