June 2021

Medical Mass Spectrometry Vol. 5 No. 1

Research Paper

Development of a method for quantiﬁcation ofserum dehydroepiandrosterone sulfate
by stable isotope-dilution MALDI-MS
1,2

1

3

4

Shoujiro Ogawa , Masahiro Nakamura , Mamoru Satoh , Takayuki Ishige ,5

61

Fumio Nomura , Yuki Sugiura , Tatsuya Higashi *Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278‒8510, Japan
2
Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University,
1 Sanzo, Gakuen-cho, Fukuyama-shi, Hiroshima 729‒0292, Japan
3
Division of Clinical Mass Spectrometry, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8677, Japan
4
Division of Laboratory Medicine, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8677, Japan
5
Division of Clinical Genetics, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8677, Japan
6
Department of Biochemistry, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160‒8582, Japan
1

Abstract

The quantiﬁcation of dehydroepiandrosterone sulfate (DHEAS) in the human circulation might be of diagnostic

help for several diseases, such as diabetes, depression, Alzheimer s disease, osteoporosis and chronic heart failure. Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) could be an eﬀective approach to quantify the serum DHEAS due to its high speciﬁcity, handleability, rapid analysis and ruggedness. Nonetheless, to the best of our knowledge, no MALDI-MS assay has been reported for the quantiﬁcation of DHEAS in biological samples. In this study, wedeveloped and validated a method to quantify DHEAS in human serum by MALDI-MS using a stable isotope-dilution tech2
nique with H4-DHEAS as an internal standard. This method enabled the reproducible quantiﬁcation of the serum DHEAS(intra- and inter-assay relative standard deviations, 9.3% or lower) with a small sample volume (20μL) and eﬀort-saving
pretreatment (only deproteinization and crystallization with 9-aminoacridine). The measurable range was 0.250‒5.00μg/
mL and the analysis time after the sample preparation was 3 min. The serum DHEAS concentrations measured by the newly-developed MALDI-MS method agreed well with those by liquid chromatography/electrospray ionization-MS/MS; thisresult demonstrated that the developed MALDI-MS method yielded reliable results.
Key words: matrix-assisted laser desorption/ionization-mass spectrometry, dehydroepiandrosterone sulfate, quantiﬁcation,
serum, stable-isotope dilution

Introduction

adrenal zona reticularis. More than 99% of DHEA is circu-

Dehydroepiandrosterone (DHEA) is an androgen synthe-

lating in the blood as the sulfated conjugate (DHEAS, Fig.

sized by the steroidogenic enzyme P450c17 within the1) and its serum/plasma concentration is reported to beabout several hundred nanograms‒several micrograms/mL* Corresponding author

1‒4)

in healthy adult males

Tatsuya Higashi. A number of studies demon-

strated that the decline in the circulating DHEAS levels is

Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba 278‒8510, Japan

5,6)

associated with several diseases, such as diabetes
7,8)

depression

Tel/Fax: ＋81‒4‒7121‒3660
E-mail: higashi@rs.tus.ac.jp

3), Alzheimer s disease , osteoporosis

9)

,

and

10)

chronic heart failure . Therefore, the determination of theserum/plasma DHEAS concentration might be of diagnos-

Received: November 26, 2020. Accepted: February 5, 2021.
Epub March 19, 2021.

tic help for these diseases. DHEAS has a long half-life (ca.

DOI: 10.24508/mms.2021.06.003

11 h) in blood and no signiﬁcant diurnal rhythm in its
22June 2021

Medical Mass Spectrometry Vol. 5 No. 1

method for this speciﬁc purpose though MALDI-MS has anumber of attractive properties, such as handleability andrapid analysis due to the absence of LC process, ruggedness(allowance for contamination of ion source), low consumption of organic solvent (environmentally preferred method)
as well as high speciﬁcity. A major cause for this is the lowquantitative capability of MALDI-MS due to the poor
reproducibility of the signal intensities. The stable isotope-dilution technique, which is widely used in LC/
ESI-MS/MS-based quantitative assays, would be useful tocorrect the ion abundance ﬂuctuation in MALDI-MS.
Recently, two MALDI-MS-based quantitative methods of
13,14), insteroids (including a secosteroid) were reported
which the stable isotope-dilution technique worked well forincreasing the quantitative accuracy.
Based on this background information, the objective of
this study was to develop a simple and rapid method for thequantiﬁcation of DHEAS in human serum by MALDI-MS.
The applicability of this method was evaluated by the analysis of serum samples of healthy subjects and by comparison to the conventional LC/ESI-MS/MS assay.

Fig. 1. MALDI-MS spectra of DHEAS and IS. (a) Standard DHEAS and IS (2.5 ng each/spot). (b) Serumsample spiked with IS; the DHEAS concentrationof this sample was subsequently determined to be
2.85 μg/mL.

Experimental
Materials and chemicals

2
DHEAS and [7,7,16,16‒ H4]-DHEAS (internal standard,

serum/plasma level unlike DHEA , which are advantages

IS, Fig. 1) were synthesized in our laboratories and the15)same as those used in a previous study . Standard solu-

of DHEAS over DHEA as the diagnostic marker.

tions of DHEAS (0.500, 1.00, 2.50, 5.00 and 10.0μg/mL)

11)

DHEAS has conventionally been measured in serum/

were prepared in ethanol. The IS was used as the ethanolic
solution of 5.00μg/mL. 9-Aminoacridine (9-AA) was pur-plasma by immunoassay in clinical practice. However,some of the commercially-available immunoassays forchased from Sigma-Aldrich Japan (Tokyo, Japan) and usedas the matrix. The saturated 9-AA solution (10 mg/mL) was

DHEAS have several limitations and drawbacks . Diﬀerent immunoassay kits sometimes provide diﬀerent mea12)

prepared in acetonitrile-water (1 : 1, v/v). All other reagents

sured values even for the same sample. Poor quality assur-

and solvents were of analytical grade or LC/MS grade.

ance of the standard solutions is considered a major causeof this discrepancy. Furthermore, there is a well-known

Serum samples

inaccuracy in the immunoassay due to cross-reaction ofSerum samples were collected from apparently healthy

antibodies with steroids that are structurally similar to
DHEAS. The mass spectrometry (MS)-based method has aadult male volunteers (n＝21) at the Chiba University Hos-

high speciﬁcity, therefore, it would be a promising alterna-

approved by the ethics committee of Chiba University (No.

tive to immunoassay for the DHEAS measurement. Many

640). Based on the approved experimental procedures, themethods using liquid chromatography (LC)/electrosprayionization (ESI)-tandem MS (MS/MS) have been devel-serum DHEAS concentrations were measured at Tokyo1‒4)

oped for the quantiﬁcation of DHEAS in serum/plasma

pital (Chiba, Japan). The experimental procedures were

University of Science. Written forms of informed consent
were obtained from all the volunteers. All the serum sam-.

ples were stored at −30°
C until used.

However, to the best of our knowledge, there is nomatrix-assisted laser desorption/ionization (MALDI)-MS
23

June 2021

Medical Mass Spectrometry Vol. 5 No. 1

MALDI-MS

LC/ESI-MS/MS method for quantiﬁcation of serum
DHEAS

MALDI-MS was performed using a Waters Synapt G1
quadrupole time-of-ﬂight (ToF) mass spectrometer (Milford,TM

To further evaluate the accuracy of the MALDI-MS

MA, USA). The ToF mass analyzer was operated in the negative-ion V mode. A Nd:YAG laser (wavelength, 355 nm andassay, the serum DHEAS concentrations measured by the
MALDI-MS method were compared with those by the LC/

energy, 350 arbitrary value) was employed at the ﬁring rate

ESI-MS/MS method.
A Waters LC-e2695 chromatograph and a Waters Quattroof 200 Hz. The laser was ﬁred for 180 s to obtain a mass
(m/z 367.21) and IS (m/z 371.23) were measured to quantify

Premier XE triple quadrupole-mass spectrometer wereused. The LC/ESI-MS/MS conditions were as follows: col-DHEAS. Masslynx software (version 4.1, Waters) was used

umn, J sphere ODS-H80 (4μm, 150×2.0 mm i.d., YMC,

for the system control and data processing.

Kyoto, Japan); column temperature, 40°
C; mobile phase,methanol-10 mM ammonium formate (4 : 3, v/v); ﬂow rate,

spectrum. The intensities of the ions derived from DHEAS

Pretreatment procedure

0.2 mL/min; capillary voltage, −2.8 kV; cone voltage,

The serum sample (20μL) was added to acetonitrile(200μL) containing IS (50.0 ng), vortex-mixed for 30 s,−50 V; collision energy, −35 eV; source temperature, 120°
C;then centrifuged at 2000×g for 10 min. The supernatant(200μL) was transferred to another test tube. After the sol-rate, 600 L/h; cone gas (N2) ﬂow rate, 50 L/h; and collisiongas (Ar) ﬂow rate, 0.19 mL/min and selected reaction moni-vent was evaporated, the residue was dissolved in the saturated 9-AA solution (20μL), 1.0μL of which was spotted[HSO4] (DHEAS) and m/z 371.2 → 96.8 (IS). Masslynx

onto the MALDI sample plate and allowed to dry (dried

software (version 4.1) was used for the system control anddesolvation temperature, 350°
C; desolvation gas (N2) ﬂow

toring (SRM) transitions, m/z 367.2 [M‒H] → 96.8
−

−

16)

droplet method ).

data processing.

Calibration curve

The serum sample (20μL) was pretreated in the sameway for the MALDI-MS method. The residue was dis-

DHEAS (5.00, 10.0, 25.0, 50.0 or 100 ng, corresponding tosolved in the mobile phase (100μL), 10μL of which was
subjected to the LC/ESI-MS/MS. DHEAS and IS eluted at

0.250, 0.500, 1.25, 2.50 or 5.00μg/mL, ﬁve points) weredissolved in the saturated 9-AA solution (20μL), 1.0μL of

5.9 and 5.8 min, respectively, under the stated conditions
and the LC/ESI-MS/MS run time per one sample waswhich was spotted onto the MALDI sample plate. The peak

7 min. The calibration samples were prepared by dissolving

intensity ratio (DHEAS/IS, y) was plotted versus theIS (50.0 ng) and a graduated amount of the standard

amount of DHEAS (ng per tube, x) with a weighting of 1/x

DHEAS (5.00, 10.0, 25.0, 50.0 or 100 ng, ﬁve points) in theto construct the calibration curve.

mobile phase (100μL). A portion (10μL) of these samples
was injected into the LC/ESI-MS/MS. The peak area ratio

Assay precision and accuracy(DHEAS/IS, y) was plotted versus the amount of DHEAS(ng per tube, x) with a weighting of 1/x to construct the cali-

repeated measurements of three pooled serum samples withbration curve.

diﬀerent concentrations on one day (n＝5) and over threedays, respectively. The precision was determined as the rel-Results and Discussion

IS (50.0 ng) and a graduated amount of the standard

The intra- and inter-assay precisions were assessed by theative standard deviation (RSD, %). The acceptance crite-

Selection of optimum mode for quantiﬁcation of
DHEAS

rion was met if the RSD values were ≤ 15% according to
the guidance from the US Food and Drug Administration .

17)

DHEAS (5.0 ng) was dissolved in the saturated 9-AA

The assay accuracy (%) was calculated by dividing thesolution, and 1/20 of the resulting sample (equivalent to
250 pg of DHEAS) was spotted onto the plate for the MAL-

mean measured concentrations by the theoretical concentrations for the DHEAS-spiked samples. The acceptance crite-

DI-MS analysis. Because DHEAS has a sulfuric acid group,
MALDI-MS was operated in the negative-ion mode, inrion was met if the calculated accuracy values were within17)
the range of 85‒115% .

−which the deprotonated molecule ([M‒H] ) was detected at
m/z 367.21 with a satisfactory intensity (signal-to-noise24June 2021

Medical Mass Spectrometry Vol. 5 No. 1

ratio of ca. 12). We also tested α -cyano-4-hydroxycinnamic
acid, 2,3-dihydroxybenzoic acid and sinapinic acid as theCalibration curve

MALDI matrices for the DHEAS quantiﬁcation. However,no characteristic ion was detected from 250 pg of DHEAS

drawing the calibration curve to correct the matrix eﬀect, ablank serum was not available. For this reason, the calibra-

when these matrices were used. Based on these results, weconcluded that 9-AA was the suitable matrix to sensitively

tion curve was constructed using a standard solution as
described in the experimental section. The MALDI-MS

quantify DHEAS by the MALDI-MS. By the collision-in−
duced dissociation (CID) of [M‒H] , only a sulfuric acid

spectrum of standard DHEAS and IS (2.5 ng each/spot; sec-

Although a blank (DHEAS-free) serum is preferable forond highest point of calibration curve) was shown in Fig.

−
group-derived product ion ([HSO4] ) was observed at m/z1(a). All the regression lines constructed three times
2

showed a good linearity (r ≥ 0.999) and the slopes were

96.88. The IS also produced the product ion at m/z 96.88−
from its [M‒H] (m/z 371.23) during the MS/MS. The SynG1 mass spectrometer could not individually detect

reproducible [0.0192±0.0002 (mean±SD, n＝3) and 1.12%(RSD)]. The y-intercept (0.0107±0.0037) was also close to

DHEAS and IS in their mixture by the MS/MS mode

zero. The applicability of this calibration curve was veriﬁed

because the product ions derived from these compounds

by the analytical recovery test, which will be described inwere the same. For this reason, in this study, the serum
DHEAS was quantiﬁed based on the intensity ratio of [M‒the next section.

H] of DHEAS and IS in the MS mode (without CID).

Assay precision and accuracy

TM

apt

−

As is obvious from Table 1, the proposed MALDI-MS
method was highly precise; the intra- (n＝5) and inter- (n＝Pretreatment of serum sample

Our method required only deproteinization and crystallization with 9-AA for the serum sample prior to the MAL-

3) assay RSDs did not exceed 9.3% and 3.2%, respectively,

DI-MS analysis. The MS spectrum obtained from a serum

Table 2 shows the accuracy (analytical recovery) of the
MALDI-MS method. The DHEAS concentrations of thefor the samples with the diﬀerent DHEAS concentrations.

sample is shown in Fig. 1(b), in which the ions derived
from DHEAS (m/z 367.21) and IS (m/z 371.23) were read-serum samples spiked with the standard DHEAS were
determined using the calibration curve described in the pre-ily identiﬁable. These ions were fully resolved from theinterfering ions derived from the 9-AA and serum matrix.
Table 1.

ceding section. As shown in Table 2, the accuracy values
Assay precision

Serum A

Serum B
a

a

RSD
(%)

Measured concentration(μg/mL)

RSD(%)

Measured concentration(μg/mL)

RSD(%)

3.56±0.33

9.3

1.91±0.04

2.1

0.85±0.02

2.4

3.67±0.08

2.2

1.85±0.06

3.2

0.85±0.01

1.2

Mean±SD.

Table 2.

Applicability of calibration curve and assay accuracy

Serum Da

Serum Ea

Serum Fa

Measured(μg/mL)

Accuracy(%)

Measured(μg/mL)

Accuracy(%)

Measured(μg/mL)

Accuracy
(%)

1.04

̶

1.06

̶

0.98

̶

Intactaa

Measured concentration(μg/mL)Intra-assay (n＝5)
Inter-assay (n＝3)
aSerum C

Spiked (＋0.25 μg/mL)

1.36

105.4

1.35

103.1

1.33

108.1

Spiked (＋0.50 μg/mL)

1.59

103.2

1.60

102.6

1.59

107.4

Spiked (＋1.25 μg/mL)

2.32

101.3

2.29

99.1

2.32

104.0

Spiked (＋2.50 μg/mL)

3.57

100.8

3.59

100.8

3.39

97.4

Mean measured concentration.

25

June 2021

Medical Mass Spectrometry Vol. 5 No. 1

ranged from 97.4 to 108.1%. This result demonstrated thatthe calibration curve using the standard solution was applicable for the quantiﬁcation of the serum DHEAS, andmoreover, the proposed MALDI-MS method was suﬃciently accurate. This method employed the stable isotope-dilution technique, which worked well in correcting
for the inﬂuence of the serum matrix.

Reliability of measured values by MALDI-MS
Because no chromatographic separation occurs during
the MALDI-MS analysis, the isomeric steroids are hardly
discriminated by the MALDI-MS, which is a drawback
13,14). Testosterone sulfate iscompared to LC/ESI-MS/MS

Fig. 2. Scatter diagram comparing the measured DHEAS
concentrations by MALDI-MS to those by
LC/ESI-MS/MS. Serum samples from apparently
healthy male subjects were analyzed (n＝21).

the isomer of DHEAS, but it was reported that its serum
concentration is too low to be detected by the LC/ESI-MS/
18)

MS . Furthermore, we could not ﬁnd any information in
the literature that an isomer other than testosterone sulfate
is present in human serum. Therefore, it is a good assump-

cation of DHEAS in human serum. This method had a satisfactory precision and provided reliable serum DHEAS con-tion that the eﬀects of the isomers on the measurement ofcentrations. To the best of our knowledge, this is the ﬁrst
validated and workable quantitative MALDI-MS assay forthe serum DHEAS are not usually signiﬁcant. The serum

the serum DHEAS. We believe that the developed MALDI-MS assay would become a method of choice for the

DHEAS concentrations of apparently healthy male subjects(n＝21) were determined to be 1.89μg/mL (mean) and0.85‒3.56μg/mL (range) [1.77μg/mL (median) and 1.56‒

DHEAS measurement in clinical laboratories.

1.91μg/mL (interquartile range)] by the MALDI-MS
method; these concentrations are in good agreement with1‒4)
the literature values . To make extra sure that the devel-

Acknowledgment

oped MALDI-MS method yields reliable measured values,
the serum DHEAS concentrations determined by the MAL-

Grant Number 17K08250 (to T. H.).

This study was supported in part by JSPS KAKENHI

Conﬂicts of Interest

DI-MS were compared to those by the LC/ESI-MS/MS. As

The authors declare no conﬂicts of interest.

shown in Fig. 2, a strong positive relationship was observed
between the measured values of the two methods (Pearson scorrelation coeﬃcient test, P＜0.01). Furthermore, the Wil-

References

coxon signed-rank test revealed that there was no signiﬁ-

1) Eisenhofer G, Peitzsch M, Kaden D, Langton K, Pampor-cant diﬀerence in the measured values between the two

aki C, et al: Reference intervals for plasma concentrations

methods (P＜0.05). These results demonstrated that the
MALDI-MS method provided accurate measured values.

of adrenal steroids measured by LC-MS/MS: Impact of

The time required for the MALDI-MS analysis per sam-

blood pressure status. Clin Chim Acta 470: 115‒124,

gender, age, oral contraceptives, body mass index andple was 3 min by our method and thus, the short analysis
time is an advantage of the MALDI-MS versus the conven-2017.
2) Damgaard-Olesen A, Johannsen TH, Holmboe SA, Søe-tional LC/ESI-MS/MS (7 min for one sample). Furthermore, MALDI-MS was easier to operate than LC/ESI-MS/

borg T, Petersen JH, et al: Reference ranges of 17-hy-MS.

tal and free testosterone determined by TurboFlow-droxyprogesterone, DHEA, DHEAS, androstenedione, toLC-MS/MS and associations to health markers in 304

Conclusionmen. Clin Chim Acta 454: 82‒88, 2016.

In this study, a stable isotope-dilution MALDI-MS
method was developed and validated for the rapid quantiﬁ-3) Cho SH, Jung BH, Lee WY, Chung BC: Rapid column-switching liquid chromatography/mass spectrometric26

June 2021

Medical Mass Spectrometry Vol. 5 No. 1

assay for DHEA-sulfate in the plasma of patients with

11) Legrain S, Massien C, Lahlou N, Roger M, Debuire B, etAlzheimer s disease. Biomed Chromatogr 20: 1093‒1097,

al: Dehydroepiandrosterone replacement administration:

2006.

Pharmacokinetic and pharmacodynamic studies in healthy4) Shackleton CHL, Kletke C, Wudy S, Pratt JH: Dehydroe-elderly subjects. J Clin Endocrinol Metab 85: 3208‒3217,

piandrosterone sulfate quantiﬁcation in serum using

2000.

high-performance liquid chromatography/mass spectrom-

12) Büttler RM, Kruit A, Blankenstein MA, Heijboer AC:

etry and a deuterated internal standard: a technique suit-

Measurement

able for routine use or as a reference method. Steroids 55:(DHEAS): A comparison of isotope-dilution liquid chro-472‒478, 1990.

matography tandem mass spectrometry (ID-LC-MS/MS)

5) Liu L, Wang M, Yang X, Bi M, Na L, et al: Fasting serumofdehydroepiandrosterone

sulphateand seven currently available immunoassays. Clin Chim

lipid and dehydroepiandrosterone sulfate as important me-

Acta 424: 22‒26, 2013.

tabolites for detecting isolated postchallenge diabetes: se-13) Qi Y, Müller M, Stokes CS, Volmer DA: Rapid quantiﬁca-

rum metabolomics via ultra-high-performance LC-MS.

tion of 25-hydroxyvitamin D3 in human serum by ma-

trix-assisted laser desorption/ionization mass spectrome-

Clin Chem 59: 1338‒1348, 2013.

try. J Am Soc Mass Spectrom 29: 1456‒1462, 2018.

6) Tagawa N, Ohta M, Nakamura N, Nakano K, Obayashi
H, et al: Serum concentrations of Δ ‒3 β -hydroxysteroids14) Nakamura M, Moritsuna M, Yuda K, Fujimura S, Sugiurain type 2 diabetes mellitus. Biol Pharm Bull 25: 1634‒

Y, et al: Quantitative MALDI-MS/MS assay for serum cor-

1638, 2002.

tisol through charged derivatization. J Pharm Biomed Anal

5

178: 112912, 2020. doi: 10.1016/j.jpba.2019.112912.

7) Zhu G, Yin Y, Xiao CL, Mao RJ, Shi BH, et al: Serum

15) Higashi T, Shibayama Y, Kawasaki I, Shimada K: Deter-

DHEAS levels are associated with the development of de-mination of salivary 17-ketosteroid sulfates using liquidpression. Psychiatry Res 229: 447‒453, 2015.

chromatography-electrospray ionization-mass spectrome-8) Michikawa T, Nishiwaki Y, Nakano M, Iwasawa S, Yama-try. J Pharm Biomed Anal 43: 1782‒1788, 2007.

da M, et al: Higher serum dehydroepiandrosterone sulfate
levels are protectively associated with depressive symp-16) Karas M, Hillenkamp F: Laser desorption ionization oftoms in men, but not in women: A community-based co-proteins with molecular masses exceeding 10,000 daltons.

hort study of older Japanese. Am J Geriatr Psychiatry 21:

Anal Chem 60: 2299‒2301, 1988.
17) US Food and Drug Administration: Guidance for indus-

1154‒1163, 2013.
9) Lee D, Kim H, Ahn SH, Lee SH, Bae SJ, et al: The asso-

try: bioanalytical method validation. https://www.fda.gov/

ciation between serum dehydroepiandrosterone sulphatefiles/drugs/published/Bioanalytical -Method-Validation-(DHEA-S) level and bone mineral density in Korean men.

Guidance-for-Industry.pdf#search=%27FDA+Bioanalysis

Clin Endocrinol 83: 173‒179, 2015.

+Guideline%27 Accessed 29th October, 2020.

10) Moriyama Y, Yasue H, Yoshimura M, Mizuno Y, Nishiya-18) Sánchez-Guijo A, Oji V, Hartmann MF, Traupe H, Wudy

ma K, et al: The plasma levels of dehydroepiandrosterone

SA: Simultaneous quantiﬁcation of cholesterol sulfate,sulfate are decreased in patients with chronic heart failure

androgen sulfates, and progestagen sulfates in human se-in proportion to the severity. J Clin Endocrinol Metab 85:

rum by LC-MS/MS. J Lipid Res 56: 1843‒1851, 2015.

1834‒1840, 2000.

27