Methods to be Reviewed by OMB in 2016

chneider

ndersen

ournal of

AOAC I

nternational

98, N

. 3, 2015

659

well as the published AOAC First Action

2012.25

method (8). A

detailed study protocol based on

2012.25

was also provided to

the study participants for additional guidance. The collaborative

study was designed to meet the requirements of a U.S. Food

and Drug Administration (FDA) Foods Program level four full

collaborative study chemical method validation (9).

Control filets of Channel catfish (

Ictalurus punctatus

) and

Atlantic salmon (

Salmo salar

) were obtained from the FDA

Center for Veterinary Medicine (CVM) Office of Research

Aquaculture Program. Residue incurred catfish and salmon were

produced at the CVM by exposing one catfish (2.2 kg) and one

salmon (2.8 kg) to individual treatment baths at 25 and 12°C,

respectively, containing a mixture of MG, CV, and BG each

with a concentration of 2 μg/L. Each treated fish was placed

in an exposure tank for 1 h, removed to a clean water tank, and

then sacrificed 1 h after the exposure period. One control catfish

and one control salmon were also collected from clean water

tanks. To prepare homogenized portions of control and incurred

muscle, skinless catfish filets (approximately 700 g) and salmon

filets with attached skin (approximately 950 g) were separately

ground with dry ice to produce fine powders. After overnight

dry ice sublimation at –20°C, homogenized samples were

sealed and stored at –80°C.

Frozen, peeled, and deveined white shrimp (

Litopenaeus

vannemei

) were purchased commercially. This product was

labeled as a product of Ecuador, farm-raised without the use of

antibiotics or growth hormones. Approximately 900 g of these

shrimp were ground with dry ice to produce a homogeneous

control matrix as described above. Residue incurred shrimp was

not available for this study. Surrogate incurred shrimp samples

were produced by fortifying 2.00 g (±0.02 g) weighed portions

of homogenized control shrimp with analytes to yield the

following concentrations: 0.85 µg/kg MG, 0.75 µg/kg LMG,

1.18 µg/kg CV, 0.76 µg/kg LCV, and 1.50 µg/kg BG.

Study samples and standard solutions were shipped to each

laboratory on dry ice, with instructions to store samples at –80°C

and standards at –20°C until analysis. All analytical standards

were obtained from Sigma-Aldrich (St. Louis, MO) including

MG oxalate, LMG, CV chloride, LCV, BG (bisulfate salt),

D5-MG picrate, D5-LMG, D6-CV trihydrate, and D6-LCV.

All were indicated as Fluka analytical grade standards, except

for LCV and BG that were available at the time only as reagent

grade.

Individual stock solutions of the dyes (MG, CV, and

BG), leuco metabolites (LMG and LCV), and internal standards

(MG-D5, LMG-D5, CV-D6, and LCV-D6) were prepared in

acetonitrile with nominal concentration 100 μg/mL. Mixed

standard solutions (1.000 µg/mL) were prepared in acetonitrile

by combining the necessary volume of each stock solution to

yield the exact final concentration for all the compounds. One

mixed standard solution was prepared for the analytes (MG,

LMG, CV, LCV, and BG) and one mixed standard solution for

the internal standards (MG-D5, LMG-D5, CV-D6, and LCV-D6).

Participants received vials of both mixed standard solutions

along with instructions to prepare fresh working solutions

from these standards on each day of analysis. Three sets of fish

samples consisting of salmon, catfish, and shrimp matrix were

also provided to each participating laboratory. Each set consisted

of 17 tubes containing weighed portions (2.00 g ± 0.02 g) of

homogenized matrix including: six tubes of negative control

matrix labeled as calibrants, one tube of negative control matrix

labeled as a QC, and 10 tubes of randomly numbered blinded

test samples. Participants were instructed to fortify the six matrix

calibrant samples prior to extractionwith the appropriate amounts

of working standard solutions to produce matrix fortified with 0,

0.25, 0.5, 1.0, 2.5, and 5.0 µg/kg of the analytes and 2.0 µg/kg of

the internal standards. Participants were instructed to fortify the

QC sample with analytes (1.0 µg/kg) and internal standards (2.0

µg/kg) after completion of the extraction procedure at the final

extract reconstitution step. The QC sample was a post-extraction

fortified calibrant that could be used to measure extraction losses

when compared to the set of pre-extraction fortified calibrants.

For further calibration comparisons in this collaborative study,

participants were also requested to prepare and analyze a set of

six solvent calibrants with concentrations to match the extracted

matrix calibrants (0, 0.25, 0.5, 1.0, 2.5, and 5.0 μg/kg as tissue

equivalents). Both the QC sample and solvent calibrant solutions

were prepared in acetonitrile with an ascorbic acid concentration

of 0.01% to match the composition of the extracted matrix

calibrants. The 10 blinded test samples included two negative

control matrix samples; six fortified samples with concentrations

0.42, 0.90, and 1.75 µg/kg (in duplicate); and two residue

incurred samples (for salmon and catfish). For the shrimp matrix,

for which residue incurred tissue was not available, two surrogate

“incurred” samples were included in the set of blinded samples,

each fortified with 0.75 µg/kg MG, 0.76 µg/kg LMG, 0.85 µg/kg

CV, 1.18 µg/kg LCV, and 1.50 µg/kg BG.

Study participants

were instructed to fortify all 10 blinded samples with internal

standard working solution (2.0 µg/kg) prior to extraction

Test

samples for each of the three matrixes were labeled with unique

letter and color coding (randomized between laboratories),

which allowed these to be matched with the corresponding

matrix calibrant tubes. The identity of the matrixes was not made

known to the participants.

Both the

2012.25

method and the collaborative study

protocol provided sufficient details to perform the LC-MS/MS

analysis; however, participants were given flexibility to choose

their analytical instrumentation and optimize the performance

of their chosen system. Requirements given to the participants

in order to select an appropriate triple quadrupole mass

spectrometer were that the instrument would provide sensitivity

to detect solvent solutions of the analytes with a concentration

of 0.5 µg/L, and that two product ions would be collected for

each analyte and one for each internal standard. In addition to

the Waters Corp. (Milford, MA) LC-MS/MS system described

in the

2012.25

method, participants were provided with

optimized source parameters for an Agilent (Santa Clara, CA)

6490 LC-MS/MS system in the study protocol.

Finally, study participants were cautioned that

triphenylmethane dyes and metabolites are light sensitive

and require efforts to reduce background contamination.

Participants were cautioned to reasonably protect samples and

solutions from excessive light exposure (e.g., place in the dark

or cover with foil when not in use), avoid black markers that are

a known source of CV, and minimize instrument carryover by

using an injection needle wash and/or injecting a blank water

sample between blinded samples.

Each participating laboratory received a customized report

spreadsheet matched to sample matrix and blinded sample

coding that automatically displayed calibration curves and

provided calculated concentrations as data was entered. On

completion of the analyses, the participants returned this

completed report sheet, as well as a summary of specific

Candidates for 2016 Method of the Year

286