Water-soluble vitamins detection

FIGURE 18.3 Extracted ion current profiles of the water-soluble vitamins detected in a green kiwi extract (see reference [138] for the details). The LC—SRM (standard reference material) chromatogram was acquired by a high-flow ESI source (TurboIonSpray source). Each analyte was identified on the basis of the retention time, the two selected SRM transitions and their relative abundance. Only the most intense SRM ion current is reported in the figure. 

Description of Method. The water-soluble vitamins Bi (thiamine hydrochloride), B2 (riboflavin), B3 (niacinamide), and Be (pyridoxine hydrochloride) may be determined by CZE using a pH 9 sodium tetraborate/sodlum dIhydrogen phosphate buffer or by MEKC using the same buffer with the addition of sodium dodecyl-sulfate. Detection Is by UV absorption at 200 nm. An Internal standard of o-ethoxybenzamide Is used to standardize the method. 

SONG w o, BEECHER G R and eitenmiller R R (2000), Modern Analytical Methodologies In Fat- and Water-soluble Vitamins. Chichester, Wiley. sprenger c, galensa r and jensen d (1999), Simultaneous determination of cellobiose, maltose and maltotriose in fruit juices by high-performance liquid chromatography with biosensor detection , Dtsch Lebensm Rundsch, 95, 499-504. 

A large number of methods have been developed for analysis of water-soluble vitamins simultaneously in pharmaceutical products (like multivitamin tablet supplements). In fact, for these products no particular sample preparations are required and the high concentrations simplify the detection, enabling the use of UV [636]. The use of MS is also reported [637]. As well, Moreno and Salvado [638] reports also the use of a unique SPE cartridge (C18) for separating fat-soluble and water-soluble vitamins, which are, then analyzed using different chromatographic systems. 

Chen, Y. T., and Ling, Y. C. (2002). Detection of water-soluble vitamins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using porphyrin matrices. J. Mass Spectrom. 37 716-730. 

E. Postaire, M. Cisse, M. D. le Hoang, and D. Pradeau, Simultaneous determination of water-soluble vitamins by over-pressure layer chromatography and photodensitometric detection, J. Pharm. Sci., 80 368(1991). 

Figure 5.8 Separation of eleven water-soluble vitamins by MECC. Peaks 1, pyridoxamine 2, nicotinamide 3 pyridoxal 4, vitamin B6 5, vitamin B2 6, vitamin B12 7, vitamin B2 phosphate 8, pyridoxamine 5 -phosphate 9, niacin 10, vitamin Bi 11, pyridoxal 5 -phosphate. Conditions buffer, 50 mM SDS in 20 mAf phosphate-borate buffer, pH 9.0 applied voltage, 20 kV detection, UV absorbance at 210 nm. (Reprinted from Ref. 20 with permission.)...

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-... 

The potential of PBI LC-MS in the analysis of various vitamins was explored by Careri et al. [99-100]. The fat-soluble vitamins A, D, and E were analysed in food and multivitamin preparations [99]. Absolute detection limits in SIM mode were 0.6-25 ng after fast leversed-phase separation using a 97% aqueous methanol as mobile phase. Mass spectra in El, positive-ion and negative-ion Cl were obtained and discussed. The mass-spectral and quantitative performance of PBI LC-MS in the analysis of eleven water-soluble vitamins was also explored [100]. Detection limits were determined in SIM mode under positive-ion Cl, and were below 15 ng for ascorbic acid, nicotinamide, nicotinic acid, and pyridoxal, around 100 ng for dehydroascorbic acid, panthothenic acid, and thiamine, and above 200 ng for biotin, pyridoxamime, and pyridoxine. Riboflavine was not detected. 

An overpressured layer chromatographic procedure,g with photodensitometric detection for the simultaneou determination of water-soluble vitamins in multivita-o min pharmaceutical preparations, was developed and evaluated. HPTLC on silica gel plates with 1- butanol-pyridine-water (50 35 15 vol/vol) as mobilegj phase was used. The quantitation was carried out withoutl ... 

Fig. 8-87. Analysis of water-soluble vitamins. - Separator column Spherisorb ODS 2 (5 pm) eluent (A) 0.1 mol/L KOAc (pH 4.2 with HOAc), (B) water/methanol/acetonitrile (50 10 40 v/v/v) gradient linear, 6% B in 30 min to 100% B flow rate 2 mL/min detection UV (254 nm) injection volume 50 pL solute concentrations 5 nmol each of ascorbic acid (1), nicotinic acid (2), thiamine (3), pyridoxine (4), nicotinic add amide (5), p-aminobenzoic add (6), cyanocobala-mine (7), and riboflavine (8).

Figure 7.7. HPLC analysis of water-soluble vitamins in a beverage powder sample (Tang ) and a baby formula (Similac ) using ion-pair reversed-phase chromatography and UV detection. Reprinted with permission from reference 17.

At present, much effort is being devoted to simultaneous separation and detection of water-soluble vitamins. Undoubtedly, multiple water-vitamin analysis using LC as a separation method is effective with RP ion pair chromatography with acidified methanol or acetonitrile water as the mobile phase. Detection is performed using combined systems such as UV absorbance and fluorescence systems, depending on the vitamins to be determined. Analysis in real food... 

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. 

Three methods of detection (UV absorbance, fluorescence, and electrochemical activity) are applicable for the analysis of water-soluble vitamins. Currently, UV absorption detection is used in many LC applications for water-soluble vitamins. A simultaneous assay of several vitamins with several wavelengths along with information of peak purity can be accomplished with a diode array absorbance detector. The detection limit of a UV detector is in the order of 1-10 ng (10-100 pmol), which is poorer than that of fluorescence and electrochemical detectors but often sufficient for analysis for many vitamins in foods and physiological samples (Table 4). The lack of selectivity of UV detection may cause problems with interfering and co-eluting contaminants especially in biological samples, thus necessitating sample purification prior to LC. 

Electrochemical detection in LC provides a sensitive assay method for certain vitamins, such as AA, folates, and flavins. AA may be easily detected with femtomolar sensitivity. Sample preparation and matrix interference problems limit the routine applicability of electrochemistry in the analysis of water-soluble vitamins currently to AA. 

Chromatographic methods, especially LC, have offered increased selectivity and sensitivity in vitamin assays. This is reflected in the many methodological publications on the topic over the past decades. Analyses for water-soluble vitamins in physiological samples are now performed routinely using these methods. The problems with these methods are related to sample preparation and to the sensitivity of the detection method that is used. Only a few chromatographic methods enable simultaneous assay of several water-soluble vitamins in physiological samples, and so separate assays are needed. Less complex samples (e.g., pharmaceuticals or fortified foods) are easier to analyze in this respect. 

An overpressured layer chromatographic procedure, with photodensitometric detection for the simultaneous determination of water-soluble vitamins in multivitamin pharmaceutical preparations, was developed and evaluated. HPTLC on silica gel plates with 1-butanol-pyridine-water (50 35 15 vol/vol) as mobile phase was used. The quantitation was carried out without derivatization [vitamin B2 (Rf value 0.30), vitamin Bg (Rf value 0.64), folic acid (Rf value 0.37), nicotinamide (Rf value 0.80), and vitamin C (Rf value 1.02)] or after spraying ninhydrin reagent [calcium panthothenate (Rf value 0.72)] or 4-demethylaminocin-namaldehyde [vitamin B12 (Rf value 1.84) and biotin (Rf value 0)]. 

Today, HPLC is used as a reference technique to analyze any type of vitamin [25]. This technique possesses simultaneous detection and determination of vitamins in one sample [17]. HPLC is often used for the simultaneous qualitative and quantitative analysis of water-soluble vitamin and fat-soluble vitamin in biological matrices such as plasma and urine [26-28]. 

Table 55. JiRf-values and detection of less well-known water-soluble vitamins, according to Ishikawa and Katsxji [70, 77]...

The ability to apply a combination of UV- and conductivity detection to analyze inorganic cations and vitamins in the same run is advantageous for the determination of water-soluble vitamins with a mixed-mode column. 

Heudi, O., Kilinc, T., and Fontannaz, P., 2005. Separation of water-soluble vitamins by reversed-phase high performance Kquid chromatography with ultra-violet detection Application to polivitamined premixes. Journal of Chromatography A. 1070 49 55. 

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