Water-soluble vitamins columns

Figure 2 Reversed-phase LC separation of eight water-soluble vitamins. Column Vydac 201HS54. Eluent gradient from 2.5% to 50% acetonitrile with 0.1 mol acetate buffer, pH 5.2 for 15 min. 1, vitamin C 2, niacin 3, pyridoxine (Be) 4, thiamin (B,) 5, nicotinamide (B3) 6, folic acid (M) 7, cyanocobalamin (B12) 8, riboflavin, (B2). (Reproduced with permission from The Separations Group, Hesperia, CA.)...

K. Johansen and P. O. Edlund, Determination of water-soluble vitamins in blood and plasma by coupled-column liquid clrromatography , 7. Chromatogr. 506 471-479 (1990). 

Water-soluble vitamins in formulations have been determined by use of ion-pair chromatography. The vitamins include several B vitamins as well as niacin, folic acid, and ascorbic acid (565). Vitamins D and Da were rapidly separated on reverse phase columns (247) as are vitamins A, D, and E in multivitamin tablets (564). Addition of silver ions to the mobile phase has been shown to increase the flexibility inherent in RPC by complexing with the unsaturated bonds and thereby decreasing the retention factor. This effect is also observed with other unsaturated drug molecules including steroids (247). Vitamin A and related compounds have... 

CF, water-soluble vitamins and sodium benzoate HPLC-UV at 210 nm. Column Nucleosil Cl 8, T = 40°C. Mobile phase HzO, ACN, triethylamine, heptanesulfonate (pH = 2.8). 

First, mobile phase and column we saw that most of the small molecule separations could be made on a C18 column in An/buffer water, with the exception of charged molecules and carbohydrates, which are too water soluble. We saw a range of polarity from fat-soluble vitamins, steroids, triglycerides, chlorinated pesticides eluting in 60-80% An/water, to carbamate, phosphate pesticides, anticonvulsants, antidepressants at 40-50% An/water, to nucleosides, nucleotides, aspirin, and water-soluble vitamins at 5-10% An/water. If you know something about the compound s structure or its solubility, you have a good clue as to what mobile phase can be used for its separation. 

Among more complex macrocycles, Li et al. [47-52] reported the preparation and characterization of stationary phases incorporating calixarenes or calix-crowns bonded to silica. With individual columns, high selectivity was observed in the separation of alkylated aromatics, aromatic carboxylic acids, sulfonamides, nucleosides, and water-soluble vitamins. In other work, Sokoliess et al. [53] have characterized calixarene- and resorcinarene-bonded stationary phases similar to those described in the previous section of this chapter. And Huai et al. [54] used an end-capped p-tert-butyl-calix[4]arene-bonded silica phase for HPLC separation of a number of organic compounds. Resorcinarenes have also found application in GC. [55-57] Recently, exotic macrocycles have been used in capillary electrochromatography, as reported by Gong et al. [58]... 

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).

The application of SCF to the extraction of vitamins has been widely reported. Thus, retinyl palmitate and tocopherol acetate have been extracted from a hydrophobic ointment with supercritical CO2 at 40°C and 196 bar for 4 min, the extract analysis being performed by SFC (137). The calibration graphs were linear from 0.5 to 2.5 pg and the recoveries were quantitative. On the other hand, water-soluble vitamins can be extracted mixing them with low substituted hydroxypropil cellulose. Portions were placed in a column to which a reversed micellar extractant was delivered (138). Extraction of vitamins A and E and their esters from tablet preparations prior to FIPLC was performed in the dynamic mode with CO2 at 40°C and 253 bar for 15 min (139). Calibration graphs were linear from 0.02 to 0.8 and from 0.005 to 0.2 mg/mL of vitamins E and A, respectively. The corresponding RSDs (six... 

Ion-exchange and reversed-phase media are most commonly used as stationary phases in the LC analysis of water-soluble vitamins. Aminopropyl-bonded silica columns (e.g., LiChrosorb NH2, /tBondapak NH2) have been used as weak anion exchangers for... 

Choosing a chromatographic column gives the possibility to perform various water-soluble vitamin and vitamin-like compound separations in one run and with better resolution. Chromatographic separation was performed using Waters Acquity UPLC BEH Shield RP,g column (100 x 2.1 mm i.d., 1.7 pm particle size) at a flow rate of 0.2 mL/min, and the injection volume was 10 pL. The mobile phase also has a significant effect on peak shape, sensitivity, and resolution. Methanol with 10 mM aqueous ammonium acetate gave the best overall performance and was deaned to be most suitable when all 14 compounds were considered. 

In a wonderfully short and effective separation, seven water-soluble vitamins (ascorbic acid, niacin, niacinamide, pyridoxine, folic acid, thiamine, riboflavin) were baseline resolved on a Cg column using an isocratic 7/93 acetonitrile/water (1% acetic acid and 5mM sodium heptanesulfonate) mobile phase [1114]. Elution was complete in 6 min. 

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. 

Although a water-soluble vitamin, biotin is sufficiently non-polar to exhibit good retention on reversed-phase columns. Depending on the pH of the mobile phase, biotin elutes from C8 and C18 columns well separated from other... 

Figure 6 Chromatogram of water-soluble vitamins from standard solution. Ion pair chromatography with a reversed-phase Cig column (Tracer Spherisorb ODS 2, 250 X 4.6 mm i.d., 5 pm) UV detection at different wavelengths mobile phase (1 mL/min) contained octanesulfonic acid (5 mM), triethylamine (0.5%), glacial acetic acid (2.4%), and methanol (15%) at pH 3.6. Peak identities (1) nicotinamide (2) pyridoxal (3) pyridoxine (4) pyridoxamine (5) folic acid (6) riboflavin (7) cyanocobalamin (8) thiamin. (From Ref. 94.)...

With a mobile phase of 0.2 M ammonium phosphate buffer (pH 5.1) in a reversed-phase system, folate, pyridoxine, nicotinamide, and thiamine could be separated, in this order, within 20 min, followed by vitamin Bn and riboflavin (17). The latter two compounds eluted after a step gradient to 30% aqueous methanol. This procedure has the advantage of completely separating at least nine coenzyme forms of water-soluble vitamins, including TPP. A similar mobile phase, composed of methanol-water (50 50) was used in connection with a LiCh-rosorb RP-18 column to separate thiamine, pyridoxine, vitamin and riboflavin in 3 min, as shown in Figure 4 (23). The detection limit at 254 nm is 5 ng (15 pmol) for thiamine and 10 to 20 ng for the other three vitamins, with a coefficient of variation of <4%. 

D Blanco, LA Sanchez, MD Gutierrez. Determination of water soluble vitamins by liquid chromatography with ordinary and narrow-bore columns. J Liq Chromatogr 17 1525-1539, 1994. 

Vitamin analyses in elemental diets are frequently required for process and quality control. Van der Horst et al. developed reversed phase methodology to determine the water-soluble vitamins, including PN, in total parenteral nutrition solutions (103). Iwase described a HPLC method to analyze the aqueous extract from an elemental pediatric diet for PN and nicotinamide (104). Chromatography involved a two-column, double-UV detector system to allow simultaneous determination of both PN and nicotinamide (104). 

Figure 9 Effect of water/methanol ratio with tetrabutylammonium phosphate reagent on retention times of water-soluble vitamins on xBondapak C18 ascorbic acid (O), niacin ( ), folic acid ( ), pyridoxin ( ), riboflavin (A), vitamin Bjj (A), niacinamide (V), thiamine (V), x not eluted from column. (From Ref. 103.)... 

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. 

Gennaro (51) proposed a method for the separation of water-soluble vitamins by means of the ion interaction reagent using octylamine < -phosphate or octylamine salicylate buffer (at pH 6.4) as the interaction reagent and the mobile phase at a flow rate of 1 mL/min, and a 2.5-p.m Spherisorb ODS C18 column (250 X 4.6 mm) as the stationary phase. The column effluent was monitored at 210 nm. Retention times of pantothenic acid obtained with octylamine t)-phos-phate and octylamine salicylate were 64.0 and 9.8 min. The method was used for the determination of pantothenic acid in a model mixture of water-soluble vitamins and also in a commercial multivitamin isotonic salt dietetic drink (Fig. 9). 

Particle beam LC-MS was investigated by Careri et al. (62) for the analysis of pantothenic acid and 10 other water-soluble vitamins. A reversed-phase HPLC method making use of volatile buffers was set up for the simultaneous separation of this mixture of vitamins using narrow-bore columns. 

AA acts as a radical scavenger, preventing the oxidation of luminol System equipped with monolithic column for simultaneous determination of six water-soluble vitamins (thiamine, riboflavin, AA, nicotinic acid, nicotinamide, and pyridoxine)... 

The selected methods are presented in tables and have been grouped by analyte of interest, type of column, and detection method, which are often the principal criteria governing method selection. Because of the water-soluble nature of the vitamins, reversed-phase chromatography is the most common mode of HPLC. Ion exchange is used occasionally. When attempting to reproduce a published HPLC method, attention should be paid to both the type of column and the manu-... 

The first grouping is a mix of fat-soluble compounds that function as hormones, co-factors, and membrane components. Fat-soluble vitamins separate on a Cis column in 80% acetonitrile/water and are usually detected at UV, 280 nm, or with fluorescence. Triglycerides are slightly less nonpolar than fat-soluble vitamins and require 60% acetonitrile/water to run on Ci8. They have poor extinction coefficients, and detection at UV, 220 nm, competes with refractive index detection in sensitivity. A phenyl column run in 50%... 

A reverse-phase HPLC assay, as part of the Association of Official Analytical Chemists report on analysis of fat-soluble vitamins, was described by DeVries et. al. (65). Analysis were made with a Merck LiChrosorb RP-18 column (Manufacturing Chemists, Inc., Cincinnati, OH) and a acetonitrile propionitrile water (79 15 6) mobile phase. Although adequate chromatography was realized, the authors were concerned that problems arose concerning influence of temperature, dissolution of sample and purification of solvents in the mobile phase. For these reasons they recommended normal-phase chromatography. Separation of vitamins D2 and Dj with their systems was not discussed. 

In this procedure, the HPLC analysis is carried out on a DuPont Zorbax ODS column using methanol, acetonitrile and water 10 2 1 at the mobile phase. This system resolves vitamins D3 from D2 and from degradation products and other fat soluble vitamins. Didecyl and dinonyl phthalate were used as the internal standards. The latter was included for use in the event that extraneous peaks interfered with the didecyl phthalate peak. Figure 11 shows a chromatogram of an extract of a multivitamin formulation with the two added internal standards. 

The quantitative analysis of the fat-soluble vitamins (A, E, D and K) and their esters by reversed-phase partition in water/alcohol solvents on Zipax columns has been reported [255]. The applicability of gas and high pressure liquid chromatography of vitamin A was discussed by Vecchi, Vesely and Oesterhelt [256] who concluded that HPLC was superior in this application. 

MARS The SPAD method was further developed into a combination of dialysis, filtration and adsorption (= molecular adsorbent recycling system). (103). The patient s blood is fed through a hollow-fibre filter and dialyzed against an albumin dialysate. The ABS (s. tab. 20.5) pass through the pores in the filter and become bonded. Plasma proteins, hormones and vitamins are not lost. The albumin dialysate is recirculated in a closed circuit where it is fed through a second dialyzer and two adsorber columns which bind the ABS. The albumin dialysate is returned to the hollow-fibre filter. It is dialyzed against a bicarbonate solution in order to remove the excess water and water-soluble substances (ammonia,... 

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