Determination of Vitamin K1 Isomers in Oil Seeds by High
Performance Liquid Chromatography-Tandem Mass Spectrometry
By Fred A. Claussen | [email protected]
Chemicals and Reagents
Abstract
Objective
An analytical procedure for the determination of cis and trans isomers of vitamin K1
(phylloquinone) in canola seed (Brassica napus) and soybean seed (Glycine max) was
developed and validated. The validated method included extraction of ground seed
with a two phase solvent system consisting of dimethyl sulfoxide (DMSO) and hexane.
Crude extracts were purified on a silica solid-phase extraction (SPE) cartridge. Purified
extracts were analyzed by high performance liquid chromatography-tandem mass
spectrometry (HPLC-MS/MS). trans-vitamin K1 was separated from the biologically
inactive cis isomer using a C30 reverse phase column. Analytes were detected using
electrospray ionization (ESI) in positive ion mode and quantified by multiple reaction
monitoring (MRM). An internal standard, d7-vitamin K1, was used to compensate for
detector source suppression and losses during sample workup. The DMSO:hexane
solvent system is described in the United States Pharmacopeia (USP) for the analysis of
fat soluble vitamins in capsule formulations. It was compared to a lipase digestion
procedure described in AOAC official methods and appearing frequently in the
general scientific literature for the determination of vitamin K1 in a variety of food
products, food ingredients and raw agricultural commodities. Our research indicates
that the lipase digestion procedure is inadequate for quantitative extraction of intrinsic
vitamin K1 from canola and soybean seed matrixes.
The objective of the research was to develop and validate an
analytical method for the determination of cis and trans isomers
of vitamin K1 in canola seed and soybean seed. The developed
analytical procedure was based on the extraction procedure
described in the USP for oil soluble vitamin capsules (1) and
HPLC-MS/MS procedures described by Huang et. al. (2).
Test Matrixes



Table 1. Single Analyst Precision (1 analyst/1 day)
Vitamin K1 Analytical Standard, Sigma-Aldrich, St. Louis, MO.
89% trans isomer, 11% cis isomer as determined by area%
HPLC
d7-Vitamin K1 (d7-K1) Internal Standard, Sigma-Aldrich, St. Louis,
MO. 70% trans isomer, 30% cis isomer, 98% deuterium isotopic
purity
Individual stock standard solutions of vitamin K1 and d7-K1 were
prepared in hexane, each at a target concentration of 100
µg/mL.


Weighed 0.5 g (±0.025 g) of
ground seed into a 15 mL plastic
centrifuge tube.
Laboratory fortified canola seed
sub-samples were spiked with 0.07
mL of a 10 µg/mL vitamin-K1
working standard solution.
Laboratory fortified soybean seed
sub-samples were spiked with 0.05
mL of a 10 µg/mL vitamin-K1
working standard solution.
All canola seed and soybean seed
samples were spiked with 0.1 mL of
a 10 µg/mL internal standard
working solution.
Brought the 25 mL volumetric flask to
volume with hexane and mixed.
Transferred a 2.5 mL aliquot of the
hexane extract to a 15 mL culture
tube.
Evaporated the hexane solution to
dryness under a stream of nitrogen
using a heating block temperature
setting of 30˚C.
The dried extract was reconstituted
with 1 mL of hexane
Loaded 1 mL of hexane sample
solution onto a silica SPE cartridge
(Waters Sep-Pak, 500 mg/6 cc) that
had been conditioned with 2 mL of
hexane.



After centrifuging at 3,000 rpm for
10 minutes, the upper hexane layer
was decanted into a 25 mL
volumetric flask.



Extraction was repeated with 2 x 6
mL of hexane by shaking for 5
minutes on a platform shaker, with
each 6 mL extract added to the 25
mL volumetric flask.







Table 2. Multiple Analyst Precision (2 analysts/2 days)





Subsequent dilutions of the stock solutions were prepared in
isopropanol to achieve concentrations needed for laboratory
fortification and instrument calibration.
All standard solutions were stored in amber glass vials in a
refrigerator (ca. 4˚C) when not needed in the laboratory.
Dichloromethane, HPLC grade, EMD, Billerica, MA
Isopropyl alcohol, HPLC grade, EMD, Billerica, MA
Formic acid, 88%, ACS, Fisher Scientific, St. Louis, MO
Methanol, HPLC grade, EMD, Billerica, MA
Dimethyl Sulfoxide, Fisher Scientific, St. Louis, MO
Hexane, HPLC grade, EMD, Billerica, MA
Deionized (DI) water was obtained from a Barnstead NANOPure
water system.
The SPE cartridge was washed with
1 mL of hexane, 2 mL of 5:95
dichloromethane (DCM):hexane
(v/v). Vacuum was applied and
used to dry the cartridge for 5
minutes.
Vitamin K1 was eluted with 2 mL of
DCM. The DCM eluate was collected
in a 15 mL glass culture tube.
The DCM eluate was evaporated to
dryness under a stream of nitrogen.
Added 1 mL of isopropyl alcohol
(IPA) to the culture tube and vortex
mixed for about 10 seconds.
Transferred the IPA sample solution
to an HPLC autosampler vial and
analyzed by HPLC-MS/MS.

Flow Rate:
0.6 mL/min.

Injection Volume:
5 µL

Colum Temp.:
30˚C

MS Source:
Electrospray

MS Polarity:
Positive

MS Mode:
Multiple Reaction Monitoring (MRM)


Mobile Phase Gradient Program
Time (min)
%A
%B
0.00
5
95
Expected Retention Times (Figures 1a-1c):
trans-Vitamin K1: 7.1-7.3 min.
cis-Vitamin K1: 7.7-7.9 min.
Retention time shifts from batch to batch of mobile phase B
were observed.
0.10
5
95
5.00
0
100
8.00
0
100
HPLC Gradient:
8.01
5
95
12.00
5
95
Linear
0.832
0.0689
7.39
8.28
Mean
(mg/kg DB)
Matrix
1.55-1.85
0.740-0.903
Canola
Seed
1.82
(n=20)
0.156
Soybean
Seed
0.979
(n=40)
0.0853
021919_19_001 Smooth(Mn,1x1)
2013-360
MRM of 2 channels,ES+
451.7>187.2
1.119e+004
Trans-Vitamin K1
7.24
362.15
100
Range
(mg/kg DB)
RSD (%)
8.53
Fortification
Level (mg/kg DB)
Matrix
Mean (n=5)
Recovery (%)
Linearity: The linear concentration range for trans-vitmain K1 was approximately 5180 ng/mL (Figure 4). The linear range for cis-vitamin K1 was approximately 0.5-20
ng/mL (Figure 5).
Recovery Range
(%)
1.50-2.05
8.71
Figure 1. HPLC-MS/MS Chromatograms
1a. Vitamin K1 Calibration Standard
ca.180 ng/mL trans-vitamin K1, ca. 20 ng/mL cis-vitamin K1, ca.
70 ng/mL trans-d7-vitamin K1, ca 30 ng/mL cis-d7-vitamin K1
Conclusions
A method for the extraction and HPLC-MS/MS analysis of cis and
trans isomers of vitamin K1 in canola and soybean seed was
developed and validated. The method demonstrated acceptable
accuracy, precision, sensitivity, selectivity and linear range. The
extraction procedure was compared to the lipase procedure
described in AOAC 999.15. Lipase digestion was found to be
inadequate for the quantitative extraction of intrinsic vitamin K1
from canola seed.
Std. Dev.
Canola Seed
1.5
102
90.4-108
Soybean Seed
1.1
105
86.6-114
0.841-1.24
Interferences: The use of d7-vitamin K1 as an internal standard proved adequate for
compensation of analyte loss during sample workup and for ESI source suppression.
However, an interfering peak was observed in canola seed extracts for the vitamin
K1 MRM transition (m/z 451.6>187.2). The peak eluted between the trans and cis
isomers, partially coeluting with the cis peak (Figure 1b). This unknown compound
was previously reported by Woolard et. al. in canola oil using a C30 bonded phase
HPLC column with post column zinc reduction-fluorescence detection (3). This peak
was not observed in soybean extracts (Figure 1C). The fact that the unknown peak
was detected using the same MRM transition as vitamin K1 and had a similar
retention time suggests that it is related compound, and could be of biological
significance.
040213_19_002 Smooth(Mn,1x1)
QKS184A12-3
MRM of 2 channels,ES+
451.6>187.2
1.357e+004
Trans-Vitamin K1
7.24
368.76
100
Compound name: Trans-Vitamin K1
Correlation coefficient: r = 0.999956, r^2 = 0.999912
Calibration curve: 0.931844 * x + 1.24726
Response type: Internal Std ( Ref 3 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
Figure 2. Product Ion Spectrum of Vitamin K1
7.59
Figure 3. Product Ion Spectrum of d7-Vitamin K1
0
6.60
6.70
6.80
021919_19_001 Smooth(Mn,1x1)
2013-360
6.90
7.00
7.10
7.20
7.30
7.40
7.50
7.60
7.70
7.80
7.90
8.00
7.24
100
6.70
6.80
6.90
7.00
7.10
7.20
7.30
7.40
7.50
7.60
7.70
7.80
7.90
8.00
8.10
8.20
min
8.30
Internal standard not shown. cis-vitamin K1 not detected
(Expected retention time =7.74 minutes).
Unknown peak appears at 7.59 minutes.
min
8.10
8.20
8.30
MRM of 2 channels,ES+
451.7>187.2
1.119e+004
100
75
50
100
25
0
%
Cis-Vitamin K1
7.74
71.12
0
6.60
6.70
6.80
021919_19_001 Smooth(Mn,1x1)
2013-360
6.90
7.00
100
7.10
7.20
Trans-d7-K1
7.17
272.27
7.30
7.40
7.50
7.60
7.70
7.80
1c. Soybean Seed Sample
7.90
8.00
8.10
8.20
458.7
MRM of 2 channels,ES+
451.7>187.2
1.308e+003
Trans-Vitamin K1
7.28
152.53
7.66
221.0
141.1
%
171.2
109.6
213.0
433.7
255.2
7.81
307.7
241.6
7.10
7.20
7.30
7.40
7.50
7.60
7.70
7.80
7.90
8.00
7.17
100
min
8.10
8.20
8.30
MRM of 2 channels,ES+
458.8>194.3
3.916e+003
0
6.60
6.70
6.80
6.90
7.00
7.10
7.20
7.30
7.40
7.50
7.60
7.70
7.80
7.90
8.00
021919_19_002 Smooth(Mn,1x1)
QSY184B32-1
8.20
min
8.30
MRM of 2 channels,ES+
451.7>187.2
1.308e+003
7.28
100
8.10
Cis-d7-K1
7.66
181.24
Cis-Vitamin K1
7.81
20.58
%
0
6.60
6.70
6.80
6.90
7.00
7.10
7.20
7.30
7.40
7.50
7.60
7.70
120
395.7
7.80
7.90
8.00
8.10
8.20
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
Ion(m/z)
Dwell
Time(s)
Cone
Voltage
Collision
Energy
Precursor
Product
Vitamin K1
ca. 451.6
ca. 187.2
0.5-1.0
40
25
d7-K1
ca. 458.6
ca. 194.2
0.5-1.0
40
25
Evaluation of AOAC 999.15 Extraction Procedure: AOAC 999.15 (4)
describes the determination of vitamin K1 in infant formulas (liquid
ready-to-feed and powdered) and milk (liquid and powdered).
Triglycerides in the samples are enzymatically hydrolyzed to free fatty
acids using purified lipase (Candida rugosa) at pH 7.8-8.2 in phosphate
buffer. The digestion is carried out at 37˚C ±2˚C for 2 hours. Free
fatty acids are precipitated with potassium carbonate and ethanol.
Vitamin K1 is extracted using hexane, the hexane is evaporated under a
stream of nitrogen and the residue is redissolved in isopropanol for HPLC
analysis. Analysis of a wide variety of crop, food and feed matrixes
using the lipase procedure has been described in the general scientific
literature (3, 5-6). Lipase treatment of canola seed was evaluated in
our laboratory and found to be non-quantitative for the extraction of
intrinsic vitamin K1 (Table 4) from canola seed when compared to the
developed method. This fact should be considered when evaluating new
seed matrixes using lipase digestion.
160
180
200
220
min
8.30
6.60
6.70
6.80
6.90
7.00
7.10
7.20
7.30
7.40
Internal standard not shown.
7.50
7.60
7.70
7.80
7.90
8.00
8.10
8.20
min
8.30
(4) AOAC International Official Method 999.15. Vitamin K1 in Milk and Infant Formulas,
Liquid Chromatographic Method. 1995. Association of Analytical Communities, International,
Gaithersburg, Maryland.
240
260
219.4
159.5 178.4
280
300
320
340
360
380
332.6
304.3
245.7
430.4
439.0
313.3
400
420
440
460
480
500
m/z 0
520
80
100
120
140
160
180
Compound name: Cis-Vitamin K1
Correlation coefficient: r = 0.999102, r^2 = 0.998205
Calibration curve: 0.81918 * x + -0.206719
Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None
200
220
240
260
280
300
320
(5) Cook, K.K.; Mitchell, E.G.; Grundel, E.; Rader, J.I. HPLC analysis for trans-vitamin K1 and
dihydro-vitamin K1 in margarines and margarine-like products using the C30 stationary phase.
Food Chemistry. 1999. 67: 79-88.
400.5
340
360
380
400
420
440
460
480
500
17.5
m/z
520
15.0
Precursor Ion: m/z 458.7, Product Ion: m/z 194.4
12.5
10.0
7.5
5.0
2.5
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
22.0
ng/mL
(6) Schimpf, K.J.; Thompson, L.B.; Schmitz, D.J. Determination of trans Vitamin K1 in Infant and
Medical Nutritional Products Using AOAC Method 999.15 with Modified Preparation and
Extraction Procedures and C30 Bonded Phse Chromatography: Single-Laboratory Validation.
2010. Journal of the AOAC International. 2010. 93 No. 2. 650-661.
AOCS | May 2013 | www.eplbas.com | 866-963-2143 | Excellence, Passion and Leadership in Agriculture




HPLC autosampler vials, 1.5 mL,
glass, snap caps, Waters
Corporation, Milford, MA
HPLC column, Carotenoid S-3 C30,
150 mm x 3 mm x 3 µm, YMC
HPLC, 2695 Separations Module,
Waters Corporation, Milford, MA
Instrument Data System, Masslynx
Version 4.1
Mass spectrometer detector, Quattro
micro™ API, Waters Corporation,
Milford, MA
Solid-phase extraction (SPE)
cartridges, silica, 500 mg/6 cc, SepPak, Waters Corporation, Milford,
MA





SPE vacuum manifold, 24 position,
Supelco (Sigma-Aldrich), St. Louis,
MO
Ultrasonic cleaner with
programmable water bath
temperature, VWR, Radnor, PA
Syringe filters, 0.45 µm, Teflon,
VWR, Radnor, PA
Syringes, 20 cc, plastic, VWR,
Radnor, PA
Centrifugal mill grinder, Retsch,
Haan, Germany
Method Validation
(3) Woolard, D.C.; Indyk, H.E.; Fong, B.Y.; Cook, K.K. Determination of Vitamin K1 Isomers in
Foods by Liquid Chromatography with C30 Bonded-Phase Column.
Journal of AOAC International. 2002. 85 No. 3: 682-691.
ng/mL
170
Figure 5. cis-Vitamin K1 Linear Range
262.1
381.1
265.0
140
141.1
109.0
Precursor Ion: m/z 451.6, Product Ion: m/z 187.3
%
0
100
234.2
283.5
123.7
0
80
337.5
321.5
155.3
7.00
10
204.5
197.5
%
6.90
Compound


min
8.30
100
6.60
6.70
6.80
021919_19_001 Smooth(Mn,1x1)
2013-360
0
451.6
MRM of 2 channels,ES+
458.8>194.3
3.916e+003
021919_19_002 Smooth(Mn,1x1)
QSY184B32-1
0

194.4
100
187.3

(2) Huang, B.; Zheng, F.; Fu, S.; Yao, B.; Ren, Y. UPLC-ESI-MS/MS for determining
trans- and cis-vitamin K1 in infant formulas: method and applications.
Eur Food Res Technol. 2012. 235: 873-879
125
6.60

150
7.74
0

(1) United States Pharmacopeia and National formulary, Oil Soluble Vitamins Capsules, 2000.
USP24/NF 19, 2321-2322.
%
%


References
Figure 4. trans-Vitamin K1 Linear Range
1b. Canola Seed Sample
Response
Soybean
Seed
0.124
Range
(mg/kg DB)


Centrifuge
Centrifuge tubes, plastic, 15 mL
Culture tubes, glass, 15 mL
Platform shaker with orbital
rotation
Vortex mixer, VWR, Radnor, PA
Vials, glass, 20 mL capacity (for
extract storage)
Nitrogen evaporation unit, NEvap, Organomation Berlin, MA
Method Validation (continued)
Method Validation (continued)
Response
1.68
RSD (%)


Internal standard calibration curves were generated for cis-vitamin K1 (ca. 0.5-20 ng/mL) and trans-vitamin K1 (ca. 4.5-180 ng/mL).
Each calibrant solution contained approximately 30 ng/mL of cis-d7-vitamin K1 and 70 ng/mL trans-d7-vitamin K1 internal standard.
A linear regression calibration curve was constructed using peak area ratios (y-axis) and the standard solution concentration ratios (x-axis).
Sample solution concentrations were calculated using the straight line equation from the calibration curve. A regression weighting function of 1/x was used.
%
Canola
Seed
Std. Dev.

Analytical balance, capable
of weighing to the nearest
0.01 mg (stock standard
preparation)
Top-loading balance, capable
of weighing to the nearest mg
(sample weighing)
Class A volumetric glassware
Transfer pipets
Pipetters, fixed and
adjustable volume
A: 0.1% Formic Acid in DI Water. B: 5:95 (v/v) DCM:MeOH
Table 3. Accuracy (2 analysts/3 days)
%
Mean (n=7)
(mg/kg DB)

MRM Program for the Detection of Vitamin K1 and d7-Vitamin K1 (Figures 2 and 3)
Limit of Quantitation (LOQ): The method LOQ for trans-vitamin K1 was established at
0.09 mg/kg. The cis-vitamin K1 LOQ was established at 0.04 mg/kg. LOQ’s were
based on peak signal to noise ratios (S/N) for seed extracts. S/N at the LOQ was
approximately 10.
Matrix
Equipment (continued)
Equipment
HPLC-MS/MS Parameters
HPLC-MS/MS Parameters
(continued)
Vitamin K1 was extracted
ultrasonically for 45 minutes. The
tubes were shaken vigorously
approximately every 10 minutes
during the extraction period.


Extraction and Cleanup Procedures:
10 mL of 40:60 dimethyl sulfoxide
(DMSO):hexane (v/v) were added
and the tube was vortex mixed, then
placed in an ultrasonic bath (60ºC).



Extraction and Cleanup Procedures:
The method was validated using commercial varieties of canola
seed (Brassica napus) and soybean seed (Glycine max). Seed
samples were ground and homogenized using a centrifugal mill
grinder to pass a 0.75 mm sieve. Ground seed was stored in a
freezer (ca. -20ºC) when not needed in the laboratory.
The chemical structure of Vitamin K1 (Phylloquinone):



Chemicals and Reagents (continued)
Accuracy and precision were based on total vitamin K1 content (sum
of cis and trans isomers). The content of the cis isomer was found to
be near or below the limit of quantitation (LOQ) and accounted for
less than 10% of the total vitamin K1 in the soy and canola seed
samples used in this research. Vitamin K1 content was expressed on
a dry weight basis (DB). Single analyst precision (Table 1) was
the relative standard deviation (RSD) obtained by a single analyst
during the first attempt to validate the analytical method. Multiple
analyst precision (Table 2) was the pooled RSD obtained by two
analysts on two different days. Accuracy (Table 3) is represented
by the pooled mean recovery of laboratory fortified samples
obtained by two analysts on three different days.
Table 4. Canola Seed Vitamin K1 Extraction
Efficiency Comparison
Method
Mean (n=7)
(mg/kg DB)
Std. Dev.
Range
(mg/kg DB)
AOAC 999.15
(Lipase)
0.980
0.0591
0.865-1.07
USP
(DMSO:Hexane)
1.68
0.124
1.55-1.85