Introduction: Vitamin K (VK) as well as vitamin D (VD) plays an important role in osteoporosis. Vitamin K1 (VK1), vitamin K2 (VK2, menaquinone-4 (MK-4), and menaquinone-7 (MK-7)) are significant for the metabolism of skeletal muscle. 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 (25(OH)D2 and 25(OH)D3) reflect circulating VD levels. More sensitive measurements remain to be developed. In the present study, a new high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of VK1, VK2 (MK-4 and MK-7), as well as 25(OH)D2 and 25(OH)D3 levels in human serum. Methods: We developed a simple LC-MS/MS method for the determination of VK1, MK-4, MK-7, 25(OH)D2, and 25(OH)D3 levels in human serum and validated the method in a study cohort of 200 patients divided into the premenopausal women group and postmenopausal osteoporosis patient group. Results: The overall precision (coefficient of variation) ranged from 2.66 to 10.11% in the specified working range (0.05–5 ng/mL) for VK1, MK-4, and MK-7. Serum VK1, MK-4, and MK-7 levels in postmenopausal women with osteoporosis were 1.187 ± 0.094 ng/mL, 0.058 ± 0.009 ng/mL, and 0.885 ± 0.064 ng/mL, respectively (mean ± standard deviation). Serum VK1, MK-4, and MK-7 levels in premenopausal women were 1.143 ± 0.103 ng/mL, 0.028 ± 0.003 ng/mL, and 1.553 ± 0.226 ng/mL, respectively. Serum 25(OH)D2 and 25(OH)D3 levels in postmenopausal women with osteoporosis were 0.757 ± 0.056 ng/mL and 11.72 ± 0.632 ng/mL, respectively. Serum 25(OH)D2 and 25(OH)D3 levels in premenopausal were 1.793 ± 0.575 ng/mL and 12.42 ± 1.069 ng/mL, respectively. Conclusion: A new LC-MS/MS method for determination of serum VK and VD levels was evaluated and validated. MK-7 in plasma decreased earlier than VD in postmenopausal osteoporosis patients. MK-7 status is significantly associated with osteoporosis and could be considered a predictable biomarker in the diagnosis of osteoporosis in postmenopausal women.

1.
Dunovska
K
,
Klapkova
E
,
Sopko
B
,
Cepova
J
,
Prusa
R
.
LC-MS/MS quantitative analysis of phylloquinone, menaquinone-4 and menaquinone-7 in the human serum of a healthy population
.
PeerJ
.
2019
;
7
:
e7695
.
2.
Klapkova
E
,
Cepova
J
,
Dunovska
K
,
Prusa
R
.
Determination of vitamins K1 , MK-4, and MK-7 in human serum of postmenopausal women by HPLC with fluorescence detection
.
J Clin Lab Anal
.
2018
;
32
(
5
):
e22381
.
3.
Berry
T
,
Moustafa
A
.
Osteoporosis and the effect of dysregulation of the transsulfuration pathway via taurine on intracellular calcium homeostasis, vitamin D absorption and vitamin K absorption
.
Clin Nutr ESPEN
.
2021
;
43
:
191
6
.
4.
Alonso
N
,
Meinitzer
A
,
Fritz-Petrin
E
,
Enko
D
,
Herrmann
M
.
Role of vitamin K in bone and muscle metabolism
.
Calcif Tissue Int
.
2023
;
112
(
2
):
178
96
.
5.
Kamao
M
,
Suhara
Y
,
Tsugawa
N
,
Okano
T
.
Determination of plasma Vitamin K by high-performance liquid chromatography with fluorescence detection using Vitamin K analogs as internal standards
.
J Chromatogr B Analyt Technol Biomed Life Sci
.
2005
816
1–2
41
8
.
6.
Suhara
Y
,
Kamao
M
,
Tsugawa
N
,
Okano
T
.
Method for the determination of vitamin K homologues in human plasma using high-performance liquid chromatography-tandem mass spectrometry
.
Anal Chem
.
2005
;
77
(
3
):
757
63
.
7.
Paroni
R
,
Faioni
E
,
Razzari
C
,
Fontana
G
,
Cattaneo
M
.
Determination of vitamin K1 in plasma by solid phase extraction and HPLC with fluorescence detection
.
J Chromatogr B Analyt Technol Biomed Life Sci
.
2009
;
877
(
3
):
351
4
.
8.
Riphagen
I
,
van der Molen
J
,
van Faassen
M
,
Navis
G
,
de Borst
M
,
Muskiet
F
.
Measurement of plasma vitamin K1 (phylloquinone) and K2 (menaquinones-4 and -7) using HPLC-tandem mass spectrometry
.
Clin Chem Lab Med
.
2016
;
54
(
7
):
1201
10
.
9.
Meinitzer
A
,
Enko
D
,
Zelzer
S
,
Prüller
F
,
Alonso
N
,
Fritz-Petrin
E
.
Development of a liquid chromatography mass spectrometry method for the determination of vitamin K1, menaquinone-4, menaquinone-7 and vitamin K1-2,3 epoxide in serum of individuals without vitamin K supplements
.
Clin Chem Lab Med
.
2022
;
60
(
7
):
1011
9
.
10.
Lee
D
,
Jee
J
,
Cho
Y
,
Jang
J
,
Yu
T
,
Kim
T
.
Serum 25-hydroxyvitamin D cutoffs for functional bone measures in postmenopausal osteoporosis
.
Osteoporos Int
.
2017
;
28
(
4
):
1377
84
.
11.
Song
Q
,
Wen
A
,
Ding
L
,
Dai
L
,
Yang
L
,
Qi
X
.
HPLC-APCI-MS for the determination of vitamin K(1) in human plasma: method and clinical application
.
J Chromatogr B Analyt Technol Biomed Life Sci
.
2008
;
875
(
2
):
541
5
.
12.
Kaneki
M
,
Hodges
S
,
Hosoi
T
,
Fujiwara
S
,
Lyons
A
,
Crean
SJ
.
Japanese fermented soybean food as the major determinant of the large geographic difference in circulating levels of vitamin K2: possible implications for hip-fracture risk
.
Nutr
.
2001
;
17
(
4
):
315
21
.
13.
Fusaro
M
,
Noale
M
,
Viola
V
,
Galli
F
,
Tripepi
G
,
Vajente
N
.
Vitamin K, vertebral fractures, vascular calcifications, and mortality: VItamin K Italian (VIKI) dialysis study
.
J Bone Miner Res
.
2012
;
27
(
11
):
2271
8
.
14.
Ahmed
S
,
Kishikawa
N
,
Nakashima
K
,
Kuroda
N
.
Determination of vitamin K homologues by high-performance liquid chromatography with on-line photoreactor and peroxyoxalatechemiluminescence detection
.
Anal Chim Acta
.
2007
;
591
(
2
):
148
54
.
15.
Sato
T
,
Schurgers
L
,
Uenishi
K
.
Comparison of menaquinone-4 and menaquinone-7 bioavailability in healthy women
.
Nutr J
.
2012
;
11
:
93
.
16.
Kuchuk
N
,
Pluijm
S
,
van Schoor
N
,
Looman
C
,
Smit
J
,
Lips
P
.
Relationships of serum 25-hydroxyvitamin D to bone mineral density and serum parathyroid hormone and markers of bone turnover in older persons
.
J Clin Endocrinol Metab
.
2009
;
94
(
4
):
1244
50
.
17.
Gutiérrez
O
,
Farwell
W
,
Kermah
D
,
Taylor
E
.
Racial differences in the relationship between vitamin D, bone mineral density, and parathyroid hormone in the National Health and Nutrition Examination Survey
.
Osteoporos Int
.
2011
;
22
(
6
):
1745
53
.
18.
Hannan
MT
,
Litman
HJ
,
Araujo
AB
,
McLennan
CE
,
McLean
RR
,
McKinlay
JB
.
Serum 25-hydroxyvitamin D and bone mineral density in a racially and ethnically diverse group of men
.
J Clin Endocrinol Metab
.
2008 Jan
93
1
40
6
.
19.
Koitaya
N
,
Sekiguchi
M
,
Tousen
Y
,
Nishide
Y
,
Morita
A
,
Yamauchi
J
.
Low-dose vitamin K2 (MK-4) supplementation for 12 months improves bone metabolism and prevents forearm bone loss in postmenopausal Japanese women
.
J Bone Miner Metab
.
2014
;
32
(
2
):
142
50
.
20.
Akbulut
A
,
Wasilewski
G
,
Rapp
N
,
Forin
F
,
Singer
H
,
Czogalla-Nitsche
K
.
Menaquinone-7 supplementation improves osteogenesis in pluripotent stem cell derived mesenchymal stem cells
.
Front Cel Dev Biol
.
2020
;
8
:
618760
.
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