• Application Note

Rapid Separation of Vitamin K1 Isomers and Vitamin K2 in Dietary Supplements Using UltraPerformance Convergence Chromatography with a C18 Column

Rapid Separation of Vitamin K1 Isomers and Vitamin K2 in Dietary Supplements Using UltraPerformance Convergence Chromatography with a C18 Column
  • Jinchuan Yang
  • Waters Corporation
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Abstract

In this application note presents the use of UltraPerformance Convergence Chromatography for a fast separation of vitamin K1 trans and cis isomers and menatetrenone (MK-4), a common form of vitamin K2, on an ACQUITY UPC2 HSS C18 SB Column.

Benefits

  •  Fast and reliable separation of vitamin K1 trans and cis isomers and MK-4 in less than three minutes. 
  •  Separation is achieved on a C18 column; no special C30 column is needed. 
  •  The use of carbon dioxide as the primary mobile phase minimizes organic solvent waste.

Introduction

Vitamin K1 (phylloquinone) is an essential human nutrient produced in plants, especially green leafy vegetables. The vitamin K1 in natural products exists mainly as the trans form, while the vitamin K1 used in food supplementation is often synthetic K1, which may contain appreciable amounts of the cis form. The trans-vitamin K1 is bioactive, while the cis-K1 is not. It is highly desirable to separate the trans- and the cis-vitamin K1 isomers to truly evaluate the nutritional value of the supplement ingredient. Available HPLC methods for the separation  of vitamin K1 isomers require C30 columns. Their typical run time is about  20 minutes, and chlorinated solvents are used in some of the methods.1-3

UltraPerformance Convergence Chromatography (UPC2) is a separation technique that leverages the unique properties (i.e., low viscosity and high diffusivity) of compressed CO2 at or near its supercritical state, as well as  sub-2 micron particle packed columns to significantly improve the separation efficiency, speed, and selectivity.4 This application note demonstrates a fast separation of vitamin K1 trans and cis isomers and menatetrenone (MK-4),  a common form of vitamin K2, by UPC2 in less than three minutes on an  ACQUITY UPC2 HSS C18 SB Column. Figure 1 shows the structures of  vitamin K1 isomers and MK-4. Comparing to current LC-based vitamin K1 trans and cis isomers analysis methods, this UPC2 method is faster, simpler  (no need to use a C30 column), and it uses less organic solvent.

Figure 1. Structures of trans- and cis-vitamin K1 and menatetrenone.

Experimental

Sample preparation

Sample preparation Vitamin K1 (Sigma-Aldrich) and MK-4 (Sigma-Aldrich) were weighed and dissolved in iso-octane (ReagentPlus, Sigma-Aldrich) to obtain a stock solution at 1 mg/mL. Intermediate and working standard solutions were obtained by serial dilution of the stock solution with iso-octane. Vitamin K1 supplement tablets were purchased from a local store and were ground into a powder and extracted with iso-octane. The supernatant was filtered with a 0.45-μm PTFE syringe filter and diluted before injection.

Conditions

UPC2 conditions

System

ACQUITY UPC2 with ACQUITY UPC2 PDA Detector

Software

Empower 3

Detection

UV at 243 nm (compensation reference 400 to 500 nm, res. 6 nm)

Column

ACQUITY UPC2 HSS C18 SB 3.0 x 100 mm, 1.8 μm

Column temp.

50 °C

Sample temp.

10 °C

Injection volume

20 μL (Full loop)

Flow rate

3.00 mL/min

Mobile phase A

Compressed CO2

Mobile phase B

Acetonitrile/methanol mixture (50/50 v/v)

Run time

4 min

ABPR pressure

1500 psi

Gradient

0.5% B for 2 min, ramp to 20% B in 1.5 min, hold at 20% B for 0.5 min

Results and Discussion

Vitamin K1 cis and trans isomers and MK-4 were baseline separated in less than three minutes by UPC2 using a single UPC2 HSS C18 SB Column (3.0 x 100 mm, 1.8 μm). The cis form eluted first, followed by the trans form, then the MK-4, as shown in Figure 2. The USP resolution between the critical pair, the cis- and the trans-K1, was 1.7 (Table 1). In the gradient program, the initial two-minute isocratic elution at 0.5% B was necessary for the baseline separation of the cis- and the trans-vitamin K1. Precise control of the mobile phase B delivery volume at 0.5% is critical for the critical pair separation. The ACQUITY UPC2 System is the only SFC system on the market that can provide this level of precision control. Following the isocratic hold, a generic gradient from 0.5% to 20% B was used in the study. This gradient range could be modified in applications depending on the retention of the actual vitamin K2 homologues of interest. MK-4 was included in this study because it is a common form of vitamin K2, and it is structurally the closest vitamin K2 to K1. Other forms of vitamin K2, such as MK-7, have longer side chains, and tend to be retained longer at column. They can therefore be easily separated from vitamin K1. The total run time was four minutes, which was at least five times faster than the typical run time for HPLC methods using C30 columns. The organic solvent consumption was less than 1 mL per injection, which is only  a fraction of the typical 15 to 30 mL of solvent used in LC methods.

Figure 2. Chromatogram overlay of vitamin K1 isomers and MK-4 standard mixture (n=10).
Table 1. Results of replicate analysis of vitamin K standard mixture (n=10).

Ten replicate analyses of a standard mixture demonstrated excellent repeatability (Table 1). The limits of quantitation (LOQ), estimated at a signal-to-noise ratio at 10, were 0.06, 0.06, and 0.04 µg/mL for the cis-vitamin K1, the trans-vitamin K1 and the MK-4, respectively (Table 2). Excellent linearity (R2>0.998) was obtained for these compounds (Table 2). Analysis of a commercial vitamin K supplement product also showed excellent repeatability and resolution (Figure3). In this product, the cis-K1 was found to account for 11.2% of the total vitamin K1 (Table 3).

Table 2. LOQ and linearity.
Figure 3. Chromatogram overlay of replicate analysis of vitamin K tablet (n=3).
Table 3. Results of replicate analysis of vitamin K supplement tablet (n=3).

Conclusion

UPC2 Technology enables a rapid separation of the cis- and the trans-vitamin K1 isomers and MK-4 on an ACQUITY UPC2 HSS C18 SB Column in less than three minutes. The analysis time is at least five times faster than the current available HPLC methods, and no special C30 column is needed. This UPC2 method has excellent separation selectivity, resolution, sensitivity, repeatability, and it uses much less solvent than HPLC methods. UPC2 can potentially  be used by food ingredient testing labs for routine vitamin K analysis with significant increases in throughput and decreases in operating cost.

References

  1.  AOAC Official Method 999.15 Vitamin K in milk and infant formulas  liquid chromatographic method. AOAC International. 2005.
  2.  Woollard DC, Indyk HE, Fong BY, Cook KK. Determination of vitamin K1  isomers in foods by liquid chromatography with C30 bonded-phase column.  J AOAC International 85(3):682-691. 2002
  3.  Huang B, Zheng F, Fu S, Yao J, Tao B, Ren Y. UPLC-ESI-MS/MS for determining trans- and cis-vitamin K1 in infant formulas: method and applications. Eur Food Res Technol.;235(5):873-879. Nov. 2012.
  4. Aubin A. Analysis of fat-soluble vitamin capsules using UltraPerformance Convergence Chromatography. Waters Application Note No. 720004394en. June, 2012.

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