2. AOACRIChemContMethods-2018Awards

662  S chneider & A ndersen : J ournal of AOAC I nternational V ol . 98, N o . 3, 2015

Results and Discussion

Table 2. MS/MS parameters for the Waters Corp. Quattro LCZ system

Method Performance AOAC First Action Method 2012.25 proved to be fairly straightforward for participants, and all were able to complete the study and submit the required data. Participants were requested to perform their three sets of extractions and analyses within 3 weeks. Two laboratories completed their analyses in the first week from sample receipt, and the majority of laboratories completed or initiated their sample analysis within the second week. One laboratory completed the analyses in the fifth week. Performance of the method was evaluated based on the results from all 14 laboratories with regard to quantification and identification of each of the five analytes. Overall, the results of the study were excellent, with trueness generally ≥90% and RSDr generally ≤10%, with HorRat <1. A few deviations from, and variations within, the study protocol were noted by study participants and served to illustrate the ruggedness of the method. Deviations included differences in standard and sample storage temperatures, varying speeds of centrifugation, and differences in the pore size and material used for the final extract filtration. One laboratory stored standard solutions at –6°C, and three laboratories stored tissue samples at –20, –50, or –70°C, instead of the recommended –20°C for standards and –80°C for tissue samples. Six laboratories did not centrifuge samples at 2000 × g and eight laboratories did not microcentrifuge at 20000 × g , but instead used a range of speeds (700 to 6000 × g for centrifuge and 10000 to 30 000 × g for microcentrifuge), which were likely a function of available laboratory equipment. Final filtration was generally completed with PVDF syringe filters with 0.45 μm pore size as indicated in Method 2012.25 . Three laboratories reported that PVDF filters with 0.22 μm pore size were used, and one laboratory reported that 0.45 μm PTFE filter vials were used. None of the variations for storage temperature, centrifuge speed, or filtration appeared to have influenced method performance. A variety of liquid chromatographic systems, including three ultra-HPLC (UHPLC) systems, were used in this study. Method 2012.25 and the study protocol provided for HPLC conditions, however, participants were given flexibility to design their own chromatographic separation to ensure that all analytes were retained sufficiently on their column. Variations were observed for injection volume, mobile phase gradient, flow rate, and column temperature. The primary concerns voiced by participants during the method familiarization phase involved the high percentage of acetonitrile (approximately 99% by volume) in reconstituted samples compared to the initial mobile phase composition of 40% acetonitrile. Participants were encouraged to adjust the gradient used and/or, given their sensitive instrumentation, decrease the injection volume in order to ensure analytes were suitably retained on the chromatographic column and detected. Five laboratories slowed down the initial (0–1 min) 40 to 90% acetonitrile gradient in Method 2012.25 (Table 1) by holding the initial acetonitrile composition at 10 or 20% for 0.5 to 3 min, then ramping up to 90% acetonitrile over 2 to 12 min. One laboratory used the mobile phase gradient described in Method 2012.25 gradient until 6 min, then dropped Ruggedness

Collision energy, eV

Cone voltage, V

Retention time, min

SRM, m/z

329 → 313 a 329 → 208 334 → 318 372 → 356 a,b 372 → 251 b 378 → 362 385 → 341 a 385 → 297 331 → 239 a

MG

35 35

43 43

5.1 5.1

MG-D5

40

30

5.1

CV

40

25

5.6

35

25

5.6

CV-D6

40

25

5.6

BG

35

35

6.0

50

35

6.0

LMG

25

25

7.8

331 → 316

20

25

7.8

336 → 239 374 → 358 a

LMG-D5

25

25

7.8

LCV

30

25

7.9

374 → 239

25

25

7.9

380 → 364 387 → 342 a

LCV-D6

35

25

7.9

LBG

30

25

10.9

387 → 281

30

25

10.9

a  Product ion transition used for quantification. b  An additional transition ( m/z 372 → 340) was used by five laboratories for either quantification or for identification.

I. Quantification

( a )  Internal standards .—MG-D5 is used as the internal standard for both MG and BG. All other analytes have their corresponding isotopically labeled internal standards (LMG-D5, CV-D6, and LCV-D6) incorporated into the method. ( b )  Calibrationcurves .—Foragivenanalyte,thequantification ion peak area ratios for analyte:corresponding internal standard (y-axis) are plotted versus concentration (x-axis) for the matrix calibrant samples. The resultant linear relationship (R 2 ≥ 0.95) is used to calculate the concentration of the analyte in test samples using the equation y = mx + b, where m is the slope and b is the y intercept of the calibration curve . Acceptable identification of an analyte can be determined according to either EU (10) or FDA (11) criteria. An analyte is considered to be present in a sample when: ( a ) Its chromatographic retention time is ±2.5% (EU) or ±5% (FDA) of the average retention time for the corresponding non-zero matrix calibrant samples ( b ) Its peak area ratio of qualitative ion:quantification ion is within the acceptable range of the corresponding average ratio for the non-zero extracted matrix calibrant samples. For the EU, this range is dependent on the peak ion ratio, ranging from ±20 to ±50 relative % (10). For the FDA, the acceptable range is ±10% absolute (11). ( c ) The S/N must be ≥3 for both SRM transitions. J. Identification

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