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 usually 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]. Additionally, fluorescence detection is suitable for the determination of free and total pantothenic acid in food, but the method may be too complex for routine analysis
Chemostat glucose-limited synthetic minimal medium containing (per liter) 0.1 g calcium chloride, 0.1 g sodium chloride, 0.5 g magnesium sulfate, 1 g potassium dihydrogen phosphate, 5 g ammonium sulfate, 500 μg boric acid, 40 μg sulfuric acid Copper, 100 μg potassium iodide, 200 μg ferric chloride, 400 μg manganese sulfate, 200 μg sodium molybdate, 400 μg zinc sulfate, 1 μg biotin, 200 μg calcium pantothenate, 1 μg folic acid, 1 mg inositol, 200 μg smoke Acid, 100 μg p-aminobenzoic acid, 200 μg pyridoxine, 100 μg riboflavin, 200 μg thiamine, and 0.08% glucose.
Prepare 10 L of medium, mix well, filter sterilize and place in autoclaved glass bottles. The bottom of the Carboy has an outlet port that leads to a short length of tubing with a Luer lock connector on the end. All inlets and outlets are covered with aluminum foil prior to autoclaving. The outflow tube is sealed with a metal clip before filling. Carboy is placed on a shelf above the chemostat area.