Biochemical and Biophysical Research Communications xxx (2017) 1e5

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Biochemical and Biophysical Research Communications

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Analysis of serotonin concentrations in human milk by high-

performance liquid chromatography with fluorescence detection
Takeshi Chiba a, *, Tomoji Maeda b, Tomohiko Tairabune a, Takashi Tomita a,
Atsushi Sanbe c, Rika Takeda d, Akihiko Kikuchi e, Kenzo Kudo a
a
Department of Clinical Pharmaceutics and Pharmacy Practice, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba-cho, Shiwa-gun,
Iwate 028-3603, Japan
b
Department of Pharmacology, Nihon Pharmaceutical University, 10281 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0862, Japan
c
Department of Pharmacotherapeutics, School of Pharmacy, Iwate Medical University, 2-1-1, Nishitokuta, Yahaba-cho, Shiwa-gun, Iwate 028-3603, Japan
d
Department of Nursing, Iwate Medical University Hospital, 19-1, Uchimaru, Morioka, Iwate 020-8505, Japan
e
Department of Obstetrics and Gynecology, School of Medicine, Iwate Medical University, 19-1, Uchimaru, Morioka, Iwate 020-8505, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in milk volume homeostasis in the
Received 26 January 2017 mammary gland during lactation; 5-HT in milk may also affect infant development. However, there are
Accepted 6 February 2017 few reports on 5-HT concentrations in human breast milk. To address this issue, we developed a simple
Available online xxx
method based on high-performance liquid chromatography with fluorescence detection (HPLC-FD) for
measuring 5-HT concentrations in human breast milk. Breast milk samples were provided by four
Keywords:
healthy Japanese women. Calibration curves for 5-HT in each sample were prepared with the standard
Serotonin
addition method between 5 and 1000 ng/ml, and all had correlation coefficients >0.999. The recovery of
Human breast milk
High performance liquid chromatography
5-HT was 96.1%e101.0%, with a coefficient of variation of 3.39%e8.62%. The range of 5-HT concentrations
Fluorescence detection estimated from the calibration curves was 11.1e51.1 ng/ml. Thus, the HPLC-FD method described here
can effectively extract 5-HT from human breast milk with high reproducibility.
© 2017 Elsevier Inc. All rights reserved.

1. Introduction 5-Hydroxytryptamine (5-HT), also known as serotonin, is a

Breastfeeding benefits infants in numerous ways [1e4], since
human breast milk not only contains normal nutrients such as
carbohydrates, fats, proteins, minerals, and vitamins, but also bio-
logically active compounds including antimicrobial substances,
growth factors, cytokines, immunoglobulins, and specific types of
immune cell [5]. Recent studies have shown that the nuclear
receptor-type transcription factor peroxisome proliferator-
activated receptor a which is known to be activated by lipid li-
gands, which is supplied by breast milk during the suckling period,
regulates fatty acid b-oxidation genes in the postnatal liver [6], and
that breastfeeding protects against sudden infant death syndrome
(SIDS), especially when practiced exclusively [7].
* Corresponding author.
E-mail addresses: chibat@iwate-med.ac.jp (T. Chiba), t-maeda@nichiyaku.ac.jp
(T. Maeda), tomohiko.tairabune@j.iwate-med.ac.jp (T. Tairabune), tatomita@iwate-
med.ac.jp (T. Tomita), asanbe@iwate-med.ac.jp (A. Sanbe), rika.takeda@j.iwate-
med.ac.jp (R. Takeda), akikuchi@iwate-med.ac.jp (A. Kikuchi), kenzkudo@iwate-
med.ac.jp (K. Kudo).
biogenic amine present in a variety of organs such as the brain,
lungs, gastrointestinal tract, liver, kidneys, and mammary glands. It
is thought to contribute to the regulation of central and peripheral
functions including sleep, mood, vascular contraction, platelet ag-
gregation, and gastrointestinal motility [8e12]. Epithelial cells in
mammary glands secrete 5-HT into milk [13], thereby controlling
milk volume homeostasis [14]. However, whether this 5-HT affects
infant development is unclear. In fact, there are few reports on 5-HT
concentrations in human breast milk [15], although this informa-
tion is important for determining 5-HT intake by newborns and
infants via breastfeeding and evaluating its effects on infant
development.
5-HT contains an indole skeleton and is intrinsically fluo-
rescent, with maximum excitation and emission wavelengths of
approximately 280 and 340 nm, respectively [10]. In the present
study, we developed a high-performance liquid chromatography
with fluorescence detection (HPLC-FD) method that exploits this
property for efficient extraction of 5-HT from human breast
milk.

http://dx.doi.org/10.1016/j.bbrc.2017.02.027
0006-291X/© 2017 Elsevier Inc. All rights reserved.

Please cite this article in press as: T. Chiba, et al., Analysis of serotonin concentrations in human milk by high-performance liquid
chromatography with fluorescence detection, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2017.02.027
2 T. Chiba et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5
2. Materials and methods
2.1. Chemicals and reagents
5-HT (molecular weight: 212.68), procaine hydrochloride (used
as an internal standard [IS]), and 1-octanesulfonic acid sodium
(used for ion-pair chromatography) were purchased from Wako
Pure Chemical Industries (Osaka, Japan). HPLC-grade methanol was
obtained from Kanto Chemical Co. (Tokyo, Japan). All other reagents
were of analytical grade.
2.2. Healthy volunteers and breast milk sample collection
Breast milk samples were provided by four healthy Japanese
women who were breastfeeding (n ¼ 3) or had recently weaned
(n ¼ 1); each subject provided written, informed consent. Of the
four women, one was primiparous and the others were multipa-
rous (Table 1). None of the subjects were taking any medications or
supplements. A total of 5e10 ml of breast milk was collected by
each subject at home in a 50-ml centrifuge tube and stored in a
freezer (20  C). The samples were mailed to our laboratory
at 20  C and immediately stored at 80  C until analysis. This
study protocol was reviewed and approved by the Iwate Medical
University Ethics Committee.
2.3. HPLC analysis
HPLC was carried out using a Prominence chromatograph
(Shimadzu, Kyoto, Japan) equipped with an LC-20AD pump, SIL-
20A autosampler, RF-10AXL fluorescence detector (excitation:
280 nm, emission: 340 nm), CBM-20A controller, and CTO-20A
column oven. A Shim-pack FC-ODS C18 column (5 mm,
150  4.6 nm internal diameter; Shimadzu) was used for reserve-
phase HPLC. A mixture (76:24) of sodium acetate buffer (pH 4.7;
0.15 mM) containing 1 mM 1-octansulfonic acid sodium and
methanol was used as an isocratic mobile phase. The mobile phase
flow rate was 1.2 ml/min. Procaine hydrochloride was used as the
IS. The analysis was performed in triplicate, and data are expressed
as the mean ± standard deviation of three measurements.
2.4. Standard addition method (SAM) for determination of 5-HT
concentration in human breast milk
5-HT was extracted from human breast milk (Fig. 1) and the
concentration was determined by the SAM. Briefly, standard 5-HT
aqueous solution was added to 300 ml of milk sample to final
concentrations of 6, 12, 60, 120, 240, 600, and 1200 ng/ml. The
spiked milk samples (400 ml total volume) were deproteinized by
mixing with 200 ml of 10% trifluoroacetic acid, vortexed, and
centrifuged at 15,000 rpm for 10 min at 4  C. A 500-ml volume of
supernatant containing 5-HT standard solution at concentrations of
5, 10, 50, 100, 200, 500, and 1000 ng/ml and 250 ml of milk was
applied to a Supel-Select SCX SPE solid extraction column (Supelco,
Bellefonte, PA, USA) preconditioned with methanol containing 1%
formic acid and 0.1% formic aqueous solution. The column was
washed with 0.1% formic aqueous solution, and the desired fraction
Table 1
Subject characteristics.
Subject no. Age Number of birth experiences Post-partum period (months)
1 32 1 18
2 40 2 24
3 33 2 7
4 34 2 3
Please cite this article in press as: T. Chiba, et al., Analysis of serotonin concentrations in human milk by high-performance liquid
chromatography with fluorescence detection, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2017.02.027
was eluted with methanol containing 5% ammonium hydroxide
solution. The eluate was evaporated in a vacuum with a centrifugal
evaporator, and the residue was dissolved in 50 ml water containing
IS (5 mg/ml); 20 ml of this solution were analyzed by HPLC. Samples
were prepared in triplicate, and the mean value of three mea-
surements was used to generate a calibration curve for 5-HT con-
centrations of 5, 10, 50, 100, 200, 500, and 1000 ng/ml based on the
ratio of 5-HT and IS peak areas. The 5-HT concentration in breast
milk was estimated from the calibration curve by extrapolation.
2.5. Extraction recovery (ER) of 5-HT from human breast milk
Average extraction recovery (ER) was calculated at three con-
centrationsdi.e., 5, 50, and 500 ng/ml (ER50, ER500, and ER500,
respectively) using the following equations:
ER5 ð%Þ ¼ ½ðRA10  RA5 Þ=RAs5   100 (1)
ER50 ð%Þ ¼ ½ðRA100  RA50 Þ=RAs50   100 (2)
ER500 ð%Þ ¼ ½ðRA1000  RA500 Þ=RAs500   100 (3)
where RA5, RA 10, RA50, RA100, RA500, and RA1000 are the ratios of
peak areas of 5-HT and IS obtained using breast milk with 5-HT
concentrations of 5, 10, 50, 100, 500, and 1000 ng/ml, respec-
tively; and RAs5, RAs50, and RAs500 are the ratios of peak areas of 5-
HT and IS obtained using standard 5-HT aqueous solutions at
concentrations of 5, 50, and 500 ng/ml.
2.6. Statistical analysis
Values are expressed as mean ± standard deviation. Calibration
curve parameters were evaluated by one-way analysis of variance
followed by Dunnett's test.
3. Results and discussion
3.1. Chromatograms and assay precision
To determine whether 5-HT was detectable by HPLC-FD, we
generated a calibration curve and estimated the limit of detection
using a 5-HT standard solution. We found that the detection limit
was about 5 ng/ml (signal-to-noise ratio ¼ 5, data not shown); the
equation y ¼ 0.007328x þ 0.001 was extrapolated from the cali-
bration curve (correlation coefficient [r2] ¼ 0.9999; Fig. 3E)
between 5 and 1000 ng/ml (Fig. 2).
Two chromatograms were obtained by HPLC-CD for each breast
milk sample: one of the sample passed through an extraction col-
umn and the other of the same sample modified by addition of 5-HT
standard solution at a concentration of 12 ng/ml. The chromatogram
of the sample from subject 1 had a sharp peak at a retention time of
22.5 min (Fig. 2); a chromatogram of the same sample modified by
adding 5-HT standard solution showed a larger peak at 22.5 min
(Fig. 2). The peak at 32.3 min had a similar size in both samples
(Fig. 2). These results indicate that the retention time of 5-HT and IS
Breastfeeding status Use of medicines or supplements
Weaned None
Breastfeeding None
Breastfeeding None
Breastfeeding None
 T. Chiba et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5 3

Fig. 1. Procedure for extraction of 5-HT from human breast milk. Standard 5-HT aqueous solution was added to 300 ml of breast milk sample to final concentrations of 6, 12, 60,
120, 240, 600, and 1200 ng/ml. Spiked milk samples (400 ml total volume) were deproteinized with 10% trifluoroacetic acid, and 500 ml of the supernatant was applied to a Supel-
Select SCX SPE solid extraction column. The residue as the final product was dissolved in 50 ml water containing IS (5 mg/ml) and analyzed by HPLC. IS, internal standard; TFA,
trifluoroacetic acid.

was 22.5 and 32.3 min, respectively. Moreover, there were no
interfering peaks in the chromatograms using the HPLC-FD method.
3.2. Determination of 5-HT concentration in human breast milk
To confirm the accuracy and reproducibility of our HPLC-FD
method, the slope, intercept, and correlation coefficient were
Fig. 2. HPLC chromatograms of 5-HT and IS in human breast milk. The chromato-
gram represented by a solid line shows peaks corresponding to 5-HT and IS (procaine
hydrochloride, 5 mg/ml) in milk obtained from subject 1. The chromatogram repre-
sented by a dotted line shows these peaks in the same sample modified by adding 5-
HT (12 ng/ml) standard solution.
calculated from 5-HT calibration curves for each of the four sam-
ples. The mean slopes for subjects 1, 2, 3, and 4 were
0.007373 ± 0.00008687, 0.007342 ± 0.00006771,
0.007347 ± 0.0001838, and 0.07426 ± 0.0002234, respectively;
these values were similar to that obtained for the calibration curve
generated using 5-HT standard solution (0.007328 ± 0.00007621)
(Fig. 3). The mean intercept values obtained from curves generated
using breast milk samples were greater than that of 5-HT standard
solution 0.001022 ± 0.001022, 0.2632 ± 0.01032, 0.3951 ± 0.01966,
and 0.2987 ± 0.02907 for subjects 1, 2, 3, and 4, respectively vs.
0.1032 ± 0.01010) (Fig. 3). Additionally, all calibration curves
generated using breast milk samples had correlation coefficients
over 0.999 (0.9995 ± 0.0007187, 0.9998 ± 0.0001723,
0.9994 ± 0.0005749, and 0.9994 ± 0.0004605 for subjects 1, 2, 3,
and 4, respectively) that did not differ from that of the 5-HT stan-
dard solution (Fig. 3). These results indicate that the linearity of the
calibration curves obtained from the breast milk samples was
similar to that of the standard solution, and confirmed the accuracy
and reproducibility of 5-HT detection by HPLC-FD.
We next evaluated milk samples modified by adding 6, 12, 60,
120, 600, and 1200 ng/ml of 5-HT standard, which corresponded to
concentrations of 5, 10, 50, 100, 500, and 1000 ng/ml 5-HT,
respectively. For 5, 50, and 500 ng/ml samples, the recovery of 5-HT
was 96.1%e101.0% with a coefficient of variation (CV) of 3.39%e
8.62%; 96.3%e99.8% with a CV of 1.03%e8.47%; and 96.1%e98.1%
with a CV of 1.00%e5.67%, respectively (Table 2). Using the mean
slope and intercept obtained from calibration curves by the SAM,
we estimated the mean 5-HT concentration in the four breast milk

Please cite this article in press as: T. Chiba, et al., Analysis of serotonin concentrations in human milk by high-performance liquid
chromatography with fluorescence detection, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2017.02.027
4 T. Chiba et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5

Fig. 3. Calibration curves of 5-HT in breast milk samples. Samples were obtained from four subjects (AeD) and compared to an aqueous 5-HT standard solution (E). The peak area
ratio of 5-HT and IS for each calibration curve is shown as the average of triplicate measurements.

Table 2
ER of 5-HT from human breast milk.a

Subject no. 5 ng/ml 50 ng/ml 500 ng/ml

Mean ± SD (%) CV (%) Mean ± SD (%) CV (%) Mean ± SD (%) CV (%)

1 96.8 ± 4.44 4.58 96.6 ± 2.33 2.41 95.6 ± 5.42 5.67

2 101.0 ± 4.62 4.58 97.3 ± 7.64 7.85 98.1 ± 0.97 1.00
3 96.1 ± 3.26 3.39 95.3 ± 1.83 1.03 95.5 ± 2.62 2.74
4 98.3 ± 8.48 8.62 99.8 ± 8.45 8.47 96.1 ± 4.70 4.89

CV, coefficient of variation; ER, extraction recovery; SD, standard deviation.

a
Data represent the average of triplicate measurements.

samples as 11.1 ± 1.51, 35.7 ± 1.83, 55.1 ± 3.23, and 40.2 ± 3.81 ng/
ml for subjects 1, 2, 3, and 4, respectively, with CVs ranging from
5.13% to 9.89% (Table 3). Thus, our method efficiently extracted 5-
HT from human breast milk with high reproducibility.
We also investigated the mechanisms underlying the effects of
5-HT in human breast milk on milk protein expression in human
mammary epithelial cells. We previously showed that 5-HT treat-
ment inhibited the expression of the differentiation marker b-
casein in human mammary epithelial cells, which was associated
with 5-HT7 receptor-mediated activation of protein tyrosine
phosphatase 1B and inhibition of signal transduction and activator
Table 3
Concentration of 5-HT in human breast milk estimated by the SAM.a
Subject no. Mean ± SD (ng/ml)
1 11.1 ± 1.51
2 35.7 ± 1.83
3 55.1 ± 3.23
4 40.2 ± 3.81
CV, coefficient of variation; SD, standard deviation.
a
Data represent the average of triplicate measurements.
of transcription 5 phosphorylation in MCF-12A human mammary
epithelial cells [16e19]. However, the 5-HT concentration required
for this effect was >30 mM, which is higher than that observed in
the present study (61e225 nM). We speculated that this discrep-
ancy between the earlier in vivo and current in vitro studies was
due to differences in protein conformation that influenced the
strength of tight junctions and interactions with other cells,
although additional studies are required to evaluate this possibility.
A previous study using LCetandem mass spectrometry showed
that 5-HT concentration in breast milk obtained from five women
ranged from 9 to 45 nM (1.9e9.5 ng/ml); however, donor infor-
mation such as postpartum period and use of medication was
lacking [15]. We therefore sought to determine 5-HT concentrations
in breast milk obtained from healthy women and found that they
ranged from 11.1 to 55.1 ng/ml (52e259.0 nM), which was higher
CV (%)
than previously reported values. Human milk contains fat- and
water-soluble vitamins including A, E, D3, K, B1, B2, B6, B12, and C,
9.89
which are supplied to the infant through breastfeeding [20].
5.13
7.17 Vitamin B6 is important for normal behavioral development [21]
9.62 and is present in human breast milk at a concentration of about
57 ng/ml [20], which is comparable to the 5-HT level observed in
this study. 5-HT has been linked to SIDS; the concentration of 5-

Please cite this article in press as: T. Chiba, et al., Analysis of serotonin concentrations in human milk by high-performance liquid
chromatography with fluorescence detection, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/
j.bbrc.2017.02.027
 T. Chiba et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e5
hydroxyindoleacetic acidda metabolic product of 5-HTdin the
spinal fluid of SIDS cases is about 200 ng/ml [22]. Since enzymatic
activity in the stomach and intestine is low during the early post-
partum period, it is possible that 5-HT in milk is degraded upon
enzymatic transfer to spinal fluid. Further investigation including
an expanded clinical study and in vivo experiments using an animal
model are needed for a more detailed understanding of the role of
5-HT in infant development.
In conclusion, the 5-HT concentration in breast milk obtained
from four healthy Japanese women was found to range from 11.1 to
55.1 ng/ml using our newly developed HPLC-FD method. The re-
sults demonstrate that 5-HT in human breast milk can be measured
by HPLC-FD with high reproducibility, providing a basis for in-
vestigations into the effects of 5-HT on infant development and
health outcome.
Funding
This work was supported in part by a Grant-in-Aid from the
Japanese Society for Promotion of Science (KAKENHI; no.
16K18953).
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.bbrc.2017.02.027.
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