CLIN.CHEM. 35/5, 874-878 (1989)
Determination of trans-Phylloquinone in Children’s Serum
Fathl Moussa,1 Luclenne
Dutour,’
Jean Ren#{233}
Didry,’
and Pierre
Aymard2
By optimizing the conditions for determining trans-phylloquinone and its metabolite, K-2,3-epoxide, in serum through a
two-step HPLC process combined with fluorometric detection
after coulometnc reduction, we have been able to develop a
method applicable to small volumes of serum (200 to 500
p.L). The limit of detection (signal-to-noise ratio of 3) was 15
ng/L for trans-phylloquinone, 30 ng/L for K-2,3-epoxide. The
trans-phylloquinone concentrations measured by this method in serum from 82 children, ages one to six years, whose
results were normal for overall coagulation tests, ranged from
40 to 880 ng/L (median 175 ng/L). We discuss these findings
and compare them with vitamin K, (20) values reported for
adults.
A
3
B
k
AdditIonal Keyphrases: vitamin K
pediatric chemistry
reference interval
chromatography, reversed phase . fluoromettic and coulometric methods
Vitamin
K (Figure 1) is required forthe post-translational gamma carboxylation ofglutamic acid residues on several
proteins
in plasma and tissue.The best known of these
vitamin
K-dependent proteins are procoagulant
Factors II,
VII, IX, and X and proteins
C and S, allof which are
involved in the formation or inhibition of thrombin. The
physiological role of the other vitamin K-dependent proteins
is less well understood
(1, 2).
The diagnosis of K hypovitaminoses is currently based on
detection of depressed coagulant
activity
of vitamin Kdependent factors or increased concentrations
of their inactive precursors in the circulation (3). However, such tests
are not specific (4).
Several high-performance liquid-chromatographic
(HPLC) methods have been publishedfordetermining
concentrationsof vitamin K1(20) in biological media (5-14).
However, the mean concentrationsreported for serum of
normal adults range from 247 (15) to 2600 ng/L (5).
By optimizing the conditions for determination by HPLC,
and incorporating fluorometric detection after post-column
coulometric
reduction
(9, 10), we have developed a method
that can detect - 15 pg of trans-phylloquinone
[the biologically active isomer of vitamin K1 (8)] and -30 pg of its
metabolite, K-2,3-epoxide, per milliliter
of serum sample.
We have applied this method to the study of trans-phylloquinonemia in children.
C
3
0
n
Fig. 1. Structuralformulas of vitamin K compounds
(A) bans.vitaininK1(20) (other synonymsusedare “frans-vitaminK1 or fransphylioquinone’).( bans-vitamin
K1(25),synthetic stnicturalanalog of vitamin
K,, used as internal standard. (
bans-K,(20)-2,3-epoxide.(1 bans-vitamin
I(2(n) or menaquinone-n
respiratory
infections,
tonsillectomy,
or adenoidectomy. None ofthem displayedany symptoms ofdigestive
or hepatic trouble, and results of their hematological tests
[prothrombin
time (PT) and activated partial thromboplastin time (APTI’),which evaluatethe total extrinsic clotting
system and the total intrinsic clotting
system,respectively]
were all within normal limits. The blood samples were
promptly dispatched
to the laboratoryfor centriftigation.
The separated serum was immediately frozen and stored at
-20 #{176}C
until analysis. To plot the calibration curves, we
used standards
prepared
in a commercial lyophilized serum
(“Biotrol 3 Taux: Taux faibles”; bioMerieux, Charbonnieres
les Bains, France).
recurrent
MaterIals and Methods
Reagents
Samples
We used two batches of vitamin
K1(20) (2-methyl-3phytyl-1,4-naphthoquinone)
from different sources (F. Hoffmann-La
Roche,
Neuilly-sur-Seine,
France,
and Sigma
Chemical Co.,St.Louis, MO). Vitamin
K1(25) and K-2,3epoxide were supplied by F. Hoffinann-La
Roche. Stock
Blood (1-1.5 mL) was sampled from 82 children (34 girls
and 48 boys, ages 1-6 years) for various blood testson the
first day of their admission to our otorhinolaryngology
departments.
The children
were hospitalized
for either
‘Hopita! Trousseau, Laboratoire de Biochimie,
26 Ave du Docteur Arnold-Netter, 75571 Paris Cedex 12, France.
2Universite
Paris-Sud, Laboratoire
de Physiologie, Rue J. B.
Clement, 92290 Chatenay-Malabry, France.
Received November 21, 1988; accepted February 17, 1989.
874 CLINICALCHEMISTRY, Vol. 35, No. 5, 1989
solutions of the different vitamins-1
g/L, in hexane-were
stored at -20 #{176}C
in the dark.The standard solutions were
diluted in absoluteethanol and stored under the same
conditions.
The concentration
of the standard
solutionof
vitamin K1 was determined
by its absorbance
at 248 nm
(molar absorptivity
=
19.9 mol’cm’
in ethanol).
The
hexane and acetonitrile
were “Uvasol” grade (Merck, Darm-
stadt, F.R.G.). The absolute ethanol wa from Carlo Erba,
Milan, Italy. The NaC1O4 for analysis came from Merck. All
the chemicals were used as obtained, without further purification. An “Oxisorb” cartridge (Messer-Griesheim
GMBA,
Ivry, France)was used to ifiter the U-grade nitrogen.
For the PT test, we used “Thrombomat” thromboplastin
with calcium forthe automated
system (bioMerieux)
and
the AP’I’F test (Automated
APTF for General Diagnosis;
Organon
Teknika
Corp., Durham,
NC 27713).
any intake
Apparatus
Results and Discussion
Two HPLC systems are required for this detennination.
The first, a normal-phase
system, used for the semipreparative step, consisted ofa Model SP 8810 pump (Spectra-Physics, Les Ulis, France) connected
to a Model 7125
injector (Rheodyne, Cotati, CA) equipped with a 100-giL
loop; the 250 mm x 4 mm silica column packed with 5-pm
particles was protected by a 30 mm x 4 mm pre-column
(Waters Chromatography, Division of Millipore, St. Quentin
en Yveline, France), dry-packed with 30- to 38-pm (diameter) particles of silica (HC Pellosil). The LC-UV Pye Unicam
detector (Philips, Bobigny, France) operating at 248 nm was
connected
to a Servotrace recorder (Sefram, Paris, France).
The fractions containing
the different K vitamins
were
collected in a fractioncollector (Microcol TDC 80; Gilson,
Procedures
Villiers-le-Bel,
France).
The second, a reversed-phase
HPLC system, used forthe
analytical step,consisted of the same pump system with a
50-pL injection loop. A 150 mm x 4 mm Novapak (C18, 4pm particles) chromatographic
column (Waters) was used
for separation tests; a 70 mm x 4.6 mm XL 3-pin octyl
cartridge (Beckman, Gagny, France) was used forroutine
assays. The post-column coulometric reduction was performed with a Model 5011 cell controlled by a Coulochem
5100A
module
(both from ESA, Cergy-St.
Christophe,
France). The fluorescenceof the reduced products was
measured
in a fluorospectrophotometer
(Model RF 530;
Shimadzu,
Kyoto,Japan) connected
to a recorder.
of oxygen into the chromatographic system, only
tubes and connections were used for its
assembly.) Because the applied potential on the two detectors of the cell from ESA was -0.8 V, we did not begin
sample injections until the current displayed by the 5100-A
module exceeded -8 p.A; this was vital for obtaining optimum efficiency of reduction. For the fluorometer, the excitation and emission wavelengths were set at 320 and 430 nm,
respectively.
stainless-steel
Extraction
of lipid fractions. Physical recovery of vitamin
K1(20)and K1(25)added to serum exceeded 90% in samples
extracted with six volumes of hexane after proteinshad
been precipitated
with two volumes ofethanol.These results
agree with thoseofothers(8).
Semi-preparative
normal-phase
HPLC. Figure
2 illustrates the separation of a mixture of cis-trans isomers of
vitamin K1(20) and K1(25) and of vitamin
K-2,3-epoxide.
The presenceof menaquinones (Figure 1) or vitamins
K2
(endogenous vitamin K synthesized by intestinal flora) was
not evaluated for lack of calibration reagents. The commercial vitamin K1(20) we used is a mixture of about 12% of cisisomer and 88% of trans-isomer. Similarly, commercial
vitamin K1(25) is a mixture of about 33% of cis-isomer and
67% of trans-isomer. A typical chromatographic
proffle ofa
serum sample is shown in Figure 3. Currently, investigators
generally
agree on the need fora first, normal-phase
HPLC
to eliminate the interfering lipids in the serum (8, 11, 12,
14). Haroon et al. (13) proposed a method ofextraction and
purification that obviatesthe first chromatographic
step;
however, their method is tedious and time-consuming
and
does not separate the cis-trans isomers of vitamin
K1.
Analytical
reversed-phase
HPLC. Figure 4 shows the
chromatographic
profiles of two untreated serum samples
(500 pL of serum). The limit of detection of the method for
trans-vitamin
K1(20) approximates15 ng/L, as determined
Procedure
After extracting
the lipid fractions of the serum, we
subjected them to normal-phase
HPLC, followed by reversed-phase HPLC. Vitamin K1(25) was used as the internal standard.
Extraction of lipid fractions: After adding 2 ng of the
internal standard
in 50 pL ofethanol to 200 or 500 pL of
serum, we precipitated
the proteins with two volumes of
absolute ethanol. We then mixed the samples with six
volumes of n-hexane. After mixing for 15 mm and then
centrifuging,
we removed the hexane (upper) layer and
evaporated it under a stream of nitrogen.
Semi-preparative
chromatography:
We dissolved the residue from the extraction in 100 p.L of mobile phase (hexane/
acetonitrile,
99.85/0.15 by volume) and injected 90 L ofthis
intothechromatograph.
The fractions containing
the transisomers of the internal
standard
and vitamin K1(20) (and
sometimes K-2,3-epoxide) were collected, combined, and
evaporated under nitrogen.
Analytical
chromatography:
We dissolvedthe residue
from the effluent
collected inthe first chromatographic
step
in 60 pL ofthe second mobile phase (NaC1O4, 5 mmol/L in
acetomtrile/ethanol,
95/5 by vol) and injected 50 L of this
into the chromatograph.
(This mobile phase had been deoxygenating by bubblinga stream ofnitrogen through it; this
was maintainedthroughouttheentire operatIon.
To prevent
I
0
8
16
win
Fig.2. Separationof vitamin K1compoundsby normal-phasechromatography
Column:250 mm x 4 mm. Silica (5 pm). Mobilephase 1.5 mL per liter
ofnhexane,flow rate 1.2 mL/min. Ultraviolet detection (X: 248 nm). Peaks: (A) cia.
K1(25), (
cis-K1(20), (C trans.K1(25), (L frans-K,(20), (E) trans-K,-2,3epoxide.Chart scale: 32 x iO absorbanceunit,as indicatedby bar
CLINICAL CHEMISTRY, Vol. 35, No. 5, 1989 875
Table 1. Precls Ion of the Method
CV, %
Av. concn.
Serum
pool
I
It
Z
12
Fig. 3. Semi-preparative chromatogram of an extract of 0.5 mL of
serum
Fractionscontaining frans-K1(25),trana.K1(20),and K1 epoxideare indicated.
Conditions as in Fig.2
k
Fig. 4. Reversed-phase chromatographic analysis of a collected fraction from the silica column
Stationaryphase:3-pm octyfcartridge.Mobilephase:acetonithle-ethanol
(95/5
by vol) containing5 mmol of sodiumperchiorateper liter. Flowrate:0.8 mL/min.
Appliedpotential: -0.8 V. K,,, 430 nm, K,,,, 320 nm.Peaks:(A) trana.K,(20),(
nt. 5th., (C) impurityaccompanyingthe nt std.(a) frans-K1(20)concentration=
140ng/L. (b) eans-K1(20) concentration= 730 ng/L
K,
ng/L
Within-run
(n =
5)
Between-run
(n
=
10)
1
100
2
250
8.9
6.6
3
400
-
7.1
4
750
5.1
-
Although
vitamins
o
of vit.
12.7
-
this does not separate the cis-trans
isomers,
K1 and K2 are easily separated.
The fluorescent
naphthohydroquinone,
obtained by reduction of the nonfluorescent naphthoquinone (9), affords a high degree ofspecificity and sufficient sensitivity for the study of small volumes
ofserum. Post-column reductioncan be done eitherchemically (13,14) orcoulometrically
(9-12). Chemical derivitization is difficult to set up, and the void volumes it generates
seem prejudicial to the sensitivity
of the method; reported
(13, 14) detection limits are all >50 ng/L. Coulometric
reduction is easier to use, cleaner, and especially more
specific, because
it is governed by the applied potential.
However, the method proposed by Langenberg and Tjaden
(9, 10) has been criticized (13, 14).
Van Haard etal. (11) improved themethod ofLangenberg
and Qjaden (9) by introducing a first, normal-phase
chromatographicstep,but they used the same mobile phase for
the analytical step, thereby retainingthe problems of adsorptiononto the working electrodes.
In fact,when we
ourselvesinitially
attempted to use the mobile phase proposed by these authors (9, 11)-NaClO4,
5 mmol/L,
in
methanol/water,
92.5/7.5
by vol-we
observed
a lack of
reproducibility,
related to a gradual declineinthe detector’s
response with the number ofinjections.
This lackof reproducibility
is,in fact,ascribable
to the passivationof the
working electrodes by adsorption of the reduction products,
because regeneration of the electrodes is accompanied
by
improved sensitivity,
as we recorded during a series of
determinations.
Moreover, the decrease in sensitivity observed at voltages under -0.5 V [20% loss according to
Langenberg and 1’aden (10)1 was undoubtedly caused by
amplification of the phenomena of adsorption, which would
tend to limit reduction of the vitamin K molecule. It was for
these reasons that we decided to reject the mixture they (10)
used in favor of a mobile phase with an acetonitrile
base.
According to Langenberg
and Tjaden,the background noise
produced by acetomtrile/water
is higher than that produced
by methanol/water (9). In our opinion, this is not caused by
the acetomtrile; conversely, the water added to the medium
may accountforthe phenomena ofadsorptionand passivationofthe electrodes
(16). The mobilephase we propose in
our methods consistsofa mixture ofNaC1O4, 5 mmol/L, in
acetonitrile/ethanol
(95/5 by vol) and offers the following
advantages:
with a signal-to-noise ratio of 3 fora zero standard. The
linearity of the method was studied by supplementing three
500-giL aliquots
of Biotrol control serum with 50, 150, and
375 pg of vitamin K1, respectively,
and analyzing them.
Within these limits, the calibration curve determined by
linear regression gives the following equation: y = 0.61x +
5.92 ng/L (r = 0.997, n = 3). Analytical
recovery after
extraction and the first chromatography
exceeded 70% as
measured in relation to the internal standard. The precision
of the method was studied by use of four pools of serum with
500-pL samples (Table1).
Published methods indicate the wide use of silica C18.
876 CLINICALCHEMISTRY, Vol. 35, No. 5, 1989
#{149}
As acetonitrile is less viscous than methanol, it is more
efficient for chromatographic separation.
#{149}
From the polarographic
point of view, under the same
experimental
conditions (electrodes,
supportingelectrolyte,
etc.), thepotential
fields
thatcan be used aremore cathodic
for acetonitrile than for methanol (15). We verified
thiswith
the porous graphite electrode we used. (The reference electrode is not specified by the supplier.) Figure 5 shows the
current-voltage
curves obtainedunder the same operating
conditionsfor the two mixtures considered. Use of our
acetonitrile
mixture
minimized
background noise and
achieved optimum
efficiency of the working electrodes.
C,,)
(2)
20
o
8
8
12
16
20
20
-in
10
(b)
A
-0.5
-1.0
-
2,0
volt,
Fig. 5. Coulometricscans of mobile phases: (A) methanol-water(92.5/
7.5 by vol) containing 5 mmol of sodium perctilorate per liter, (B)
acetonitille-ethanol (95/5 byvol)containing5 mmolof sodium perchlorate per liter
0
8
8
12
16
20
20
0
0
8
12
-in
Figure 6 shows the fluorescence
curves depending
on the
potential. For vitamin K1 the maximum signal is obtained
at -0.8 V; for epoxide, at -1.2 V. Contrary to what happens
in the methanol/water mixture (10), there is no decrease in
the fluorescence
intensity at the more cathodic potentials,
the adsorption
phenomena being negligible. In the methanol/water mixture, the half-wave potential (E’) approximates -0.3 V; in the acetonitrile/ethanol
mixture,
-0.6 V.
This cathodic shift is due to the lower proton-donor capacity
ofthe acetonitrile/ethanol
mixture.
#{149}
Adding ethanol to acetonitrile
increases the detector’s
response twofold. This is mainly because ethanol, by increasing the solubility ofvitamin K, decreases its capacity
ratio; also, acetonitrile is an aprotic solvent, and the ethanol
acts as proton donor to facilitate the reduction of the
naphthoquinone.
There is sufficient
final concentration of
supporting electrolyte
forthe reduction
current
to be governed essentially by the diffusion phenomena. Figure 7, a
and b, shows the results obtained with a Novapak column
(C18, 4 jim) at two different voltages. The stationary phase is
highly selective, entailing big retentionvolumes and overly
long analysis times. We obviated this by using a C8 column
Fluorescence
(B)
(A)
0.1
0.5
0.8
1.0
1.2
volt.
Fig. 6. Relationship between the fluorescence intensity and the potential appliedto the electrodes of the electrochemicalcell
(A) K1(20);(
K1(20)-2.3-epoxide
Fig. 7. Chromatogramsof a mixtureof (A) vitamin K1 epoxide,(B)
K1(20),and (C) K1(25),the internalstandard
(a)stationary
phase:Novopak
C18column,
flowrate1.6 mL/min, applied potential
-0.8 V. (b) Same, but applied potential-1.2 V. Retention
volume(V,) ofpeakA
= 5.9OmL, V,ofB= 11.62mL,and VrOf C= 37.5OmL(C)Stationaryphase:3pm octyl cartridge, flow rate 0.8 mL/min,appliedpotential-1.2 V. V, of A = 1.90
mL, V, of B = 2.60 mL, and V, of C = 5.20 mL D is an unknown impurity
accompanying the vitamin K1(25). Mobile phase: acetonitnle-ethanol(95/5 by
vol) containing
5 mmolof NaCIO4per liter
to reduce the hydrophobiclinkages between the stationary
phase and the solutes.
Figure 7c shows the results obtained
with a fast column.
The method of determination
proposed by MummahSchendel and Suttie (12) is similar to that of van Haard et
al. (11) and ours; however, these authors used a mobile
phase with an ethanol base (too viscous). Moreover, their
method is not very practicable, because they used radioactive vitamin K1 as the internal standard.
trans-Phylloquinone in Children’s Serum
Figure 8 shows trans-phylloquinone
concentrations,
categorized by age and sex, for the 82 children studied. Because
of the small amount of serum available,
we did not study K2,3-epoxide. The distribution (cf thehistogramin Figure 9)
followsa normal logarithmic rule, as confirmed by Shapiro’s
test. This agrees with the results observed in adults by
Lambert et al. (14). The observed concentrations in serum
ranged from 40 to 880 ngfL. When we compared the median
values for the 48 boys (196 ngIL) and the 32 girls (149 ng/L),
we found no meaningful sex-related difference (t = 1.51, 80
degrees
of freedom). Apparently, there is no correlation
between age and the concentrations of trans-phylloquinone
in serum. The significant F-value for 1 and 80 degrees of
freedom for numerator and denominator, respectively, at
the 5% level of significance was 3.9, and we obtained an Fvalue of 0.79. The overall median was 175 ng/L (95% limits
for one measurement:
33 to 920 ng/L; 95% limits for the
median:
140 to 210 ngfL). Although
close to those reported
by Shearer et al. (6) and Lambert et al. (14), our values are
lower than those indicated for adults (14). Remember
that
our values were obtained for children hospitalized for ear,
nose, or throat trouble, and whose diets in the days preceding the sampling were not monitored. Also, all these chilCLINICALCHEMISTRY, Vol. 35, No. 5, 1989 877
conc.
of methods of determination of PIVKA-ll, a “proteininduced
in vitamin K absence,”will undoubtedly
make it possible to
define vitamin
K requirements
better by studying the
correlation between the concentrations of this protein in
serum and those of vitamin K. This is the objective we are
pursuing
in our laboratory.
(nq/L)
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We thank Christine Beri-ivin fortranslation
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*131 (oocthsl
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0
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between serum frans-K1(20)concentrationsin 82
childrenaccording age and sex
Fig. 8. Relationship
number
25
30
25
20
‘5
References
1. Suttie JW. The metabolic role of vitamin K. Fed Proc Fed Am
Soc Exp Biol 1980;39:2730-5.
2. Goodman LS, Gilnian AG, Murad F. Pharmacological basis of
therapeutics. 7th ed. New York: Macmillan 1985;1582-6.
3. Kries RV, Shearer MJ, Gobel U. Vitamin K in infancy. Eur J
Pediatr 1988;147:106-12.
4. Lane PA, MD, Hathaway WE. Vitamin K in infancy. J Pediatr
1985;106:351-9.
5. Lefevere MF, De Leenheer AP, Clayes AE. High performance
liquid chromatographic
assay of vitamin K in human serum. J
Chromatogr 1979;186:749-62.
6. Shearer MJ, Rahim S, Stimniler L. Plasma vitamin K1 in
mothers and theirnewborn babies. Lancet 1982;ii:460-3.
7. Pietersma De Bruyn ALJM, Van Haard PMM. Vitamin K1 in
the newborn. ClinChim Acts 1985;150:95-101.
8. Takami U, Suttie JW. High pressure liquid chromatographic
reductive electrochemical detection analysis of serum transphylloquinone. Anal Biochem 1983;133:62-7.
9. Langenberg JP, Tjaden UR. Determination of (endogenous)
vitamin K1 in human plasma by reversed phase high performance
liquid chromatography
using fluorometric detection after post-
column electrochemical reduction. J Chromatogr 1984;305:61-72.
10. Langenberg JP, Tjaden UR. Improved method for the determination of vitamin K1 epoxyde in human plasma with electrofluorometric reaction detection. J Chromatogr 1984;289:377-85.
11. Van Haard PMM, Engel R, Pietersma De Bruyn AL.JM.
Quantitation of trans-vitamin
K1 in small serum samples by offline
10
0.i
tOO
200
300
400
500
600
700
multidimensionalliquidchromatography.
000
900
(ng/t)
1986;157:221-30.
12. Mummah-Schendel
centrations
dren studied had normal valuesforPT and APTF, even with
serum vitamin K concentrations of 40 ngfL. However, these
two tests (PT and APTT’) are not sensitive enough to detect a
Clin Chem 1986;32:1925-9.
K hypovitaminosis;
nor should we overlook the
contrary
to what is observed in infants, menaquin-
ones may play a role in children and adults.
At present an isolated concentration of trans-phylloquinone in serum is difficult to interpret.
The recent elaboration
878 CLINICALCHEMISTRY, Vol. 35, No. 5, 1989
Clin Chim Acta
LL, Suttie
JW. Serum phylloquinone
conadult population. Am J Clin Nutr
Fig. 9. Frequencydistribution curves forserum trans-K1(20) concentrations in 82 children
moderate
fact that,
and Valerie Jour-
dam forsecretarial
assistance.
0.
in a normal
1986;44:686-9.
13. Haroon Y, Bacon DS, Sadowski JA. Liquid chromatographic
determination ofvitaminK1 in plasma with fluorometric detection.
14. Lambert WE, De Leenheer AP, Baert FJ. Wet chemical postcolumn reaction and fluorescence detection analysis ofthe reference
internal of endogenous
serum vitamin K1(20). Anal Biochem
1986;158:257-61.
15. Bard AJ. Electro-analytical
chemistry: a series of advances. Vol
3. New York: Marcel Dekker, 1969:65-110.
16. Chao F. Transfert d’#{233}lectrons
et reactions superficiellea. In:
Ecole d’#{233}lectrochimie
du C.N.R.S., Reactions electrochimiques
applications. Vol 1. Lea Houches: C.N.R.S., 1978:59-85.