June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Research Paper

Highly-sensitive analysis of vitamin D3 metabolites by liquid chromatography/
electrochemistry/electrospray ionization-mass spectrometry
1

1,2

1,3

1

1

Hajime Mizuno , Yuta Kobayashi , Jun Zhe Min , Toshimasa Toyo oka , Kenichiro Todoroki *Laboratory of Analytical and Bio-Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka,
52‒1 Yada, Suruga-ku, Shizuoka 422‒8526, Japan
2
Present address: Kyoto R&D center, Maruho Co., Ltd., 92 Chudoji Awatacho, Shimogyo-ku, Kyoto 600‒8815, Japan
3
Present address: Pharmaceutical Analysis, College of Pharmacy, Yanbian University, Yanji 133002, Jilin Province, China
1

Abstract

A highly-sensitive analytical method using a liquid chromatography (LC)/electrochemistry (EC)/electrosprayionization (ESI)-mass spectrometry (MS) is developed to analyze vitamin D3 (VD3) and its active metabolites, 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in blood. LC/ESI-MS, which is used for thegeneral sensitive analysis of biological compounds, has a low ionization eﬃciency for low polarity molecules such as VD3

metabolites. The LC/EC/ESI-MS method, which connects the electrochemical detector (ECD) between the LC and ESIMS, enables simultaneous and sensitive detection of VD3 and its metabolites. The sample separated by LC is oxidized byan electrochemical cell, then the oxidant is ionized by ESI and detected by a mass spectrometer. Based on the results of an-alyzing VD3 metabolites, ion peaks of the oxidized products generated by EC are detected, and their peak intensities are 10
times or greater than the unoxidized metabolites. This proposed method for the VD3, 25(OH)D3, and 1,25(OH)2D3 analysis
2achieves the low detection limit of 1 nmol/L, 1 nmol/L, and 20 nmol/L, respectively, a good linearity (r ＞0.9903), and agood repeatability within＜10% (n＝5). Analysis of serum samples spiked with VD3 metabolites detect their oxidants peaks

with high sensitivity. This result indicates that this LC/EC/ESI-MS method is also useful for biological sample analysis andallows for quantiﬁcation of trace amounts of endogenous fat-soluble metabolites other than vitamin D3 metabolites.
Key words: Vitamin D3, 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, electrochemistry, electrospray ionization, LC/EC/MS

Introduction

1,25-dihydroxyvitamin D3 (1,25(OH)2D3), are obtained byVitamin D2 (VD2) and vitamin D3 (VD3) are some of thefat-soluble vitamins and essential nutrients. VD2 is synthe-

the hydroxylation reaction from VD2 and VD3. These
metabolites play a signiﬁcant role in the calcium homeosta-

sized by plants, whereas VD3 is synthesized by exposure tosis and are useful biomarkers for bone metabolic diseases .

UV (sunlight) in the epidermis of animals. The active
metabolites, 25-hydroxyvitamin D3 (25(OH)D3) andFurthermore, the metabolites deﬁciency has been linked to2)
3)various diseases such as diabetes , cancer , autoimmu-

1)

4)
5)
nity , and cardiovascular . In order to quantify the concentrations of VD3 and its metabolites in the human blood sam-

* Corresponding author
Kenichiro Todoroki

ple, a highly-sensitive analytical method is required.
However, liquid chromatography (LC)/electrospray (ESI)-

Laboratory of Analytical and Bio-Analytical Chemistry, Schoolof Pharmaceutical Sciences, University of Shizuoka, 52‒1

mass spectrometry (MS), which is used for general sensitiv-

Yada, Suruga-ku, Shizuoka 422‒8526, Japan
Tel: ＋81‒54‒264‒5656, Fax: ＋81‒54‒264‒5654

ity analysis of biological compounds, has a low ionizationeﬃciency for low polarity molecules such as VD3 metabo-E-mail: todoroki@u-shizuoka-ken.ac.jp

Received: January 15, 2020. Accepted: March 12, 2020.

lites. To increase the sensitivity, atmospheric pressure

Epub April 16, 2020.

chemical ionization (APCI), which is a suitable ionization
technique for nonpolar molecules, has been used for ioniza-

DOI: 10.24508/mms.2020.06.004
58

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Medical Mass Spectrometry Vol. 4 No. 1

5‒8). In addition,chased from Fisher Scientiﬁc (Waltham, MA, USA). Meth-

Ogawa et al. reported that the ionization eﬃciency of ESI
was improved by labeling VD3 metabolites with a func-

anol of LC/MS grade was purchased from the Kanto

tion during the VD3 metabolites analysis

Kagaku Co. (Tokyo, Japan). All other reagents and solvents-dimethylaminotional cationic reagent (DAPTAD: 4-(4′phenyl) 1,2,4 triazoline 3,5 dione) by a derivatization reac-

were of analytical reagent grade. Water (H2O) was puriﬁedtion. However, APCI is limited to low polar (fat-soluble)
substances and the derivatization is limited to target sub-(ELGA LabWater, High Wycombe, UK).

stances, and is not suitable for comprehensive molecularSample preparation

using a PURELAB ﬂex ultrapure water puriﬁcation system

9,10)Standard solutions of VD3, 25(OH)D3, and 1,25(OH)2D3

analyses.
On the other hand, an electrochemical detector (ECD)

were made by dissolving in acetonitrile to 1 μmol/L each.

has been used as a detector for the LC analysis of vitamin

The serum samples were prepared by dissolving 5 pmol of

11)

D and its metabolites . In this study, we developed an LC/
EC/ESI-MS method which enables the simultaneous and

VD3, 25(OH)D3, and 1,25(OH)2D3 in 50μL of humanpooled serum (L-Consera I EX, Nissui Pharmaceutical Co.,sensitive detection of VD3 and its metabolites by ESI-MS
analysis combined with electrochemical oxidation. Analyti-Ltd., Tokyo, Japan). To remove proteins in the serum,

cal methods in which an ECD is connected to a mass spec-

10 min (10,000 g, 5°C) was performed. The supernatant was

12,13)

450 μL of acetonitrile was added, and centrifugation for, however, most ofevaporated using a centrifugal evaporator, and the residue

them focused on the analysis of drug metabolites by EC
14,15)and the reduction of disulﬁde bonds in prooxidation

was redissolved with 50μL of acetonitrile to 1μM of VD3,

trometer have been reported before

25(OH)D3, and 1,25(OH)2D3 each.

16,17)

teins and peptides. This is the ﬁrst study to improve the

LC/EC/ESI-MS analysis

mass spectrometric detection sensitivity of VD3 metabolites

The LC/EC/ESI-MS analysis was performed by connec-

using an EC oxidation reaction. To detect EC oxidized
fat-soluble metabolites by ESI-MS, a coulometric cell capable of oxidizing almost all molecules passing through thetion to an EC detector (CoulArray Detector, Model 5600A,
Dionex) between the LC and ESI-MS. The schematic image

electrodes was used. Since porous graphite is used for theof this method is shown in Fig. 1. In order to separate thecell electrode, a highly reproducible oxidation reaction ispossible even at high ﬂow rates during the sample analy-

analytes, the LC conditions were described as follows: thesis . The sample separated by LC is oxidized by an electrochemical cell, then the oxidant is ionized by ESI and1.7μm, 2.1 mm I.D. × 100 mm, Waters, Milford, MA,
USA) at the ﬂow rate at 0.2 mL/min and column tempera-

detected by a mass spectrometer. This method not only
enables the highly-sensitive analysis of fatty and low polar-

ture of 40°
C. The LC was isocratic elution and the mobile
phase used 100 mmol/L formic acid and 5 mmol/L ammo-

ity molecules without any pretreatment such as derivatiza-

nium formate in 90% acetonitrile. The eluted analytes fromtion, but also the simultaneous analysis of polar molecules
as the normal ESI-MS is possible when the ECD voltage isthe column were oxidized by the EC. The applied voltage

turned oﬀ.

temperature and injection volume were 10°
C and 5μL,

column was an ACQUITY UPLC BEH C18 (particle size

13)

to the EC analytical cell was 1,300 mV. The autosampler
respectively. To prevent a high voltage aﬀect in the EC cell,

Materials and Methods

the grounding wire was connected between the ESI ion

Materials

source and EC cell. The oxidized analytes were ionized anddetected by a high-resolution mass spectrometer (Q Exac-

L-Menthol and Vitamin D3 was purchased from theTokyo Chemical Institute (Tokyo, Japan). 25-Hydroxy vita-

tive, Thermo Fisher Scientiﬁc, Waltham, MA, USA). The

min D3(calcidiol) and 1,25-dihydroxyvitamin D3(calcitriol)

MS conditions were as follows: ESI positive ionization,

were purchased from Toronto Research Chemicals (North

spray voltage; 3.5 kV, sheath gas ﬂow rate; 10 (arbitrary
units (au)), auxiliary gas ﬂow rate; 10 au, capillary tempera-

York, ON, Canada) and Cayman Chemical Company (Annture; 350°
C, heater temperature; 300°
C, s-lens RF level;
50%, resolution power; 70,000, and scan range; m/z 200‒

Arbor, MI, USA), respectively. LC/MS grade acetonitrile(MeCN; Optima LC/MS), formic acid (FA; Optima LC/
MS), and ammonium formate (Optima LC/MS) were pur-3,000. Mass accuracy of detecting ion peaks was within
59June 2020

Fig. 1.

Medical Mass Spectrometry Vol. 4 No. 1

The schematic image and photo of LC/EC/ESI-MS used in this study. An electrochemical reaction cell (EC cell) isconnected between the LC and MS ion source.

3 ppm. The MS/MS spectra (m/z 50‒500) were obtained bya data dependent acquisition program in the mass spectrom-

this reaction optimization used only the VD3 standard. Fig.
1S shows a plot of the detected peak height of the proton-eter. The normalized collision energy (NCE) and the resolu-

ated VD3 oxidant ion (m/z 383.3313) for each acetonitrile,tion power were 20% and 35,000, respectively. In order topreserve the mass accuracy, the mass spectrometer was cali-

formic acid, ammonium formate concentration and applied
the EC voltage conditions. For connection to a mass spec-

brated using a calibration solution containing n-butylamine,trometer, volatile ammonium formate was used as the salt
13)
additive . From the maximum peak intensity in each ﬁg-

caﬀeine, MRFA, and Ultramark 1621 before the analysis.

ure, the optimal values for each concentration in the mobile

Data analysis

phase were determined as follows: 90% acetonitrile,

Monitoring of the EC cell and control of the applied voltage was performed using the Coul Array Data Station Pro-

5 mmol/L ammonium formate, and 100 mmol/L formicgram (version 3.06, Dionex) software. The obtained LC/
EC/ESI-MS data were evaluated using Xcalibur software

the maximum intensity values when 1,300 mV or higher(Thermo Fisher Scientiﬁc). The Xcalibur analyzed theselected applied voltage of the EC cell was 1,300 mV inexact m/z values and the ion chromatograms of the VD3

this study.

acid. The oxidized VD3 ion peak at m/z 383.3313 showed
was applied in the EC cell. Based on these results, themetabolites and their oxidant ion peaks detected from the

LC/EC/ESI-MS analysis for VD3, 25(OH)D3, and1,25(OH)2D3

obtained data.

Results and Discussion

In order to conﬁrm the improved sensitivity of the VD3

In order to oxidize VD3, 25(OH)D3, and 1,25(OH)2D3 in

metabolites, each 1μmol/L of VD3, 25(OH)D3, and1,25(OH)2D3 was analyzed by the LC/EC/ESI-MS using

the EC analytical cell, the acetonitrile, formic acid, andthe optimized conditions. Figs. 2(a) and (b) show theammonium formate concentrations of the mobile phase andextracted ion chromatograms of the protonated VD3 ion at

the voltage applied to the EC cell were optimized. Sincem/z 385.3470 and VD3 oxidant ion at m/z 383.3313. When

25(OH)D3 and 1,25(OH)2D3 are both metabolites of VD3

no voltage was applied to the EC cell, only a peak at m/z
385.3470 was detected at 17.5 min. However, after a volt-

Optimization of EC oxidation conditions

and their basic chemical structures were almost the same,60June 2020

Fig. 2.

Medical Mass Spectrometry Vol. 4 No. 1

Extracted ion chromatograms of VD3 (m/z 385.34) and oxidized VD3 (m/z 383.33).

(a): No applied EC voltage, and (b): applied EC voltage (1,300 mV). The MS/MS fragment spectra of VD3 (c) and oxidized VD3(d). Each precursor ion is m/z 385.34 (c) and m/z 383.33 (d), respectively.

Fig. 3.

Extracted ion chromatograms of VD3 (a), 25(OH)D3 (b), and 1,25(OH)2D3 (c).

Each upper chromatogram is no applied EC voltage and the lower chromatogram is the applied EC voltage (1,300 mV). Obtained
calibration curves of VD3 (c), 25(OH)D3 (d), and 1,25(OH)2D3 (e). The solid lines show the calibration curves for the EC oxidants,
and the dotted lines show the unchanged forms.

61June 2020

Medical Mass Spectrometry Vol. 4 No. 1

385.3470 disappeared and a new ion peak at m/z 383.3313

times higher. These results suggested that the sensitivity of
the ESI-MS detection was improved by improving the pro-appeared at 17.5 min. The mass diﬀerence between the two

ton aﬃnity of VD3, and its metabolites were dehydroge-

peaks was 2.0157 Da, which was the same as the m/z value

nated by the EC

of exactly two hydrogen atoms being removed from the ion
at m/z 385.3470. Figs. 2(c) and (d) show the MS/MS frag-the calibration standard solutions (n＝5) with concentrations

ment spectra of both of these ions. The ion at m/z 385.3470

ranging from 0.5 to 200 nmol/L (0.5, 1, 2, 5, 10, 150, andhad the same fragment pattern as VD3. A similar fragment
pattern was obtained for the ion at m/z 383.3313. Based on

200 pmol/L) which were prepared by diluting the stock
solutions. Peak heights were used for making the calibra-

these results, it was found that the ion at m/z 383.3313 wastion curves. The relative standard deviations (RSD) of thean oxidized form of VD3.
Figs. 3(a)‒(c) show the ion chromatograms obtained bypeak heights at each concentration of VD3, 25(OH)D3, andanalyzing the VD3, 25(OH)D3 and 1,25(OH)2D3 standard
samples. When a voltage was applied to the EC cell, ion(LODs) were determined from the signal to noise ratio of 3
to 20. The calibration curves as shown in Figs. 3(d)‒(f)peaks of m/z 399.3262 and m/z 415.3211, which were theshowed that VD3 and its metabolite oxidants were goodoxidized forms of 25(OH)D3 and 1,25(OH)2D3, werewith R values of 0.9986 (oxidized VD3), 0.9908 (oxidizedage (1,300 mV) was applied to the EC cell, the peak at m/z

13,18). Each calibration curve of VD3,25(OH)D3, and 1,25(OH)2D3 was obtained by analysis of

1,25(OH)2D3 were all 10% or less. The limit of detections2

detected at 3.8 min and 4.5 min, respectively. Furthermore,

25(OH)D3 and 0.9903 (oxidized 1,25(OH)2D3). Their

25(OH)D3 and 1,25(OH)2D3 were similarly oxidized by EC

LODs were 1 nmol/L (oxidized VD3), 1 nmol/L (oxidized
25(OH)D3), and 20 nmol/L (oxidized 1,25(OH)2D3), respec-

to VD3 since almost no peaks at m/z 401.3405 and m/z
417.3362 were detected in the Fig. 3 chromatogram. Com-

tively. In the combination method of DAPTAD derivatiza-

paring the native forms of the detected ion peak intensities

tion and SRM, the detection sensitivity of 25(OH)D3 andwith the oxidized forms, all the oxidative VD3, 25(OH)D3,

1,25(OH)2D3 was about 30 times higher in this method

and 1,25(OH)2D3 ion peaks were detected at more than 10

The derivatization method is suitable for highly sensitive
analysis of VD3 and its metabolites, however cannot ana-

Table 1.

lyze other metabolites other than VD3 simultaneously. In

LOD values of VD3, 25(OH)D3, and 1,25(OH)2D3
by LC/EC/ESI-MS and other previous methods

VD3

25(OH)D3

1,25(OH)2D3

ESI

20

20

ND

EC/ESI

1

1
7)
8)
‒
2.8 3.820
7)8)
1.2 ‒3.8

APCI/tandem MS

a

6)

60

ity. Table 1 shows a comparison of the LOD values of the
VD3 metabolites obtained in this study with the general
method using the LC/APCI/tandem quadrupole MS and6‒8)selected reaction monitoring (SRM). It was clariﬁed thatthe sensitivity was dramatically improved by oxidizing
with the EC and ionizing with the ESI compared to the con-a: APCI data were obtained by QQQ-SRM. LOD values of VD3,25(OH)D3, and 1,25(OH)2D3 obtained by LC/EC/ESI-MS and6‒8)other previous methods .

Fig. 4.

.

order to achieve comprehensive metabolite analysis, it isnecessary to analyze intact metabolites with high sensitiv-

LOD (nmol/L)

Ionization
method9,10)ventional method. Furthermore, it was close to the LODs in

Extracted ion chromatograms of VD3, 25(OH)D3, and 1,25(OH)2D3 in the serum added samples.

Each upper chromatogram is no applied EC voltage and the lower chromatogram is the applied EC voltage (1,300 mV).

62

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Medical Mass Spectrometry Vol. 4 No. 1

the target analysis by SRM using APCI. This result indicates that metabolites other than VD3 can be simultane-nents.

ously detected with a detection sensitivity comparable to
the QQQ using SRM mode because a full scan measure-Acknowledgment

ment is performed in this study. Furthermore, this method

grant number 16K21276 (to H.M.).

enables simultaneous detection of various metabolites otherthan the targeted VD3-related metabolites. The blood vita-

Conﬂict of Interest

This work was supported by JSPS KAKENHI under

min D3 concentration is the range of 50 to 125 nmol/L for

The authors have no conﬂicts of interest directly relevant25(OH)D3, and the clinical test reference value for vitaminto the content of this article.

D deﬁciency is 30 nmol/L or less as serum concentration of2)
25(OH)D3 . The method developed in this study has suﬃ-

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