Analytical Sciences

Quantitative Determination of Total L-Carnitine in Infant Formula, Follow-up Formula, and Raw Materials by Liquid Chromatography with Tandem Mass Spectrometry

Isao NADAOKA,* Emi HATAKEYAMA,* Chihiro TANADA,** Tasuku SAKAMOTO,** Shinichi FUKAYA,* Takashi AKIBA,* Koichi INOUE,** Yutaka YAMANO,* and Toshimasa TOYO’OKA**

*Department of Infant Milk and Analytical Technology, Research and Development Center, Wakodo Co., Ltd., 2-2 Wakaba, Chofu, Tokyo 182-0003, Japan
**Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga, Shizuoka 422-8526, Japan

We developed a rapid and useful routine screening assay for total L-carnitine content in various infant formulas and materials by liquid chromatography coupled with a tandem mass spectrometric method (LC-MS/MS) and alkaline hydrolysis. For separation of L-carnitine, a multi-mode octadecylsilane (ODS) column was used that contained ODS ligands, anion ligands, and cation ligands to avoid using ion-pairing agents. The stable isotope L-carnitine-d₃ (m/z 165 → 103/85) was used in electrospray MS/MS in the multiple reaction monitoring mode with the ion transitions of m/z 162 → 103/85 for detection and quantitation of L-carnitine. Alkaline hydrolysis of short/medium chain (C2 – C15) acyl-L-carnitines in infant formula was analyzed by LC with quadrupole time-of-flight mass spectrometry (QTOF-MS). The majority of short/medium chain acyl-L-carnitines were hydrolyzed to free L-carnitine. The overall standard deviations for L-carnitine in infant formula, follow-up formula and raw materials ranged from 2.1 to 4.0. The overall mean recoveries ranged from 90.2 to 94.2%.

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