6. AOACSPIFANMethods-2018Awards

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1130  Mottier et al. : J ournal of AOAC I nternational Vol. 98, No. 4, 2015

( c )  Identification and confirmation .—Sodium fluoroacetate is identified and confirmed when the following criteria are fulfilled (1). ( 1 ) The ratio of the chromatographic retention time of the analyte to that of its IS, i.e., the relative retention time, corresponds to that of the averaged relative retention time of the calibration solutions within a ±2.5% tolerance. ( 2 ) The peak area ratios from the different transition reactions recorded for the analyte and its IS are within the tolerances fixed by the EU criteria (1) as shown in Table 2015.03E . ( d )  Time of analysis .—Following this procedure, 20 samples can be analyzed within 24 h. H. Calculations and Expression of Results ( a )  Calculation .—Calculate the mass fraction, w , of sodium fluoroacetate in microgram per kilogram of sample (µg/kg), using the equation: = peak area of the analyte in the sample (transition = peak area of the IS in the sample (transition reaction used for quantification); I = intercept of the regression line for the transition reaction used for quantification; S = slope of the regression line for the transition reaction used for quantification; m is = mass of IS added to the test portion, in ng (i.e., 10 ng for powdered sample and 50 ng for liquid sample); m a = mass of the test portion, in g (i.e., 1 g for powdered sample and 5 g for liquid sample). ( b )  Expression of results .—Report the result of sodium fluoroacetate in µg/kg with one significant figure. Nondetected amount must be expressed as <1 µg/kg. I. Performance Characteristics The method was validated using samples provided by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Infant formulae considered are described in Table  2015.03F . Validation was performed according to the protocol described in SANCO/12571/2013 (2): precision data were obtained by spiking each sample at 1 and 10 µg/kg concentration levels (respectively corresponding to 1 × LOQ and 10 × LOQ level). At least two operators were involved in these experiments, each performing five replicates at the mentioned fortification levels on two different days (leading to a total of 10 separate experiments for each fortification level). Nonfortified samples were analyzed as well to verify absence of the pesticide before fortification trials. Table 2015.03C. LC gradient used for analysis of sodium fluoroacetate Time, min A, % B, % 0 10 90 2.0 10 90 3.0 60 40 4.5 60 40 4.6 10 90 8.0 10 90 reaction used for quantification); A is a is is a m m S I A A x w = −      where A a

Table 2015.03B. HPLC conditions for the analysis of sodium fluoroacetate Mobile phase A Water containing 5 mM ammonium formate and 0.01% formic acid, C ( i )( 1 )

Acetonitrile, C ( i )( 2 )

Mobile phase B Injection volume 

20 µL

Column 

Waters Acquity UPLC BEH Amide, 2.1 × 100 mm, 1.7 µm

Column oven temp.

45°C

Flow rate

0.45 mL/min

Needle wash

In flush port for 20 s using acetonitrile–water (1 + 1) solution, C ( i )( 3 ) HPLC flow is directed into the MS detector between 1.0 and 2.5 min LC gradient is described in Table 2015.03C

Diverter valve

Gradient

along with each series of routine samples. Water is used instead of milk. Proceeded exactly as described in E ( a ) and ( b ). F. Instrumental Conditions ( a )  LC-MS/MS analysis .—Where a specific instrument is cited, an alternative may be used provided it has the same or better characteristics. As well, an alternative HPLC column may be used provided it allows a retention time of the eluting analyte that is at least twice the retention time corresponding to the void volume of the column. ( 1 )  HPLC conditions.— Using an Agilent 1200 SL HPLC system ( see Table 2015.03B ). See Table 2015.03C for LC gradient. Using these conditions, the compound elutes at approximately 1.7 min ( see Figures 2015.03B – E ). ( 2 )  MS parameters. —MS parameters (Tables 2015.03D and  E ) are obtained by separately syringe-infusing standard solution (approximately 1 µg/mL) of each unlabeled and labeled compounds (syringe flow rate of 10 µL/min) along with the HPLC flow at 0.45 mL/min using a T connector. The HPLC flow is constituted with 10%A, C ( i )( 1 ), and 90% B, C ( i )( 2 ). ( b ) Instrument check test .—Before routine analysis, ensure that the LC-MS/MS apparatus is working in conditions such as the method remains fit for purposes. This involves to inject a low concentration calibrant [e.g., STD 2, C ( h )] to check that ( a )  Sequence setup .—Inject solutions in the following order: acetonitrile (as blank solvent) at least three times, standard solutions, C ( h ), acetonitrile at least three times, reagent blank, E ( c ), extract solutions, E ( b ), and standard solutions, C ( h ), again. Inject acetonitrile after each three to four extract solutions to check for any carry-over. ( b )  Calibration .—Draw a calibration curve by plotting peak area ratio of the analyte and its IS (= y axis) against concentration ratio of the analyte and its IS (= x axis). Calculate the slope and intercept by linear regression. Check the linearity of the calibration [regression coefficient R 2 should be higher than 0.98 and relative standard deviation of the average of response factors (= y/x ) should be <15%]. sensitivity of the instrument is adequate. G. Operating Procedure and Determination