Analysis of Fat-Soluble Vitamins Using UltraPerformance Convergence Chromatography (UPC2)

The analysis of fat-soluble vitamins (FSV) formulations, often from oil-filled and powder-filled capsules, or pressed tablets, can be a challenging task. Most often, analysis of these formulations employs a normal phase chromatographic method using traditional normal phase solvents (hexane, tertiary butyl alcohol, ethyl acetate, dichloromethane, and others) that can be expensive to procure and dispose. Other analytical chromatographic techniques for these analyses include reversed phase liquid chromatography, gas chromatography, thin layer chromatography, and colorimetric techniques. The use of UltraPerformance Convergence Chromatography™ (UPC2®) in fat-soluble vitamin analysis provides a single viable technique that is cost-effective, sustainable, and a green technology alternative that lowers the use of organic solvents, provides fast analysis times, and maintains chromatographic data quality. A series of FSV formulations were analysed using the ACQUITY UPC2™ System. The examined formulations contained vitamin A only, vitamins A + D3, vitamin E, vitamin D3 only, vitamin K1 only, and vitamin K2 only, as shown in Table 1. Results from these experiments show that UPC2 has the potential to replace many of the separation methods in use today as the sole technique with no compromises.

Results And Discussion
Vitamin A
This formulation of vitamin A was labelled as derived from fish liver oil and contained soy oil, gelatin, glycerin, and water as inactive ingredients. Two primary forms of vitamin A palmitate (cis and trans isomers, 1.325 and 1.394 minutes, respectively) were noted and resolved well from the small excipient peaks, as shown in Figure 1, which elute in the range of 2.0 to 2.5 minutes. This separation was accomplished using a gradient of carbon dioxide and methanol (containing 0.2% formic acid) 97:3 to 90:10 over 3 minutes with an Active Back Pressure Regulator (ABPR) setting of 2176 psi. Further details are contained in Table 2. Using this separation method, vitamin A acetate, palmitate, and retinol were easily resolved, as seen in Figure 2.

Vitamin A + D3
Similar to the previous example, this formulation of vitamins A + D was also labelled as derived from fish liver oil and contained soy oil, gelatin, glycerin, and water as inactive ingredients. Again, two forms of vitamin A palmitate (cis and trans isomers, 2.626 and 2.851 minutes, respectively) were noted before the bulk of excipient peaks.
To fully resolve vitamin D3 (cholecalciferol, 6.862 minutes) from the major excipient materials and a number of other compounds contained in the formulation, shown in Figure 3, it was necessary to use a longer column that provided enough separation efficiency to accomplish this goal. The system provided enough sensitivity to easily detect the vitamin D3 peak, as shown in Figure 3 inset.

This separation was accomplished using a gradient of carbon dioxide and methanol (containing 0.2% formic acid), 99:1 to 90:10 over 10 minutes. Further details are outlined in Table 3.

Vitamin D3
Using identical separation conditions as those used for vitamins A + D3, as shown in Table 3, vitamin D3 (cholecalciferol, 6.867 minutes) was easily resolved from the capsule excipient material, which was labeled as primarily sunflower oil, shown in Figure 4 and Table 3.
Vitamin E
A very rapid gradient analysis (~ 90 second run time) that easily provided baseline resolution of the four tocopherol isomers (d-alpha, d-beta, d-gamma, d-delta) was developed for the vitamin E capsule, shown in Figure 5. This separation was accomplished using a gradient of carbon dioxide and methanol, 98:2 to 95:5 over 1.5 minutes. Further details are shown in Table 4.

Vitamin K1
The vitamin K1 tablets generated two fully resolved (Rs > 2.0), distinct peaks with a simple isocratic method consisting of 99% CO2 and 1% methanol/acetonitrile 1:1, shown in Figure 6. UV spectra (collected simultaneously as the UV at 246 nm channel) of both peaks were similar, indicating that the peaks were related, as displayed in Figure 7. Although not confirmed (individual standards of each of the isomers were not available at time of analysis), it is likely that the two peaks are stereoisomers of phylloquinone (vitamin K1). Further details are shown in Table 5.
Vitamin K2
Vitamin K2 consists of menaquinone (MK) forms MK-3 through MK-14. The various forms of vitamin K2 have side chain lengths comprised of a variable number of unsaturated isoprenoid units. This tablet formulation showed one predominant peak and several smaller ones, as seen in Figure 8, using an isocratic separation of 95:5 CO2 /methanol, and was identified as MK-7 (data not shown). This result is consistent with the capsule label claim, which indicated that this formulation should have contained predominantly MK-7. Further method details are shown in Table 6.

Conclusions
•    Waters’ ACQUITY UPC2 System was able to successfully analyse six different formulations of fat-soluble vitamins.
•    Each of the FSV formulations were analysed rapidly with components of interest resolved from excipient materials.
•    Isomers of vitamins A, E, and K1 were successfully resolved from each other.
•    This system can greatly streamline FSV analysis by enabling laboratories to use a single technique on a single system to analyse a wide range of FSV formulations.