Vitamin B5 exists in food in three bioactive forms [1]: pantothenic acid, coenzyme A (CoA) and acyl carrier protein (ACP). Calcium or sodium pantothenate are the forms commonly used as infant formula supplements [5]. Total quantification of vitamin B5 requires the release of pantothenic acid from CoA and ACP. Since it consists of pantoic acid linked by an amide bond to β-alanine, chemical hydrolysis cannot be used. The only alternative to extract free pantothenic acid from CoA is digestion with multiple enzymes (pepsin, alkaline phosphatase, pantothenate); however, this treatment fails to release the vitamin from ACP [34,35]. For the extraction of free pantothenic acid from milk and calcium pantothenate from infant formula, acid deproteinization followed by centrifugation and filtration is commonly used calcium pantothenate
Ion-suppressed RP (trifluoroacetic acid, formic acid, phosphate buffer) on C18 [34–36] and C8 columns [37] are commonly used chromatographic modes. The poor selectivity and sensitivity of the UV detector (very weak absorbance at 204 nm due to the carbonyl group) makes LC-UV unsuitable for the determination of low vitamin B5 concentrations in non-formula foods. Several researchers have used multiwavelength UV detection (200, 205, and 240 nm) [37], fluorescence detection (post-column derivatization of β-alanine with o-phthalaldehyde in the presence of 2-mercaptoethanol) [34] and mass spectrometry (MS) with ES ionization [35,38,39]. The latter method provided a limit of quantitation (LOQ) sufficient for the determination of pantothenic acid greater than 0.024 mg/100 mg, such as pantothenic acid in starchy foods [35]. In addition, fluorescence detection is suitable for the determination of free and total pantothenic acid in food, but this method may be too complex for routine analysis [34].