Supplemental information
Quantitative analysis of serotonin secreted by human embryonic
stem cells-derived serotonergic neurons via pH-mediated online
stacking-CE-ESI-MRM
Xuefei Zhong1, Ling Hao1, Jianfeng Lu2, Hui Ye3, Su-Chun Zhang2, Lingjun Li1,4,5*
1
School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI
53705, USA
2
Department of Neuroscience and Department of Neurology, School of Medicine and Public
Health, Waisman Center, University of Wisconsin, Madison, WI 53705, USA
3
China Pharmaceutical University, Nanjing, 210009, P. R. China
4
Department of Chemistry, University of Wisconsin, Madison, 1101 University Avenue,
Madison, WI 53706, USA
5
School of Life Sciences, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin
300072, China
*Corresponding author
Tel.: (608) 265-8491
Fax: (608) 262-5345
E-mail: lingjun.li@wisc.edu
Serotonin (5-HT) calibration standards were prepared by spiking 0, 20, 40, 80, 120 μL of
stock solution (0.2 μg/mL in water) into 1.6 mL of blank K-R solution respectively. The
calibration standard solution or collected washing solution was then spiked with 22 ng of d4serotonin (internal standard) and 320 µL of ammonium hydroxide. Liquid-liquid extraction
was performed by adding 500 µL of ethyl acetate into the cell media, vortexing the mixture
for 30 sec, followed by 10 min ultrasonic bath. After centrifugation (14,000×g, 5 min),
approximately 450 µL of upper organic phase was collected. This liquid-liquid extraction
procedure was repeated three times and the combined organic phase was evaporated by a
SpeedVac concentrator. The extracted samples were reconstituted in 20 µL of 50% methanol
and 1% ammonium hydroxide prior to CE-ESI-MS/MS analysis.
The CE separation was carried out by an Agilent HP G1600AX 3D-CE system and the
MS data was recorded by an Agilent 1100 MSD Trap mass spectrometer (Agilent
Technologies, Inc. Santa Clara, CA). The interfacing between CE and MS was originally
developed by Maxwell et al [1, 2] and adapted to fit the ESI source of the Agilent 1100 MSD
Trap mass spectrometer as depicted in Figure S1. 30 kV was applied across a 65 cm long, 50
µm inner diameter bare fused silica capillary (Polymicro Technologies, Phoenix AZ) for
separation. 50% methanol and 0.2 % formic acid was used as background electrolyte. The
modifier solution containing 50% methanol and 0.2% formic acid was infused at 0.3 µL/min
during separation. The s.s. ESI emitter was grounded and the distance between emitter tip and
the spray shield was around 1.5 cm. The MS source parameters were set as follows: ESI
voltage (spray shield) - 4.2 kV, nebulizer gas disabled, dry gas, 300 degrees, 1.0 L/min.
Positive ESI and MRM mode were used for MS data acquisition. Transition channels m/z
177 m/z 160 and m/z 181  m/z 164 were used for monitoring signal responses from
serotonin and d4-serotonin respectively. The precursor ion isolation window was set as 3 Th,
and collision energy applied for both precursor ions was 0.76 V. The ion accumulation time
of the ion trap was set as 10 mS, average number was set as 3. Ratios of the peak areas of
serotonin and d4-5HT were calibrated against standard curves for accurate quantification.
The peak area ratios of extracted ion electropherograms (EIEs) of the transition channels
m/z 177 m/z 160 and m/z 181  m/z 164 were used for construction of the calibration
curve. Three CE-MS technical replicates were performed for each calibration standard
solution (0, 2.5, 5, 10, 15 ng/mL serotonin spiked in K-R solution) and the samples. The
calibration curve is shown in Figure S2. Intra-day coefficient of variance (CV) of the 2.5
ng/mL serotonin standard solution was 4.0% (n=3), and intra-day CV of the 10 ng/mL
serotonin standard solution was 4.9% (n=3). The linearity of the calibration curve (r2) was
0.9996. Representative EIEs of the serotonin and the internal standard are shown in Figure
S3, including calibration blank (Figure S3A), 10 ng/mL serotonin calibration standard (Figure
S3B), serotonin released by hESCs-derived serotonergic neurons in depolarizing K-R
solution (Figure S3C) and K-R solution (Figure S3D), cell releasate of the dorsal hindbrain
derived-neurons in depolarizing K-R solution (Figure S3E) and K-R solution (Figure S3F)
which served as biological control samples. In both calibration blank (Figure S3A) and
biological control samples (Figure S3E-F), serotonin was not detectable (S/N < 3). The
temperature fluctuation and evaporation of volatile components in the separation buffers
during the CE runs might cause minor shift in the measured CE migration time.
Figure S1. Photo of instruments used for the CE-ESI-MRM experiments. CE separation
was carried out using an Agilent HP 3D CE system. The subsequent MS detection was
performed on an Agilent 1100 LC/MSD Trap instrument. The inset is an enlarged view of the
CE-ESI-MS interface. The stainless steel (s.s.) ESI emitter was grounded and - 4.2 kV was
applied on the spray shield. The distance between the sprayer emitter tip and the MS inlet
was around 1.5 cm.
Figure S2. Calibration curve of serotonin meausrement by CE-ESI-MRM.
Figure S3. Extracted ion electropherograms of serotonin (red, m/z 177 −>160) and
internal standard d4-serotonin (blue, m/z 181 −>164). (A) calibration blank; (B) 10
ng/mL serotonin calibration standard; cell releasate from (C) hESCs-derived
serotonergic neurons in depolarizing K-R solution and (D) hESCs-derived serotonergic
neurons in K-R solution; cell releasate from (E) dorsal hindbrain derived-neurons in
depolarizing K-R solution and (F) dorsal hindbrain derived-neurons in K-R solution. 22
ng/mL internal standard d4- serotonin were spiked in 1.6 mL K-R solution. CE conditions
were the same as used for Figure 2E.
[1] Maxwell, E. J., Zhong, X. F., Zhang, H., van Zeijl, N., Chen, D. D. Y., Electrophoresis
2010, 31, 1130-1137.
[2] Zhong, X. F., Maxwell, E. J., Chen, D. D. Y., Anal. Chem. 2011, 83, 4916-4923.