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

Chol-08/Carn-07 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric

Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS)

This analytical method is applicable to the analysis of both free and total L-carnitine and

choline in infant formula and adult/pediatric nutritional formula.

An analytical method for the analysis of L-carnitine and total choline in infant formula

and adult/pediatric nutritional formula is needed to meet the Standard Method Performance

Requirements (SMPR) of the Stakeholder Panel on Infant Formula and Adult Nutritionals

(SPIFAN) (1,2). A method was developed that can analyze these compounds simultaneously

using strong cation exchange (SCX) chromatography since both are quaternary ammonium

compounds, and can be retained under acidic conditions. This method can be used to quantify

the free or total choline and carnitine content of a sample. The free portion is analyzed using

water extraction, while the total extraction employs acid assisted microwave hydrolysis. The

total amount can include contribution from bound sources such as phosphatidylcholine or

acetylcarnitine. Both extraction methods employ LCMSMS analysis with electrospray

ionization (ESI). Calibration standards are included through each extraction procedure for

greater assurance with quantification, and the acidic conditions of the total extraction allow

direct injection after dilution with acetonitrile for fast analysis. A single lab validation (SLV)

was performed with this method using the SPIFAN SLV kit of materials. All requirements of

the SMPR were met, except the ability to differentiate L- and D-carnitine. Verification for

recovery of the bound forms of choline and carnitine commonly found in these matrices was also

completed.