AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

sensitivity. Figures 2015.10A and 2015.10B show typical extracted ion chromatograms (XICs) from National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1849a for choline and carnitine. H. Quantification and Confirmation The quantification of choline and carnitine was accomplished by the generation of calibration curves using the peak area ratio of the chosen transition (Table 2015.10B ) versus the corresponding deuterated internal standards. Least-squares regression analysis using a linear model with 1/ x 2 weighting was used for both analytes. Confirmation was achieved through the analysis of ion ratios between samples and reference standards for at least one additional transition listed in the table. The concentration of each analyte in a sample was calculated by the following equation:

Table 2015.10B. Compound transitions

Transition (Q1/Q3)

Collision energy, V

Retention time, min

Compound Use/type

Carnitine

Quantitation 162.0/103.0 25

1.7 1.7 1.7 1.7 2.1 2.1

Carnitine-d 3

Internal standard

165.0/103.0 25

Carnitine Confirmation 162.0/84.4 Carnitine Confirmation 162.0/59.1

29 27 25 25

Choline

Quantitation 104.2/60.0

Choline-d 4

Internal standard

108.2/60.0

Choline

Confirmation 104.2/45.2

25

2.1

hold at a power of 1000 W, ending in a cool down (1). The contents of the vessels were transferred into 50 mL polypropylene tubes with water and diluted to a volume of 25 mL with water. A 0.5 mL aliquot of the sample solution was mixed with 0.5 mL acetonitrile in a microcentrifuge tube, and then filtered through a 0.45 μm PTFE syringe filter into a silanized injection vial. Aliquots of 0.5 mL of the working standard and reagent blank solutions were mixed with 0.5 mL of acetonitrile directly in the silanized injection vials. G. LC/MS/MS Parameters A Shimadzu Prominence LC system equipped with an Agilent Zorbax 300-SCX column (3.0 × 50 mm, 5 μm) was used. A flow rate of 1.0 mL/min was maintained over the 4.2 min total run time. The mobile phase conditions were 100% mobile phase A until 1.0 min, ramped to 100% mobile phase B by 1.5 min, and ramped back to 100% phase A by 3.0 min. A column temperature of 40°C, and an autosampler temperature of 5°C was maintained. A1 μL injection was used. Autosampler rinse settings were adjusted to eliminate carryover as much as possible. An ABSciex API 4000 mass spectrometer with positive ESI was used in multiple reaction monitoring (MRM) mode. The MS/MS overall settings used are described in Table 2015.10A . The MS/MS settings may need to be modified except for ionization, mode, and gas types to obtain optimum chromatography and

where R = results expressed in mg/100 g, C = concentration of the analyte in the injected solution in ng/mL, V = volume of the initial extract in mL, S = sample weight in grams, and D = dilution factor, the inverse of any dilution made. All results were calculated on a RTF or reconstituted basis of 25 g diluted to 225 g with water, except for SRM 1849a that was calculated back to powder basis. References: (1)  J. AOAC Int . 96 , 1068(2013)

DOI: 10.5740/jaoacint.13-128 J. AOAC Int . 99 , 204(2016) DOI: 10.5740/jaoacint.15-0144 AOAC SMPR 2012.010 J. AOAC Int. 96 , 488(2013) DOI: 10.5740/jaoac.int.SMPR2012.010 AOAC SMPR 2012.013 J. AOAC Int. 96 , 492(2013) DOI: 10.5740/jaoac.int.SMPR2012.013

Posted: February 19, 2016

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