Pantothenic acid detection

UV absorption occurs only below 220nm, thereby it is affected by the interference from mobile phase and from artifacts in complex foods. A multiwavelength UV detection has been experimented successfully for unambiguous evaluation of pantothenic acid [609]. However, UV detection presents a low sensitivity, compared to other techniques, like FLD or MS. FLD is applied by using a postcolumn derivatization. Pantothenic acid is converted to 3-alanine by hot alkaline hydrolysis and a reaction with OPA [610]. Also MS is successfully applied to increase the sensitivity of pantothenic acid analysis. 

Thiamine, riboflavin, nicotinamide, pyridoxine, and folic acid can be determined together by using DAD, but pantothenic acid and biotin do not have adequate sensitivity for UV detection in complex matrices. 

Table 24 HPLC Methods for Quantitating Pantothenic Acid in Foods (C18 Columns UV Absorbance Detection)... 

The atom efficiency of a kinetic resolution is increased if the starting material is not an ester but a lactone. Indeed, kinetic resolutions of lactones are used on an industrial scale. Fuji/Daiichi Chemicals produces D-pantothenic acid on a multi-ton scale based on such a resolution. D-Pantolactone is hydrolysed at pH 7 by a hydrolase from Fusarium oxysporum yielding D-pantoic acid with an ee of 96% while L-pantoic acid was barely detectable. The immobilized Fusarium oxysporum cells were recycled 180 times and retained 60% of their activity, demonstrating the great stability of this catalytic system [47-50]. 

Deficiency of water-soluble vitamins is far less precarious than a deficit of fat-soluble vitamins. While the first condition is generally rare, it can nevertheless often be observed in severe alcoholism. In liver cirrhosis, it was possible to detect a reduced amount of vitamins B2, Bg, Bi2, C and niacin or pantothenic acid in the liver as well as hypofunction of vitamins Bi, B2, Bg, C and folic acid. Hypovitaminosis may develop due to the reduced formation of specific transport proteins or the decreased acti-... 

Several B vitamins, including folic acid, niacin, pyridoxine, and pantothenic acid, are routinely determined using microbiological assays, details of which can be found in the AOAC Official Methods of Analysis. Standard methods for thiamine determination using fluorimetric detection are also detailed in the AOAC methods in addition, LC techniques are now being used routinely for thiamine and other B vitamins, e.g., riboflavin. 

The method of choice for the determination of most vitamins is HPLC due to its high separation capability, its mild analytical conditions, and the possibility to use various specifically adapted detection methods, e.g., LTV, fluorescence, or MS detection. All fat-soluble vitamins and most water-soluble vitamins have chromophores suitable for UV detection. Separation of vitamers and stereoisomers can be achieved. If a higher sensitivity is required HPLC with fluorescence detection can be used, either directly (e.g., vitamins A and E) or after derivatization (e.g., thiamine). A further improvement in sensitivity and specificity has been achieved by introducing HPLC with mass spectrometric detection in vitamin analysis. Due to the structural information retrievable, e.g., molecular mass, fragmentation pattern, this is the method of choice for analysis of samples with complex mixtures or low vitamin concentrations. Examples for the use of HPLC-MS in vitamin analysis include the determination of 25-hydroxy-D3 and pantothenic acid. However, one drawback of mass spectrometry is the need for an isotopically labeled reference compound for reliable quantification. Due to the structural complexity of many vitamins, these reference compounds are often expensive and difficult to synthesize. An interesting unique application is the determination of vitamin B12 by HPLC-IPC-MS, which is possible due to its cobalt content. 

Analytical conditions are shown in Table 20.4. A urine sample was directly injected into this system. Urinary pantothenic acid is separated in the reversed phase column and hydrolysed to fl-alanine and pantoic acid. The product P-alanine reacts with o-phthaldialdehyde (OPA) and 3-mercaptopropionic acid (3-MPA), and is derivatized to l-alkylthio-2-alkylisoindole which is detected by fluorescence (Takahashi et al. 2009). 

Determination of urinary pantothenic acid by HPLC-fluorimetric method. A 20 pL of urine sample was injected into HPLC system shown in Figure 20.3. Urinary pantothenic acid was detected as a sharp peak of fluorescent compound l-alkylthio-2-alkylisoindole by fluorescence and its retention time was at 14 min (Takahashi et al. 2009). 

The recent dramatic development of mass spectrometry has developed gas chromatography-mass spectrometry (GC-MS) and LC-MS methods. Although pantothenic acid is not volatile enough for direct GC, pantothenic acid can be detected by MS after conversion to volatile compounds such as trimethylsilyl derivatives. Derivatization to volatile compounds from pantothenic acid requires an internal standard and the pantothenic acid homologue hopantothenic acid or radioisotope labelled [ C3, N]-pantothenic acid are used as internal standards for measurement by GC-MS (Banno et al. 1990 Rychlik 2000). 

Other widely used detectors for HPLC include refractive index (RI), fluorescence and evaporative light-scattering (ELS). The use of Rl and ELS detectors for pantothenic acid analysis in multivitamin dietary supplements has not been reported. The main reason is that the two detectors are not selective and thus cannot resolve pantothenic acid from other components existing in a multivitamin dietary supplement. Although fluorescence detection can be highly selective depending on the application, pantothenic acid does not have fluorescence excitation and emission and so fluorescence detection cannot be used for pantothenic acid analysis unless derivatization methods are applied (Pakin et al. 2004 Takahashi et al. 2009). Derivatization adds more complexity to analytical method and should not be used unless neeessary. For deteetion and quantitation of pantothenic add in multivitamin dietary supplements with HPLC/UHPLC, a highly selective detector such as MS should be the instrument of choice. 

Thiamine, biotin, pantothenic acid, riboflavin and vitamin B12 are involved in propionic acid fermentation. Biotin forms the prosthetic group of methyl-malonyl-CoA transcarboxylase pantothenate is a constituent of CoA thiamine is not the coenzyme (co-carboxylase) of the enzyme carboxylase like in other organisms, for acetaldehyde has not been detected in propionibacteria (although traces were recently found), but it may function as a component of dehydrogenases in oxidative phosphorylation of a-keto acids. Riboflavin is a constituent of FAD and FMN. Propionibacteria can synthesize vitamins B2 and B in considerable amounts (see below), but the other three vitamins must be supplied. Some strains can grow in synthetic media without thiamine (Silverman and Workman, 1939 Delwiche, 1949), in some other strains thiamine can be replaced by / -aminobenzoic acid. 

A similar quantitative analysis of six water-soluble vitamins (B, B2, Bg, C, nicotinamide, and pantothenic acid) in a pharmaceutical formulation using CZE in uncoated fused silica capillaries with UV detection was described by Fotsing et al. (91). Eor the B-group vitamins, a good compromise among resolution, analysis time, and analyte stability was obtained by use of 50 mM borax buffer pH 8.5. A capillary wash with sodium hydroxide was necessary between successive runs to minimize absorption of excipients from the pharmaceutical formulation to the capillary surface, otherwise giving rise to a progressive decrease of the electro-osmotic flow. 

Pantothenic acid and its salts as well as its degradation products such as pantoic acid and P-alanine do not exhibit significant absorption above 220 nm. As a result, the limitation of the direct HPLC assays lies in the lack of a selective detection wavelength. Detection is mainly performed by UV absorption at low wavelength. Analysis using UV detection below 220 nm has inherent problems because of the limited number of common mobile-phase solvents that have appropriate cutoff and because of dissolved oxygen that has to be removed via sonica-tion under vacuum. 

Pantothenic acid and pantothenates may also be analyzed following derivatization to extend the chromophore and hence allow UV detection at higher wavelengths or fluorometric detection. Hudson et al. (63) have attempted to analyze the vitamin as a P-alanine-fluorescamine complex. The derivatization procedure was lengthy and required extensive sample cleanup before the hydrolysis step due to the interference of riboflavin, niacinamide, and some minerals such as zinc, copper, manganese, and molybdenum. Although these interferences were eliminated, the method did not yield reproducible results. 

Pantothenic acid/calcium pantothenate in pharmaceutical products and vitamin premixes was also analyzed using low-wavelength ultraviolet (UV) detection (64,66). The vitamin was extracted from tablets or powdered premixes with 0.005 M NaH2P04 buffer (pH 4.5) and separated from other water-soluble vitamins on an aminopropyl-bonded silica column (LiChrosorb NH2) eluted with an acetonitrile-0.005 MNaH2P04 buffer (pH 4.5) (87 13, v/v) and detected at 210 nm. Quantitative recoveries (>95%) and relative standard deviations 0.79% to 2.2% were obtained for multivitamin tablets, vitamin premixes, fortified yeasts, and raw materials. The limit of sensitivity was approximately 1 mg/g sample. The results were compared with those obtained by the standard microbiological procedure. Low levels of calcium pantothenate (<3 mg per tablet) were more precisely analyzed by the HPLC procedure than by the microbiological method. 

Figure 10 Determination of pantothenic acid in starting infant formula. Column Supersphere CIS (5 (im, 250 X 4.6 mm) mobile phase 0.25 M NaH2P04 buffer (pH 2.5)-acetonitrile (97 3, v/v) flow rate 1 mL/min detection UV 197 nm peak identification (1) pantothenic acid. (From Ref. 53.)...

A reversed-phase method was used for the detection and quantification of C-coenzyme A esters released from rat liver mitochondrial proteins (by treatment with thiols) modified with [l- " C]pantothenic acid (59). For quantitative analysis, a 119 x 4 mm Supersphere 100 CH-18 column was used. It was equilibrated for 10 min at a flow rale of 1 mL/min in solvent A (0.02 M KH2PO4... 

A number of pharmaceutical preparations containing pantothenate have been analyzed after trimethylsilylation of the compound (73). The latter can be carried out using a 2 1 1 (v/v/v) mixture of bis(trimethylsilyl)acetamide (BSA), jv-trimethylsilyl imidazole (TMSIM), or trimethylchlorosilane (TMCS) in dimethyl sulfoxide (at room temperature, for 10 min). The resulting product is analyzed on a nonpolar stationary phase (Fig. 23). The detection limit is 2 to 4 ng. Bis(trimethylsilyl)trifluoroacetamide (BSTFA) can be used instead of BSA. TMCS is required only in the derivatization of pantothenates, but pantothenic acid can be also derivatized using a simpler 4 1 (v/v) mixture of BSTFA with IMSIM. 

Aliquots of this solution were analyzed by GC-MF using a wide-bore fiised-silica column (DB-T7, 15 m X 0.53 mm) with a flow rate of helium of 15 vaL min, and the injection port, column oven, and separator temperatures of 250°C, 200°C, and 250°C, respectively. The MS detector was operating in the electron impact mode at 70 eV, the ionization current was 100 pA and the temperature of the ion source was 200°C. The stable fragment ions [M-CHs] selected for multiple ion detection were at m/z 420,434, and 448. They were produced from the trimethylsilyl derivatives of pantothenic acid, hopantenic acid, and 5-[2,4-dihydroxy-3,3-dimethyl-l-oxobutyl)amino]pentanoic acid (internal standard), respectively (Fig. 30). 

The calibration curves were linear in the range 5 to 100 ng/mL of plasma and the detection limits were ca. 1 ng/mL plasma samples. The average recoveries of pantothenic acid and hopantenic acid were 92.9 4.6% and 95.5 5.1%, respectively. 

The plasma samples were purified on an anion exchange resin MCI GEL CA08P (170 X 10 mm. Cl ). DL-Pantothenic acid and DL-hopantenic acid were eluted with 1 M NaCl solution and free acids were extracted with ethyl acetate from the eluate acidified with 6 M HCl in the presence of ammonium sulfate and esterified with 1.5 M HCl in methanol at room temperature for 1 h. The resulting methyl esters were trifluoroacetylated with trifluoroacetic anhydride in dichloro-methane at room temperature for 30 min and analyzed by GC-MF. Mass spectrometry with selected-ion monitoring was employed for the simultaneous determination of the enantiomers of pantothenic acid and hopanthenic acid. The stable mass fragments were detected at m/z 257 and 271, being the base peaks of pantothenic acid and hopantenic acid, respectively (Fig. 31). 

The calibration curves were linear in the 50 to 2000 ng/mL range. The recoveries of L-pantothenic acid and L-hopantenic acid were 93.5 5.5% and 92.4 8.3%, respectively. The detection limits of pantothenic acid and hopantenic acid were 5 ng/mL and 12 ng/mL, respectively. 

Pantothenic acid, also known as vitamin B5, is widely distributed in food, since it is a component in the coenzyme A structure. Therefore, it is essential to all organisms and its deficiency is imcommon. In addition, being part of this coenzyme, for the total vitamin B5 determination, an enzyme hydrolysis is necessary prior to analysis. Foods richest in pantothenic acid are organ meats, egg yolk, and whole grains. RP separations are employed to analyze pantothenic acid, which does not possess any specific UV—Vis absorption. To overcome this problem, either fluorescence detection or MS detection is employed. 

---