June 2023

Medical Mass Spectrometry Vol. 7 No. 1

Research Paper

Comparative study of stable isotope-labeled internal standard substances
for the LC-MS/MS determination of the urinary excretion of melatonin
Hayato Shimazaki, Shingo Ogawa, Hiroki Takano, Seiya Ohki, Momoka Fukae,
Tomomi Furihata, Hiromi Shibasaki, Akitomo Yokokawa*Laboratory of Clinical Pharmacy and Experimental Therapeutics, School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences, 1432‒1 Horinouchi, Hachioji, Tokyo 192‒0392, Japan
Melatonin (MEL) is mostly metabolized by cytochrome P450 1A2 (CYP1A2) to 6-hydroxylated melatonin

Abstract(6-O-MEL) and then excreted in urine as a sulfate conjugate (6-S-MEL) or glucuronide conjugate. Therefore, the MEL
secretion and CYP1A2 activity can be evaluated on the basis of the total excreted amount of 6-O-MEL, which can be obtained via deconjugation. Two internal standard methods can be used for the quantiﬁcation of the total excreted amountof 6-O-MEL via liquid chromatography/tandem mass spectrometry: adding stable isotope-labeled 6-O-MEL (6-O-MEL2
H4) after deconjugation or adding stable isotope-labeled 6-S-MEL (6-S-MEL- H4) before deconjugation. This study com-2pares these two internal standard methods. Adding 6-O-MEL- H4 after deconjugation increased the deconjugation rate of2

6-O-MEL with increasing the amount of deconjugating enzyme, of which 5920 units (400 μL) were required to reach a2plateau. In contrast, when adding 6-S-MEL- H4 before deconjugation, the deconjugation rate of 6-O-MEL did not change

upon increasing the deconjugating enzyme amount from 1480 to 5920 units; therefore, 1480 units (100 μL) of deconjugat-

ing enzyme were suﬃcient for the determination. Urine obtained from healthy subjects (n＝18, 0.07‒9.00 μg) was measured
using both internal standards, yielding a linear correlation coeﬃcient of 0.9976 in their relationship. The method of adding
6-S-MEL- H4 before deconjugation can automatically correct the quantitative value of the deconjugation rate; therefore,2

only a small amount of deconjugating enzyme is required, providing a cost-eﬀective method for the quantiﬁcation of thetotal excreted amount of 6-O-MEL in urine.
Key words: deconjugating enzyme, melatonin, LC-MS/MS, stable isotope, internal standards

Introduction

secretion increasing at night to promote sleep and decreasing during the day. This secretion process plays an import-

Melatonin (MEL), an endogenous hormone secreted by
the pineal gland, is involved in sleep regulation and exhib-

ant role in maintaining circadian rhythms. In fact, it has

its antioxidant, anti-inﬂammatory, and immunomodulatory

been suggested that disruption of the MEL secretion. MEL secretion depends on light exposure, withrhythms may lead to sleep disorders, cognitive decline, and1‒3)

eﬀects4‒6)

cardiac disease*Corresponding author.

MEL is mainly metabolized to 6-hydroxymelatonin (6-O-

Akitomo Yokokawa

MEL) by cytochrome P450 1A2 (CYP1A2) in the liver afterLaboratory of Clinical Pharmacy and Experimental Therapeutics,

secretion and then excreted in urine as the sulfate conjugate
6-sulfatoxy melatonin (6-S-MEL) and the glucuronate conju-

School of Pharmacy, Tokyo University of Pharmacy and Life
Sciences, 1432‒1 Horinouchi, Hachioji, Tokyo 192‒0392, Japan7)
gate 6-hydroxymelatonin glucuronide (6-G-MEL) in respec8,9)
tive percentages of 70‒90% and 10‒30% . Since the total

Tel: ＋81‒42‒676‒5803, Fax: ＋81‒42‒676‒5805
E-mail: yokokawa@toyaku.ac.jp

excreted amount of 6-O-MEL in urine reﬂects the amount

Received: January 10, 2023. Accepted: February 22, 2023.

of MEL secreted, MEL secretion could be evaluated by
10)
quantifying the excreted amount of 6-O-MEL . Moreover,

Epub March 29, 2023.
DOI: 10.24508/mms.2023.06.004
ORCID: 0000-0002-6039-2953

the measurement of the total excreted amount of 6-O-MEL
53

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Medical Mass Spectrometry Vol. 7 No. 1

could be used to evaluate the activity of CYP1A2, which
11,12). Thereshows large inter- and intraindividual variability

add excess deconjugating enzyme. Furthermore, adding
2
6-S-MEL- H4 before deconjugation would allow to performfore, the development of an accurate and simple method tomeasure the total excreted amount of 6-O-MEL is highly

the quantiﬁcation even for deconjugation reaction below

desirable.

the amount of enzyme could be reduced. In this study, we2
aimed to clarify whether the addition of 6-O-MEL- H4 or

100% because the internal standard is also a conjugate, andAlthough 6-S-MEL can be measured directly via liquid
chromatography/tandem mass spectrometry (LC-MS/MS)

6-S-MEL- H4 as an internal standard would aﬀord equiva2

as an indicator of the total excreted amount of 6-O-MEL ,this method is not accurate because 6-G-MEL cannot belent results and to reduce the amount of enzyme used in thedetermination of the total excreted amount of 6-O-MEL.

quantiﬁed. In contrast, hydrolysis of 6-S-MEL and 6-GMEL in urine to 6-O-MEL via deconjugation reaction

Materials and Methods

13)

Chemicals and materials

enables the measurement of all conjugates and, in turn, theaccurate determination of the total excreted amount of 6-O-

N - [2 - (6 - Hydroxy - 5 - methoxy - 1H - indol - 3 - yl)ethyl]acet amide (6-O-MEL), N-[2-[5-methoxy-6-(sulfooxy)-1H-indol-

MEL in urine via gas chromatography‒mass spectrometry
14‒17). Although both analytical techniquesor LC-MS/MS

3-yl]ethyl]acetamide sodium (6-S-MEL), 6-O-MEL- H4, and2
6-S-MEL- H4 were purchased from Toronto Research2

can detect small amounts of 6-O-MEL, LC-MS/MS is par-Chemicals (Toronto, ON, Canada). The deconjugating
enzyme, β-glucuronidase/arylsulfatase from Helix pomatia

ticularly useful because it has good sensitivity and does not
16,17). Furthermore, the use of a stable
require derivatization
isotope-labeled compound as an internal standard increases(Roche-Glu/Sul), was purchased from Roche Diagnostics
GmbH (Mannheim, Germany). All other reagents were pur-the accuracy of the measurements by automatically compensating the losses due to preprocessing.
The internal standard used in LC-MS/MS measurementschased from Kanto Chemical (Tokyo, Japan).

based on a deconjugation reaction is a stable isotope-

Preparation of standards

Standard solutions of 6-O-MEL and 6-O-MEL- H4 were pre2labeled deconjugated substance, which is added after
deconjugation, or a stable isotope-labeled conjugated sub-pared in methanol at concentrations of 24.5 and 67.0 ng/mL,
respectively. 6-S-MEL and 6-S-MEL- H4 were prepared inmethanol at concentrations of 86.0 and 78.0 ng/mL, respec2

stance added before deconjugation. Generally, the former
method is commonly used

16,17)

because both sulfate andglucuronate conjugates can be applied. Meanwhile, theaddition of a stable isotope-labeled conjugated substancetively.

before deconjugation allows to automatically correct the

Deconjugation reaction

measured value even if the deconjugation reaction is not
100% because the conjugated substance is used as the inter-

Urine samples were thawed and centrifuged at 3000 rpm
for 15 min, and the supernatant (0.2‒1 mL) was aliquoted.

nal standard. In the case of MEL, two methods can be used:2
adding the stable isotope-labeled 6-O-MEL (6-O-MEL- H4)

Then, distilled water was added to reach a volume of 1 mL,
and 1 mL of 0.5 M acetate buﬀer (pH 4.0) was added. If
2
6-S-MEL- H4 was used as the internal standard, it was added

after deconjugation or adding the stable isotope-labeled 6-S2
MEL (6-S-MEL- H4) before deconjugation. However, it isstill unclear which of the two approaches would be betterat this point. For the deconjugation reaction, 74‒7400 units ofRoche-Glu/Sul were added as the deconjugating enzyme, andsuited for the accurate determination of the total excreted
amount of 6-O-MEL.

incubation was performed at 37°
C for 60 min. After incubation, the reaction was stopped by rapid cooling under ice-

Motivated by this background, we recently developed acold conditions. If 6-O-MEL- H4 was used as the internal
2

standard, it was added at this point (Fig. 1).

method for the measurement of the total excreted amount2of 6-O-MEL based on the addition of 6-O-MEL- H4 after

deconjugation . 6-O-MEL- H4 can be used as an internal
17)

2

Extraction and sample preparation

standard regardless of the type of conjugate because it isadded after deconjugation. However, since the deconjuga-

Extraction from the urine sample was performed by solid phase extraction using Oasis HLB cartridges (Waters,

tion rate aﬀects the quantitative value, it was necessary to

Milforld, MA, USA). After deconjugation, the urine sample
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Medical Mass Spectrometry Vol. 7 No. 1

Fig. 1.

Urine sample preparation procedure.

The order of addition of 6-O-MEL- H4 and 6-S-MEL- H4 is shown.
22

2
6-O-MEL- H4 were monitored at m/z 249.1 → 190.1 and m/z

was injected into an Oasis HLB cartridge (1 cc Vac Cartridge, 30 mg sorbent) pre-equilibrated with methanol

253.1 → 193.1 with a collision energy of 12 eV.

(0.5 mL) and distilled water (0.5 mL). The samples were

Partial validation of the method

then washed with 5% methanol solution (0.5 mL) and

The calibration curve was prepared using ﬁve concentrations of 6-S-MEL (1.72, 2.58, 4.30, 8.60, and 17.20 ng),

eluted with methanol (300μL). After extraction, 10 mM
ammonium acetate/0.2% formic acid solution (130μL) was
added, and then 5μL was injected into the LC-MS/MS sys-

adding 3.90 ng of 6-S-MEL- H4 as the internal standard.
After adding the standard solution to 1 mL of water, decon2

tem.

jugation reaction was performed to obtain 6-O-MEL for

LC-MS/MS conditions

measurement. This calibration curve was repeated six times
to conﬁrm linearity. The accuracy and precision were evalu-

LC-MS/MS

analyses were performed using an
ACQUITY UPLC H-class and Xevo TQD triple quadrupole
mass spectrometer (Waters, Milford, MA, USA). Chro-ated using stored urine from healthy subjects. In the method2adding 6-S-MEL- H4 as the internal standard, 15 urine sam-matographic

anples were divided into three groups. One group (n＝5) was

ACQUITY UPLC BEH C18 column (2.1 mm×100 mm,
1.7 μm) and an ACQUITY UPLC BEH C18 VanGuard pre-not spiked, one group (n＝5) was spiked with 13.01 ng/mL
of 6-S-MEL, and the other group (n＝5) was spiked withcolumn (2.1 mm×5 mm, 1.7 μm). The sample manager tem-

26.01 ng/mL of 6-S-MEL. Each of these samples was mea-

perature was 15°
C, the column temperature was 35°
C, andsured after 1 day for intraday accuracy and precision andthe ﬂow rate was 0.2 mL/min. The mobile phase was
10 mM ammonium acetate solution/0.2% formic acid solu-

after 6 days for interday accuracy and precision.

tion and methanol. The gradient was set to 10% (0 min),
90% (10‒13 min), and 10% (13.1‒18 min) of methanol.

Urine sample collection for comparison of the two internal standard methods

separations

wereconducted

withand the sauce temperature was set at 500°
C. Positive ion

To compare the two internal standard methods, 2-h urine
samples (10:00‒12:00) from nine subjects (22‒41 years,

mode of multiple reaction monitoring was selected forquantiﬁcation. Precursor and product ions of 6-O-MEL and

one female and eight males) and 8-h urine samples (6:00‒
14:00, 14:00‒22:00, 22:00‒6:00) from three subjects (22‒Electrospray ionization was used as the ionization method,55

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Medical Mass Spectrometry Vol. 7 No. 1

46 years, three males) were used. After collection, the urine
volume was measured and the samples were stored at −20°C

drugs after deconjugation reactions. This suggests that if
the deconjugation of 6-S-MEL to 6-O-MEL proceeds, theuntil measurement. This study was approved by the Human
Subjects Review Board of the Tokyo University of Phar-deconjugation of 6-G-MEL to 6-O-MEL also progresses
simultaneously. Therefore, the stable isotope-labeled com-macy and Life Sciences (approval number: 18-02), andinformed consent was obtained from all subjects.

2
pound 6-S-MEL- H4 is suitable as an internal standard. If
β -glucuronidase/arylsulfatase is present in insuﬃcient

Results and Discussion

quantity, the deconjugation reactions of the glucuronate andsulfate conjugates do not proceed. Therefore, the concentra-

LC-MS/MS method developmenttion of glucuronide conjugates also can be calculated byusing an internal standard for sulfate conjugates and cor-

In the measurement of total urinary excretion by LC-MS/
MS, a stable isotope-labeled deconjugated substance is gen16,17)

erally added after deconjugation

recting for the deconjugation-reaction eﬃciency. In this2
study, the use of 6-S-MEL- H4 as an internal standard for. Since the quantitativevalue may be lower than the true value if the deconjugating

LC-MS/MS determination based on the deconjugation reac-

reaction does not proceed to 100%, an excess amount of
enzyme is normally required to ensure that the deconjugat-tion was investigated and compared with a previously2
reported method using 6-O-MEL- H4 as an internal stan-ing reaction reaches completion. Meanwhile, when a stable
isotope-labeled conjugated substance is added before

dard

deconjugation, the quantitative value is corrected by theinternal standard. Therefore, the measurement can be suﬃ-

Optimization of the deconjugation reaction

17)(Fig. 1).

ciently accurate even though the deconjugation reaction isFirst, the amount of deconjugating enzyme Roche-Glu/2
2
Sul required when using 6-O-MEL- H4 and 6-S-MEL- H4 as

not 100% and the amount of enzyme is not enough for theinternal standards was evaluated. The temperature and time

deconjugation reaction.
In the case of 6-O-MEL, the stable isotope-labeled com-of the reaction followed our previously reported method .

17)

Enzyme additions that maximized the deconjugation rate of
6-O-MEL were examined in the range of 74‒7400 units

pounds of both conjugates excreted in urine, i.e., the sulfate
conjugate 6-S-MEL and the glucuronic acid conjugate 6-G-

using 8-h urine samples from three healthy subjects. In the2method of adding 6-O-MEL- H4 after deconjugation, the7)

MEL , are potential candidates as the internal standard. In
general, the reactivity of the sulfate conjugate toward
deconjugating enzymes is lower than that of the glucuron-deconjugation rate of 6-O-MEL reached a plateau whenide conjugate. For example, Azuma et al. reported that the

Furthermore, the lowest standard deviation (S.D.) value of

glucuronide conjugate of acetaminophen is more readily

6.53% was achieved for this amount of enzyme, which was

deconjugated than the corresponding sulfate conjugate
18)
during the reaction using β -glucuronidase/arylsulfatase .

set as the required enzyme amount for the reaction (Fig. 2(a)).

5920 units (400μL) of deconjugating enzyme were added.

17)This result is consistent with our previous report . In the2case of the method of adding 6-S-MEL- H4 before deconju-

Furthermore, Azuma et al. quantiﬁed the conjugates of 19

Fig. 2.

Comparison of the enzyme addition amount.

(a) 6-O-MEL- H4 was used as an internal standard and (b) 6-S-MEL- H4 was used as an internal standard.
2

2

56

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Medical Mass Spectrometry Vol. 7 No. 1

gation, the deconjugation rate of 6-O-MEL reached a pla-

MS analysis of the deconjugation reaction with the 1480
2
units of 6-S-MEL- H4 aﬀorded single and sharp peaks ofteau at 93.77% when 1480 units (100 μL) were added (Fig.

6-O-MEL and 6-O-MEL- H4 in the urine sample at a retention time of 5.6 min, and no inﬂuence of endogenous sub22(b)). The S.D. was 8.49% at 1480 units (100 μL) and19.86% at 740 units (50 μL), indicating that the necessaryamount of enzyme was 1480 units (100 μL). The LC-MS/

stances was observed (Fig. 3).

Partial validation of the method

To verify whether the method of adding 6-S-MEL- H4
2

before deconjugation was comparable to that of adding
2
6-O-MEL- H4 after deconjugation, partial validation of theformer was performed by constructing a calibration curveand conﬁrming the accuracy and precision.
The calibration curve yielded a correlation coeﬃcient of
0.99949±0.00066, a slope of 0.6721±0.0258, and an intercept of −0.031±0.087 (Table 1). These values were comparable to those previously reported for the method of adding
2
6-O-MEL- H4 after deconjugation (correlation coeﬃcient:

0.99971±0.00020, slope: 0.437±0.023, intercept: −0.012±
17)

0.027) , conﬁrming that calibration curves with intercepts
close to zero and high linearity can be obtained for bothmethods.
The accuracy and precision of the method of adding
2
6-S-MEL- H4 before deconjugation were evaluated by preparing urine samples at three diﬀerent concentrations. As aresult, the relative error (R.E.) ranged from −5.92% to3.01% and the relative standard deviation (R.S.D.) was
within 9.78% (Table 2), which were in accord with thoseFig. 3.

LC-MS/MS chromatograms of 6-O-MEL and2
2
6-O-MEL- H4 in urine samples and 6-O-MEL- H4
in the standard sample.

Table 1.

(a) Chromatogram of 6-O-MEL in the urine sample obtained after deconjugation reaction, (b) chromatogram of
2
6-O-MEL- H4 in the urine sample obtained after adding
2
6-S-MEL- H4 and performing deconjugation reaction,2and (c) chromatogram of 6-O-MEL- H4 in the standard
2
sample obtained after deconjugating 6-S-MEL- H4.

Table 2.

Linearity of the calibration curve of the method2of adding 6-S-MEL- H4 as the internal standard
before deconjugation

Concentration(ng)
1.30‒13.01

Correlationcoeﬃcient(Mean±S.D.)

Slope(Mean±S.D.)

Intercept(Mean±S.D.)

0.99949±0.00066 0.6721±0.0258 −0.031±0.087

S.D.: Standard deviation.

2
Accuracy and precision in the method adding 6-S-MEL- H4 as the internal standard before deconjugation

Added(ng/mL)

Expected(ng/mL)Found(Mean±S.D., ng/mL)

R.E.
(%)

R.S.D.
(%)

11.93±0.32

̶

2.68

Intraday

0

(n＝5)

13.01

24.94

25.69±1.06

3.01

4.11

26.01

37.95

37.66±1.00

−0.76

2.65

12.21±1.20

̶

9.78

Interday

0

(n＝6)

13.01

25.22

24.54±1.67

−2.69

6.79

26.01

38.22

35.96±2.29

−5.92

6.36

S.D.: Standard deviation, R.E.: relative error, R.S.D.: relative standard deviation.

57

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Medical Mass Spectrometry Vol. 7 No. 1

cost-eﬀective method because it allows reducing the2corresponding to the method of adding 6-O-MEL- H4 after

deconjugation (R.E.: −3.60% to −0.47%, R.S.D. withinamount of deconjugating enzyme, providing a new

17)

6.80%) . Therefore, the method could be performed withapproach for the evaluation of MEL secretion and CYP1A2

high accuracy and precision using either of the internal

activity in the future.

standards.

Acknowledgments

Comparison of the two internal standard methods

This research did not receive any speciﬁc grant fromfunding agencies in the public, commercial, or not-for-proﬁt

Finally, the feasibility of using both methods, i.e., adding
2
2
6 O-MEL- H4 after deconjugation and adding 6-S-MEL- H4

sectors.

before deconjugation, to analyze urine samples was examined using 2-h urine samples from nine healthy subjects andConﬂict of Interest

8-h urine samples from three healthy subjects (n＝18). The
excreted amount of 6-O-MEL measured using each method

The authors declare that they have no known competing
ﬁnancial interests or personal relationships that could haveranged from 0.07 to 9.00 μg; the correlation coeﬃcient ofappeared to inﬂuence the work reported in this paper.

the graph obtained by plotting the measured values of bothmethods was 0.9976. The obtained equation has a correla-

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