Rapid Determination of Acylcarnitine Metabolic Diseases by Trifluoroacetic Acid-Doped Extraction Coupled with Nanoelectrospray Ionization Mass Spectrometry

Newborn screening for acylcarnitine-related inherited metabolic diseases (IMDs) is a critical test after birth. Conventional extraction methods require shaking with heating, centrifugation, nitrogen blowing, redissolution, etc., and the total time is more than 1 h. Herein, a small amount of trifluoroacetic acid (TFA)-doped extraction method for acylcarnitines coupled with nanoelectrospray ionization mass spectrometry was developed. This simplified approach successfully quantified 21 acylcarnitines in both serum and dried blood spot samples through a fast, three-step process requiring only 7 min, achieving nearly 1-2 orders of magnitude in sensitivity enhancement compared with conventional methods. The performance was further verified by the recommended liquid chromatography-tandem mass spectrometry procedure. Furthermore, the TFA-doped extraction technique was tested on serum and whole blood samples from six healthy individuals. Mechanistic studies using inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy showed that TFA promotes the coprecipitation of proteins and inorganic salts. These findings suggest that TFA-doped extraction with nanoelectrospray ionization mass spectrometry is a rapid, sensitive alternative for acylcarnitine screening, highlighting its considerable potential for clinical newborn IMD screening applications.