Methods to be Reviewed by OMB in 2016

668

chneider

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ournal of

AOAC I

nternational

. 98, N

. 3, 2015

would not produce statistically relevant determinations of MDL

and LOQ.

The method accuracy generally yielded analyte trueness

greater than 90%. In order to determine the extent of correction

provided by the combination of extracted matrix calibrants

and internal standard correction, participants were requested

to concurrently analyze a set of solvent calibrants and a single

post-extraction fortified matrix calibrant (QC, 1.0 µg/kg) along

with the study samples. Internal standard corrected peak area

ratios for the 0.90 ng/g fortified sample results generated by

10 laboratories were converted to concentrations using the three

different methods of calibration: (

) extracted matrix calibration

curves (the quantitative method used in this collaborative study),

(

) single point calibration against the post-extraction fortified

matrix QC calibrant, and (

) solvent calibration curve (Table 5).

For the single point QC calibration method, the 0.90 µg/kg

fortified samples were normalized relative to the 1.0 µg/kg QC

calibrant.

There was generally good agreement among the three

calibration methods for all analytes except for BG, where

the post-extraction fortified QC matrix calibrant and solvent

calibration curve overpredicted the concentration of the residue

level and often led to poorer repeatability. Differences in method

performance betweenBGandMG-D5may account for variations

in the analyte accuracy for BG. BG quantification was studied

in greater detail with respect to internal standard correction and

matrix effects in a complementary single-laboratory validation

study of this method (18). While the post-extraction fortified

QC matrix calibrant produced the highest calculated recoveries

generated by the three methods, there was generally little

difference between the average recoveries generated by the

extracted matrix calibration curves and the solvent calibration

curves for the analyteswithmatched isotopically-labeled internal

standards (MG, LMG, CV, and LCV). The variance in analyte

recovery using solvent calibration curves was also generally the

largest. A more extensive investigation of this phenomenon has

been reported (18). In that work, method accuracy determined

from data collected by a single laboratory for MG, LMG, CV,

and LCV was found to be generally comparable regardless of

which calibration method was used, as long as internal standard

correction was applied. The post-extraction fortified calibrant

trueness for BG matched the collaborative study results with

enhanced recoveries (129–163%); however, in the single-

laboratory validation, very low recoveries were found for

BG using solvent based calibrants (0–64%). From the results

of both studies, and the procedure described by First Action

2012.25

(8), it is clear that acceptable method trueness for all

analytes is achieved only when extracted matrix calibrants with

internal standard correction are used for quantitative analysis.

Qualitative Results

Analyte identification was achieved by comparison of

peak area ratios of the qualitative:quantification product ion

transitions of test samples to the average value of the ratios

obtained from extracted calibrant samples (0.25–5 µg/L). These

results are summarized in Table 6. Acceptability criteria for

both the EU (±20–50%, based on ratio found; 10) and the FDA

(±10% absolute; 11) were applied to the data, and the results

were compared. It is interesting to note that while there were

individual cases where one or the other approach provided higher

identification percentages, on the whole, the two approaches

provided comparable results. Evaluation of retention times for

identification revealed two laboratories that had some difficulty

in meeting the stricter EU standard (±2.5%) on a total of six or

13 samples, respectively. All samples, however, met the FDA

retention time standard (±5%).

In general, identification was successful for the overwhelming

majority of samples. Blank samples did occasionally meet

identification criteria, particularly for CV and LCV, as

evidenced by the higher percentage of identifications listed

for those blank samples (Table 6). Although these samples did

meet the requirement of having signals greater than three times

the instrument noise, the calculated concentrations for most

blank samples was below 0.05 µg/kg. Thirteen of the 84 blank

samples met identification criteria and had a calculated analyte

concentration >0.05 µg/kg; calculated concentrations for those

individual blank samples are reported in Table 6. Five of those

blank samples have analytes with concentrations at or above

the MDL for the particular analyte/matrix pair: 0.08 μg/kg

LMG in salmon, 0.14 μg/kg CV in salmon, 0.30 μg/kg CV

in shrimp, and 0.12 and 0.13 μg/kg LCV in catfish. None of

the blank samples have analyte concentrations that exceed the

CCα for the analyte/matrix pair. In general, concentrations of

the identified analytes in the blank samples are well below the

1 µg/kg level of concern. True false-positive samples may be a

result of instrument carryover or trace contamination with ink

from commonly used laboratory marking pens. For best results,

it is advisable to inject water samples between test samples to

identify and minimize interference (8), and to avoid the use of

laboratory marking pens when labeling samples.

Use for Screening

The authors of the first action method proposed that this

method could be used as a screening method by estimating

the concentration of residues in an unknown sample by

comparison to a single point extracted matrix calibrant spiked

at 0.5 µg/kg. In that analytical strategy, unknown samples that

yielded corrected peak areas greater than those generated for

the 0.5 matrix calibrant would require a secondary analysis

with a full calibration curve (8). From the results of the

14 participating laboratories, peak area data (internal standard

corrected) for each 0.5 µg/kg extracted matrix calibrant was

tabulated and compared to the appropriate (matching analyte

and matrix) corrected peak area for the 10 blinded unknown

samples analyzed by each laboratory. The percentage of blinded

samples that yielded peak areas greater than the peak area of

the 0.5 µg/kg calibrant is summarized in Table 7. None of

the negative control samples yielded peak areas greater than

the 0.5 µg/kg calibrant, and all of the 1.75 µg/kg fortified

samples had responses greater than the 0.5 µg/kg calibrant. For

the 0.42 µg/kg fortified samples, 14% (58 of the 420 analyte

measurements; five analytes × three matrixes × duplicate

samples × 14 laboratories) yielded peak areas greater than the

0.5 µg/kg calibrant. Of these, 22 of the analyte measurements

(5%) yielded peak areas >20% of the 0.5 µg/kg calibrant,

corresponding to concentrations of 0.62 to 1.14 µg/kg based

on the single calibrant estimation. Table 7 highlights the screen

results/analyte; however, it should be noted that one sample often

yielded incorrect screen results for more than one residue. For

example, catfish at the 0.42 µg/kg level yielded several samples

Candidates for 2016 Method of the Year

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