Next Article in Journal
8(meso)-Pyridyl-BODIPYs: Effects of 2,6-Substitution with Electron-Withdrawing Nitro, Chloro, and Methoxycarbonyl Groups
Next Article in Special Issue
Investigation of New Psychoactive Substances (NPS), Other Illicit Drugs, and Drug-Related Compounds in a Taiwanese Wastewater Sample Using High-Resolution Mass-Spectrometry-Based Targeted and Suspect Screening
Previous Article in Journal
Recent Configurational Advances for Solid-State Lithium Batteries Featuring Conversion-Type Cathodes
Previous Article in Special Issue
Proteomic Signature of Extracellular Vesicles Associated with Colorectal Cancer
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 12
10.3390/molecules28124580
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry

by
Shih-Shin Liang
1,2,3,4,*,
Po-Tsun Shen
5,
Yu-Qing Liang
1,
Yi-Wen Ke
1,
Chieh-Wen Cheng
6 and
Yi-Reng Lin
7,*
1
Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
2
Institute of Biomedical Science, College of Medicine, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
3
Research Center for Precision Environmental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
4
Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan
5
Protein Chemistry Core Laboratory, Core Instrument Center, National Health Research Institutes, Miaoli 35053, Taiwan
6
Bachelor Program in Industrial Technology, College of Future, National Yunlin University of Science and Technology, Yunlin 64002, Taiwan
7
Department of Biotechnology, School of Environment and Life Sciences, Fooyin University, Kaohsiung 83102, Taiwan
*
Authors to whom correspondence should be addressed.
Molecules 2023, 28(12), 4580; https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28124580
Submission received: 28 April 2023 / Revised: 25 May 2023 / Accepted: 29 May 2023 / Published: 6 June 2023
(This article belongs to the Special Issue Mass Spectrometry in Biomarkers Discovery)
Browse Figures
Versions Notes

Abstract

:
Herein, we used isotopic formaldehyde and sodium cyanoborohydride via reductive amination to label two methyl groups on primary amine to arrange the standards (h2-formaldehyde-modified) and internal standards (ISs, d2-formaldehyde-modified) of tryptophan and its metabolites, such as serotonin (5-hydroxytryptamine) and 5-hydroxytryptophan. These derivatized reactions with a high yield are very satisfactory for manufacturing standards and ISs. This strategy will generate one or two methyl groups on amine to create different mass unit shifts with 14 vs. 16 or 28 vs. 32 in individual compounds for biomolecules with amine groups. In other words, multiples of two mass units shift are created using this derivatized method with isotopic formaldehyde. Serotonin, 5-hydroxytryptophan, and tryptophan were used as examples to demonstrate isotopic formaldehyde-generating standards and ISs. h2-formaldehyde-modified serotonin, 5-hydroxytryptophan, and tryptophan are standards to construct calibration curves, and d2-formaldehyde-modified analogs such as ISs spike into samples to normalize the signal of each detection. We utilized multiple reaction monitoring modes and triple quadrupole mass spectrometry to demonstrate the derivatized method suitable for these three nervous biomolecules. The derivatized method demonstrated a linearity range of the coefficient of determinations between 0.9938 to 0.9969. The limits of detection and quantification ranged from 1.39 to 15.36 ng/mL.

1. Introduction

Neurotransmitters are chemical molecules that are released from axon terminals with neural impulses [1]. These chemical molecules include glutamate [2], 5-hydroxytryptamine (serotonin, 5-HT) [3], dopamine, noradrenaline [4], epinephrine [5], adenosine [6], etc. Neurotransmitters have multiple functions in the nervous system, i.e., dopamine plays an important role in regulating neuroendocrine functions, motivation, emotion, and locomotor activity [7], and serotonin affects human beings’ mood via maintaining high concentrations in the blood [8].
In the syntheses of neurotransmitters from neural precursors, tryptophan (TRP), an original component, is catalyzed using tryptophan hydroxylase (TPH) to generate 5-hydroxytryptophan (5-HTP) [9], and serotonin is generated from 5-HTP, which is catalyzed by aromatic L-amino acid decarboxylase (AAAD) [10]. The other important bioactive metabolite, melatonin, is produced by serotonin reacted with N-acetyl transferase and 5-hydroxyindole-O-methyltransferase [11]. The other neurotransmitter, dopamine, is used by tyrosine to be catalyzed by AAAD [12,13]. TRP is an essential amino acid for protein synthesis and has three pathways in TRP metabolism, including the serotonin-melatonin pathway [10,14], indole-3-acetic acid pathway [10], and kynurenine pathway [15,16,17]. An imbalance in the levels of TRP and its metabolites will cause neuronal damage and impair physiological and neurological functions [18,19]. The TRP metabolite of serotonin is also an important neurotransmitter, and regulates numerous human behaviors, emotion, appetite, memory and learning [20]. Therefore, all the metabolites in the pathways reflect behavioral and physiological significances.
Nowadays, metabolomics demonstrate a novel vision and the possibility of screening metabolites with powerful high-throughput instruments [21,22,23,24]. Among the separated metabolomics system, high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) is widely used in many research fields [25,26,27,28]. Tryptophan metabolic pathways produce important bioactive substances, and therefore, TRP and its metabolites are important in cell metabolism. Therefore, we selected the serotonin pathway as an example, with TRP, 5-HTP, and serotonin as target molecules, to develop a high-performance liquid chromatography–tandem mass spectroscopy quantitative platform. However, in this technique, each sample detection needed internal standards (ISs) to normalize the intensity of individual samples [15]. In the previous literature, the quantitation of TRP, kynurenine, kynurenic acid and serotonin occurred via reverse-phase column (TSK-Gel ODS-80 Ts) coupled with UV-Vis spectroscopy, fluorescence, and mass spectrometry [29]. The results showed that TRP, kynurenine, kynurenic acid and serotonin had fluorescence and UV absorbance functional groups without any dyes labeling. The other method involves the use of capillary electrophoresis coupled with mass spectrometry to determine serotonin, TRP, and 5-HTP in human plasma. The results demonstrated that the concentration levels were below 100 ppb (sub-μM) [30]. The HPLC/MS/MS instrument was set up with two mass analyzers to select m/z of the precursor ion at the first mass analyzer and to select m/z of the product ions at the secondary mass analyzer after fragmentation. This detection mode of tandem MS is named multiple-reaction monitoring (MRM) and has advantages, such as improvement of sensitivity and selectivity. In rat brain samples, MRM detection mode was utilized to monitor serotonin and TRP [31,32]. However, in traditional HPLC/MS analyses, a better analytical strategy is the use of isotope internal standards such as ISs to normalize the intensity of signals in every sample.
Previously, we developed a derivatized method to generate ISs beneficial for HPLC–MS/MS analyses [33,34,35,36,37,38,39]. Herein, we used isotopic formaldehyde and sodium cyanoborohydride to generate two methyl groups on the amine functional group of TRP, 5-HTP, and serotonin. This modification, with cheap and easily obtained materials, showed signal enhancement and helped target molecules to be monitored [40]. Eventually, we establish the derivatized method by estimating calibration curves, the limit of detection (LOD), the limit of quantification (LOQ), intra-day precision, and inter-day precision.

2. Results

2.1. Reductive Amination of Tryptophan and Its Metabolites

The TRP pathway shows that TRP can be metabolized using TPH to be transformed into 5-HTP and serotonin, an important neurotransmitter. The conventional method to determine the concentrations of TRP, 5-HTP, and serotonin uses HPLC–MS/MS, and ISs are needed for each sample detection. In general, isotopic analogs, such as deuterium replacing hydrogen, are used to spike the samples during pretreatment to recognize the individual signals of each sample [15,41].
Herein, we developed a modified method to generate TRP, 5-HTP, and serotonin with h2-formaldehyde and d2-formaldehyde to generate h2-formaldehyde-modified TRP, 5-HTP, and serotonin (as standards) and to react with d2-formaldehyde-modified TRP, 5-HTP, and serotonin (as ISs). Figure 1 shows the schematic diagram of this derivatized method (reductive amination), in which isotopic formaldehyde reacts and reduces using NaBH3CN.

2.2. MS/MS Fragmentation and MRM Transition Setting

The multiple-reaction-monitoring (MRM) transitions of TRP and its metabolites were determined using MS/MS fragmentation. All unmodified and isotopic formaldehyde-modified compounds were determined using tandem MS. An individual compound was infused into tandem MS and fragmented by colliding gas. The MS/MS spectrum of unmodified serotonin (Figure 2A) showed a precursor ion at mass-to-charge ratio (m/z) = 177, and its fragments showed m/z values of 160 and 115 (product ions, the structure of m/z 115 refers to the website of The Human Metabolome Database, https://hmdb.ca/metabolites/HMDB0000259 (accessed on 30 May 2023)). Serotonin was modified using reductive amination via two methyl groups, and the molecular weight was transformed into 204 (h2-modified serotonin, m/z 205) and 208 (d2-modified serotonin, m/z 209). As shown in Figure 2B, 205 > 58 and 205 > 160 were fragmented from the precursor ions of h2-modified serotonin. Meanwhile, as shown in Figure 2C, 209 > 62 and 209 > 160 were fragmented from the precursor ions of d2-modified serotonin. The difference between 205 > 58 and 209 > 62 supported that reductive amination achieved isotopic modification. Similarly, unmodified TRP with an m/z value of 205 was fragmented to generate product ions at m/z = 118 and 146 (Figure 2D). The fragmentations of h2-modified TRP and d2-modified TRP were attributed to 233 > 102, 233 > 118 and 233 > 146 (Figure 2E), and 237 > 106, 237 > 118 and 237 > 146 (Figure 2F), respectively. Finally, the MRM transitions setting from unmodified 5-HTP was assigned according to 221 > 134 and 221 > 162 (Figure 2G). h2-modified 5-HTP exhibited the MRM transitions of 249 > 134 and 249 > 162 (Figure 2H); meanwhile, d2-modified 5-HTP exhibited the MRM transitions of 253 > 134 and 253 > 162 (Figure 2I). The MRM transitions of serotonin, tryptophan, and 5-HTP and their modified analogs are listed in Table 1, including the molecular formula, molecular weight, LOD, LOQ, and coefficient of determination (R2).

2.3. Signal Enhancements of Modified Serotonin, TRP, and 5-HTP

The peak areas of the MS spectra between the unmodified and modified isotopic formaldehyde were compared to determine the signal enhancement in modified samples. We selected serotonin as an example, as shown in Figure 3. Identical amounts of unmodified, h2-derivatized, and d2-derivatized serotonin were mixed to extract the MS/MS spectra of the unmodified serotonin (MRM transitions: m/z = 221 > 162 and 221 > 134, retention time [rt] = 10.50 min, absolute intensity = 6.0 × 106), h2-derivatized serotonin (MRM transitions: m/z = 249 > 162 and 249 > 134, rt = 10.91 min, absolute intensity = 8.2 × 106), and d2-derivatized serotonin (MRM transitions: m/z = 253 > 162 and 253 > 134, rt = 10.91 min, absolute intensity = 1.6 × 107). According to the same procedures, we compared the integrated areas (average value, n = 4) of the unmodified and d2-derivatized analogs of serotonin, TRP, and 5-HTP. The datasets showed a signal enhancement of 1.19-fold (serotonin), 18.87-fold (TRP), and 2.40-fold (5-HTP) upon derivatization. The integrated and statistical results, including the average integrated area, relative standard deviation, and signal enhancement, are shown in Table 2. We have previously discussed the factor that enhances signals via reductive amination [40]. We estimated that the factor is the amine group showing a higher electronic density after modification with two methyl groups, causing the lone pair in the amine group to show a more basic quality.

2.4. Method Validation

To estimate the applicability of this derivatized method for serotonin, TRP, and 5-HTP, we used the modified three compounds to generate h2-formaldehyde-derivatized analogs to construct calibration curves and calculate R2, LOD, and LOQ. The h2-derivatized serotonin, TRP, and 5-HTP standards required to construct the calibration curve were prepared using 180 μL at concentrations of 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100, 200, 500, and 1000 ng/mL (ppb). Meanwhile, the d2-derivatized ISs were spiked with an identical 20 μL of 2.0 ppm d2-modified serotonin, TRP, and 5-HTP. Each calibration curve was extracted from the normalized MS/MS spectra of the h2-modified serotonin, TRP, and 5-HTP using their d2-modified analogs. The R2 of the calibration curves ranged from 0.9938 to 0.9969 for h2-modified analogs. The statistical data are listed in Table 1. The validation parameters of the LODs and LOQs for the modified analogs were 2.35 and 14.54 ng/mL for serotonin, 1.96 and 4.36 ng/mL for tryptophan, and 1.39 and 15.36 ng/mL for 5-HTP, respectively. A concentration of 1 ng/mL was injected as a low-concentration specimen to estimate the LOD and LOQ. The data of the intra-day and inter-day concentrations are shown in Table 3.
The other validations to assess isotopic d2-modified analogs are compatible with serotonin, TRP, and 5-HTP quantitative analysis. We spiked different ratios of isotopic amount, including 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, and 1:20. We selected serotonin as an example to demonstrate the effect of different h2:d2 ratios in detecting serotonin, while showing the corresponding ratios of signal area integrations.
Figure 4A–C show the ratios of h2-modified to d2-modified serotonin, including 1:1, 1:20, and 20:1. The dynamic linear range for the quantitative analyses of the serotonin, TRP, and 5-HTP estimated an R2 of 0.9831 (Figure 4D), 0.9998 (Figure 4E), and 0.9927 (Figure 4F), respectively.

3. Materials and Methods

3.1. Materials

Serotonin (hydrochloride form), sodium cyanoborohydride (NaBH3CN), sodium acetate, and tryptophan were obtained from Sigma–Aldrich (St. Louis, MO, USA). The LC–MS grade solvents, including acetonitrile (MeCN), methanol (MeOH), and 0.22 μm PTFE filters, were bought from Merck (Seelze, Germany). Sodium hydroxide and hydrochloric acid were purchased from J.T. Baker (Phillipsburg, NJ, USA) to adjust the buffer pH. Formaldehyde-D2 solution (20% solution in D2O) was purchased from Isotec Corp. (Miamisburg, OH, USA), and formaldehyde-H2 solution (36.5–38% in H2O), 5-HTP and formic acid (FA, 98–100%) were purchased from Sigma (St. Louis, MO, USA). Deionized H2O with a resistance of 18.2 MΩ was obtained using a Millipore water system.

3.2. Instrumentation

The instruments for separation and detection, including ultrahigh-performance liquid chromatography coupled with a tandem mass spectrometer (UHPLC–MS/MS, Thermo Fisher Scientific Inc., Waltham, MA, USA), were equipped with a Thermo Finnigan Acella 1250 UHPLC system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The MS system was a triple quadrupole MS (Thermo Fisher Scientific Inc., Waltham, MA, USA). The inlet system of a microelectrospray ionization (ESI, Thermo Fisher Scientific Inc.) ion source was set in the positive ion mode with an applied voltage of 3000 V, and the vaporizing and capillary temperatures were set at 300 °C and 350 °C, respectively. The auxiliary gas and sheath gas pressures were set to 10 units and 30 units, respectively. The collision energy was adjusted to 25 V with high-quality argon gas. The MS survey scan mode of MRM was employed to select the precursor and product ions for fragmentation with the MRM transitions setting, as shown in Table 1. The separated system used a 10-μL sample injected into a loop using an Acella 1250 autosampler and separated by a Shiseido CAPCELL PAK MG II C18 column (i.d. 1.5 mm × 150 mm, 3 μm, Tokyo, Japan). The flow rate of UHPLC was set at 200 μL/min with mobile phases of 0.1% FA in water (A) and 0.1% FA in 100% MeCN (B). The linear gradient was set as follows: 2% (B) for 1 min; 2–40% (B) for 5 min; 40–98% (B) for 3 min, followed by holding at 98% (B) for 2 min. The software to control and adjust the UHPLC and MS instrument systems was Xcalibur (version 2.2, Thermo Finnigan Inc., San Jose, CA, USA), and Xcalibur was also utilized to acquire MS data. The statistics analyses used the Xcalibur Thermo LCquan software (version 2.7, Thermo Finnigan Inc., San Jose, CA, USA).

3.3. Preparation of Derivatized Standards and Internal Standards

Serotonin, 5-HTP, and TRP were weighed and dissolved in dimethyl sulfoxide at 100 mg/mL (100,000 ppm) and stored in a refrigerator at −80 °C as stock solutions. A mixture of serotonin, 5-HTP, and TRP was dissolved, and the concentration was adjusted to 100 ppm with 50% MeCN (v/v) solution. The reductive amination procedures were used in a mixture of 20 μL of 100 ppm blended with 160 μL of 50 mM sodium acetate buffer (pH 5.6) and 10 μL of 4% h2-formaldehyde to generate standards. To derivatize for the generation of serotonin, 5-HTP, and TRP ISs, h2-formaldehyde was replaced by 4% h2-formaldehyde. After 5 min, 10 μL of NaBH3CN was spiked into each tube to reduce the derivatized serotonin, 5-HTP, and TRP. A diagram of reductive amination is illustrated in Figure 1.

3.4. Development of the Detection Method for Serotonin, 5-Hydroxytryptophan, and Tryptophan

We used MRM for the detection of h2-formaldehyde- and d2-formaldehyde-modified serotonin, 5-HTP, and TRP analogs. Individual serotonin, 5-HTP, and TRP analogs, including unmodified, h2-formaldehyde-generated, and d2-formaldehyde-generated compounds, were infused into the MS instrument using a syringe and fragmented using argon gas in an MS collision chamber. The ions, including the selected precursor and fragmented product ions, of the unmodified and modified analogs are shown in Figure 2. The fragmentation of the MS spectra was extracted to arrange the MRM transitions listed in Table 1.

3.5. Evulation of the Derivitized Method

The calibration curves for serotonin, 5-HTP, and TRP were constructed using the concentrations of h2-modified analogs: 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL (ppb). The serotonin, 5-HTP, and TRP calibration curves also determined the intra-day and inter-day LODs and LOQs. In each sample solution (180 μL), an equal volume (20 μL, 2 ppm) of d2-modified serotonin, 5-HTP, and TRP analogs was added as ISs to each Eppendorf tube. The statistical data for the intra-day and inter-day reproducibility of serotonin, 5-HTP, and TRP were calculated for triplicate experiments (n = 3), respectively. The LOD and LOQ were determined using low-concentration samples (1 ng/mL of serotonin, TRP, and 5-HTP samples, [n = 6]). To validate this modified method for serotonin, 5-HTP, and TRP, we used h2-formaldehyde and d2-formaldehyde products mixed at different ratios of 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, and 1:20 to demonstrate the linear range of the quantitative analysis via a reductive amination method. The results are shown in Figure 4. Figure 4A–C show the ratios of 1:1 (1), 1:20 (20-fold), and 20:1 (1/20, −20), respectively.

4. Conclusions

A convenient and affordable derivatized method was developed, easily compatible with HPLC–MS/MS. The derivatized method used the isotopic formaldehyde-modified serotonin, 5-HTP, and TRP for quantification. The derivatized method showed labor-saving qualities, a high yield, and, most importantly, promoted signal enhancement. We demonstrated three orders of dynamic linear ranges, and the individual coefficients of determination were 0.9938 and 0.9969. Intra-day and inter-day data indicated a stable reproducibility, and the LOD and LOQ ranged between 1.39 and 15.36 ng/mL.
Therefore, we considered that this reductive amination-based derivatized method could screen the serotonin pathway, including TRP and its metabolites, serotonin, and 5-HTP.

Author Contributions

Conceptualization, S.-S.L. and P.-T.S.; methodology, Y.-Q.L. and Y.-W.K.; software, Y.-Q.L. and Y.-W.K.; validation, Y.-Q.L. and Y.-W.K.; formal analysis, Y.-Q.L. and C.-W.C.; investigation, S.-S.L. and Y.-R.L.; resources, S.-S.L.; data curation, S.-S.L., P.-T.S. and Y.-R.L.; writing—original draft preparation, S.-S.L. and Y.-R.L.; writing—review and editing, S.-S.L., P.-T.S. and Y.-R.L.; visualization, S.-S.L. and Y.-R.L.; supervision, S.-S.L. and Y.-R.L.; project administration, S.-S.L. and Y.-R.L.; funding acquisition, S.-S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Science and Technology Council (Taipei, Taiwan), grant number MOST 110-2113-M-037-012 and MOST 109-2113-M-037-008.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

We inform that all the published data were obtained and involved in this research.

Acknowledgments

We are thankful for the assistance of the Center for Resources, Research and Development of Kaohsiung Medical University.

Conflicts of Interest

The all authors declare that they have no conflict of interest in this study.

Sample Availability

Not applicable.

References

  1. Stjarne, L. Basic mechanisms and local modulation of nerve impulse-induced secretion of neurotransmitters from individual sympathetic nerve varicosities. Rev. Physiol. Biochem. Pharmacol. 1989, 112, 1–137. [Google Scholar] [PubMed]
  2. Hillyer, J.F.; Estevez-Lao, T.Y.; Mirzai, H.E. The neurotransmitters serotonin and glutamate accelerate the heart rate of the mosquito Anopheles gambiae. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2015, 188, 49–57. [Google Scholar] [CrossRef]
  3. Seyedabadi, M.; Fakhfouri, G.; Ramezani, V.; Mehr, S.E.; Rahimian, R. The role of serotonin in memory: Interactions with neurotransmitters and downstream signaling. Exp. Brain Res. 2014, 232, 723–738. [Google Scholar] [CrossRef]
  4. Wang, K.H.; Penmatsa, A.; Gouaux, E. Neurotransmitter and psychostimulant recognition by the dopamine transporter. Nature 2015, 521, 322–327. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Hinz, M.; Stein, A.; Uncini, T. Urinary neurotransmitter testing: Considerations of spot baseline norepinephrine and epinephrine. Open Access J. Urol. 2011, 3, 19–24. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Fang, H.; Pajski, M.L.; Ross, A.E.; Venton, B.J. Quantitation of dopamine, serotonin and adenosine content in a tissue punch from a brain slice using capillary electrophoresis with fast-scan cyclic voltammetry detection. Anal. Methods 2013, 5, 2704–2711. [Google Scholar] [CrossRef] [Green Version]
  7. Wang, M.; Lee, F.J.; Liu, F. Dopamine receptor interacting proteins (DRIPs) of dopamine D1-like receptors in the central nervous system. Mol. Cells 2008, 25, 149–157. [Google Scholar]
  8. Young, S.N. How to increase serotonin in the human brain without drugs. J. Psychiatry Neurosci. 2007, 32, 394–399. [Google Scholar]
  9. Cote, F.; Thevenot, E.; Fligny, C.; Fromes, Y.; Darmon, M.; Ripoche, M.A.; Bayard, E.; Hanoun, N.; Saurini, F.; Lechat, P.; et al. Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc. Natl. Acad. Sci. USA 2003, 100, 13525–13530. [Google Scholar] [CrossRef] [Green Version]
  10. Roth, W.; Zadeh, K.; Vekariya, R.; Ge, Y.; Mohamadzadeh, M. Tryptophan Metabolism and Gut-Brain Homeostasis. Int. J. Mol. Sci. 2021, 22, 2973. [Google Scholar] [CrossRef]
  11. Hoglund, E.; Overli, O.; Winberg, S. Tryptophan Metabolic Pathways and Brain Serotonergic Activity: A Comparative Review. Front. Endocrinol. 2019, 10, 158. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  12. Azam, B.; Wienecke, J.; Jensen, D.B.; Azam, A.; Zhang, M. Spinal Cord Hemisection Facilitates Aromatic L-Amino Acid Decarboxylase Cells to Produce Serotonin in the Subchronic but Not the Chronic Phase. Neural Plast. 2015, 2015, 549671. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  13. Alachkar, A.; Brotchie, J.M.; Jones, O.T. Locomotor response to L-DOPA in reserpine-treated rats following central inhibition of aromatic L-amino acid decarboxylase: Further evidence for non-dopaminergic actions of L-DOPA and its metabolites. Neurosci. Res. 2010, 68, 44–50. [Google Scholar] [CrossRef] [PubMed]
  14. Troche, G.; Henry-Lagarrigue, M.; Soppelsa, F.; Legriel, S.; Yehia, A.; Bruneel, F.; Bedos, J.P.; Spreux-Varoquaux, O. Tryptophan pathway catabolites (serotonin, 5-hydroxyindolacetic acid, kynurenine) and enzymes (monoamine oxidase and indole amine 2,3 dioxygenase) in patients with septic shock A prospective observational study versus healthy controls. Medicine 2020, 99, e19906. [Google Scholar] [CrossRef]
  15. Eser, B.; Ozkan, Y.; Sepici Dincel, A. Determination of Tryptophan and Kynurenine by LC-MS/MS by Using Amlodipine as an Internal Standard. J. Am. Soc. Mass. Spectrom. 2020, 31, 379–385. [Google Scholar] [CrossRef]
  16. Sadok, I.; Gamian, A.; Staniszewska, M.M. Chromatographic analysis of tryptophan metabolites. J. Sep. Sci. 2017, 40, 3020–3045. [Google Scholar] [CrossRef]
  17. Ghebregzabher, M.; Rufini, S.; Castellucci, M.G.; Lato, M. Analysis of Some Tryptophan and Phenylalanine Metabolites in Urine by a Straight-Phase High-Performance Liquid-Chromatographic Technique. J. Chromatogr. 1981, 222, 191–201. [Google Scholar] [CrossRef]
  18. Wonodi, I.; Schwarcz, R. Cortical kynurenine pathway metabolism: A novel target for cognitive enhancement in Schizophrenia. Schizophr. Bull. 2010, 36, 211–218. [Google Scholar] [CrossRef] [Green Version]
  19. Schwarcz, R.; Pellicciari, R. Manipulation of brain kynurenines: Glial targets, neuronal effects, and clinical opportunities. J. Pharmacol. Exp. Ther. 2002, 303, 1–10. [Google Scholar] [CrossRef] [Green Version]
  20. Schloss, P.; Williams, D.C. The serotonin transporter: A primary target for antidepressant drugs. J. Psychopharmacol. 1998, 12, 115–121. [Google Scholar] [CrossRef]
  21. Tan, B.; Chen, J.; Qin, S.; Liao, C.; Zhang, Y.; Wang, D.; Li, S.; Zhang, Z.; Zhang, P.; Xu, F. Tryptophan Pathway-Targeted Metabolomics Study on the Mechanism and Intervention of Cisplatin-Induced Acute Kidney Injury in Rats. Chem. Res. Toxicol. 2021, 34, 1759–1768. [Google Scholar] [CrossRef]
  22. Lai, Y.; Liu, C.W.; Chi, L.; Ru, H.; Lu, K. High-Resolution Metabolomics of 50 Neurotransmitters and Tryptophan Metabolites in Feces, Serum, and Brain Tissues Using UHPLC-ESI-Q Exactive Mass Spectrometry. ACS Omega 2021, 6, 8094–8103. [Google Scholar] [CrossRef] [PubMed]
  23. Attwa, M.W.; AlRabiah, H.; Mostafa, G.A.E.; Bakheit, A.H.; Kadi, A.A. Assessment of In Silico and In Vitro Selpercatinib Metabolic Stability in Human Liver Microsomes Using a Validated LC-MS/MS Method. Molecules 2023, 28, 2618. [Google Scholar] [CrossRef] [PubMed]
  24. Attwa, M.W.; AlRabiah, H.; Mostafa, G.A.E.; Kadi, A.A. Development of an LC-MS/MS Method for Quantification of Sapitinib in Human Liver Microsomes: In Silico and In Vitro Metabolic Stability Evaluation. Molecules 2023, 28, 2322. [Google Scholar] [CrossRef]
  25. Younis, I.Y.; Khattab, A.R.; Selim, N.M.; Sobeh, M.; Elhawary, S.S.; Bishbishy, M.H.E. Metabolomics-based profiling of 4 avocado varieties using HPLC-MS/MS and GC/MS and evaluation of their antidiabetic activity. Sci. Rep. 2022, 12, 4966. [Google Scholar] [CrossRef] [PubMed]
  26. Gong, L.L.; Yang, S.; Zhang, W.; Han, F.F.; Xuan, L.L.; Lv, Y.L.; Liu, H.; Liu, L.H. Targeted Metabolomics for Plasma Amino Acids and Carnitines in Patients with Metabolic Syndrome Using HPLC-MS/MS. Dis. Markers 2020, 2020, 8842320. [Google Scholar] [CrossRef] [PubMed]
  27. Islam, J.; Shirakawa, H.; Nguyen, T.K.; Aso, H.; Komai, M. Simultaneous analysis of serotonin, tryptophan and tryptamine levels in common fresh fruits and vegetables in Japan using fluorescence HPLC. Food Biosci. 2016, 13, 56–59. [Google Scholar] [CrossRef]
  28. Wang, Y.; Feng, F. Evaluation of the Hepatotoxicity of the Zhi-Zi-Hou-Po Decoction by Combining UPLC-Q-Exactive-MS-Based Metabolomics and HPLC-MS/MS-Based Geniposide Tissue Distribution. Molecules 2019, 24, 511. [Google Scholar] [CrossRef] [Green Version]
  29. Lesniak, W.G.; Jyoti, A.; Mishra, M.K.; Louissaint, N.; Romero, R.; Chugani, D.C.; Kannan, S.; Kannan, R.M. Concurrent quantification of tryptophan and its major metabolites. Anal. Biochem. 2013, 443, 222–231. [Google Scholar] [CrossRef] [Green Version]
  30. Peterson, Z.D.; Lee, M.L.; Graves, S.W. Determination of serotonin and its precursors in human plasma by capillary electrophoresis-electrospray ionization-time-of-flight mass spectrometry. J. Chromatogr. B 2004, 810, 101–110. [Google Scholar] [CrossRef]
  31. He, B.; Bi, K.; Jia, Y.; Wang, J.; Lv, C.; Liu, R.; Zhao, L.; Xu, H.; Chen, X.; Li, Q. Rapid analysis of neurotransmitters in rat brain using ultra-fast liquid chromatography and tandem mass spectrometry: Application to a comparative study in normal and insomnic rats. J. Mass. Spectrom. 2013, 48, 969–978. [Google Scholar] [CrossRef] [PubMed]
  32. Zheng, X.; Kang, A.; Dai, C.; Liang, Y.; Xie, T.; Xie, L.; Peng, Y.; Wang, G.; Hao, H. Quantitative analysis of neurochemical panel in rat brain and plasma by liquid chromatography-tandem mass spectrometry. Anal. Chem. 2012, 84, 10044–10051. [Google Scholar] [CrossRef] [PubMed]
  33. Shen, P.T.; Wu, Y.Y.; Chang, Y.T.; Cheng, C.W.; Huang, M.F.; Chen, Z.Y.; Shiue, Y.L.; Liang, S.S. Quantification of the beta(2)-Adrenergic Feed Additives Ractopamine and Salbutamol by Reductive Amination-Assisted Modification. J. Food Prot. 2019, 82, 696–702. [Google Scholar] [CrossRef] [PubMed]
  34. Shen, P.T.; Lin, Y.R.; Chen, B.H.; Huang, M.F.; Cheng, C.W.; Shiue, Y.L.; Liang, S.S. A standard addition method to quantify histamine by reductive amination and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry. Eur. J. Mass. Spectrom. 2019, 25, 412–418. [Google Scholar] [CrossRef]
  35. Huang, M.F.; Lin, Y.R.; Chang, Y.T.; Shiue, Y.L.; Liang, S.S. Reductive amination assistance for quantification of oseltamivir phosphate and oseltamivir carboxylate by HPLC-MS/MS. J. Chromatogr. B 2018, 1087, 23–28. [Google Scholar] [CrossRef]
  36. Lin, Y.R.; Wu, C.E.; Huang, M.F.; Wu, Y.Y.; Huang, J.H.; Liu, M.C.; Chen, Z.Y.; Shiue, Y.L.; Liang, S.S. Development of Doxorubicin Quantification by Reductive Amination. Curr. Anal. Chem. 2017, 13, 270–276. [Google Scholar] [CrossRef]
  37. Lin, Y.R.; Huang, M.F.; Wu, Y.Y.; Liu, M.C.; Huang, J.H.; Chen, Z.Y.; Shiue, Y.L.; Wu, C.E.; Liang, S.S. Reductive amination derivatization for the quantification of garlic components by isotope dilution analysis. Food Chem. 2017, 230, 1–5. [Google Scholar] [CrossRef]
  38. Tsai, D.C.; Liu, M.C.; Lin, Y.R.; Huang, M.F.; Liang, S.S. A novel reductive amination method with isotopic formaldehydes for the preparation of internal standard and standards for determining organosulfur compounds in garlic. Food Chem. 2016, 197, 692–698. [Google Scholar] [CrossRef]
  39. Liang, S.S.; Wang, T.N.; Chiu, C.C.; Kuo, P.L.; Huang, M.F.; Liu, M.C.; Tsai, E.M. Reductive amination-assisted quantitation of tamoxifen and its metabolites by liquid phase chromatography tandem mass spectrometry. J. Chromatogr. A 2016, 1434, 64–69. [Google Scholar] [CrossRef]
  40. Liu, M.C.; Lin, Y.R.; Huang, M.F.; Tsai, D.C.; Liang, S.S. Mass spectrometry signal enhancement by reductive amination. Int. J. Mass. Spectrom. 2015, 387, 16–23. [Google Scholar] [CrossRef]
  41. Cong, M.; Song, J.; Chen, F.; Cao, C.; Wang, S. A surrogate analyte-based LC-MS/MS method for the determination of 5-hydroxytryptamine, kynurenine and tryptophan. Bioanalysis 2020, 12, 129–142. [Google Scholar] [CrossRef] [PubMed]
Molecules 28 04580 g001 550
Figure 1. Schematic diagram of the reductive amination for serotonin, tryptophan, and 5-HTP. The modification used isotopic formaldehyde and reduction with sodium cyanoborohydride (NaBH3CN).
Figure 1. Schematic diagram of the reductive amination for serotonin, tryptophan, and 5-HTP. The modification used isotopic formaldehyde and reduction with sodium cyanoborohydride (NaBH3CN).
Molecules 28 04580 g001
Molecules 28 04580 g002 550
Figure 2. The mass spectra of fragmented ions to set the multiple-reaction-monitoring (MRM) methods, the ion-pairs of mass-to-charge ratios (m/z) of the precursor, and product fragments. (A) Unmodified serotonin (m/z = 177) and its product ions at m/z = 115 and 160, (B) h2-derivatized serotonin (m/z = 205) and its product ions at m/z = 58 and 160, (C) d2-derivatized serotonin (m/z = 209) and its product ions at m/z = 62 and 160, (D) unmodified tryptophan (TRP) (m/z = 205) and its product ions at m/z = 118 and 146, (E) h2-derivatized TRP (m/z = 233) and its product ions at m/z = 102, 118, and 146, (F) d2-derivatized TRP (m/z = 237) and its product ions at m/z = 106, 118, and 146, (G) unmodified 5-HTP (m/z = 221) and its product ions at m/z = 134 and 162, (H) h2-derivatized 5-HTP (m/z = 249) and its product ions at m/z = 134 and 162, and (I) d2-derivatized 5-HTP (m/z = 253) and its product ions at m/z = 134 and 162.
Figure 2. The mass spectra of fragmented ions to set the multiple-reaction-monitoring (MRM) methods, the ion-pairs of mass-to-charge ratios (m/z) of the precursor, and product fragments. (A) Unmodified serotonin (m/z = 177) and its product ions at m/z = 115 and 160, (B) h2-derivatized serotonin (m/z = 205) and its product ions at m/z = 58 and 160, (C) d2-derivatized serotonin (m/z = 209) and its product ions at m/z = 62 and 160, (D) unmodified tryptophan (TRP) (m/z = 205) and its product ions at m/z = 118 and 146, (E) h2-derivatized TRP (m/z = 233) and its product ions at m/z = 102, 118, and 146, (F) d2-derivatized TRP (m/z = 237) and its product ions at m/z = 106, 118, and 146, (G) unmodified 5-HTP (m/z = 221) and its product ions at m/z = 134 and 162, (H) h2-derivatized 5-HTP (m/z = 249) and its product ions at m/z = 134 and 162, and (I) d2-derivatized 5-HTP (m/z = 253) and its product ions at m/z = 134 and 162.
Molecules 28 04580 g002
Molecules 28 04580 g003 550
Figure 3. Signal enhancement of serotonin via reductive amination and a comparison of the peak areas of the unmodified, h2-modified, and d2-modified analogs: unmodified serotonin (m/z = 177, retention time [rt] = 10.50 min, y-axis intensity = 6.0 × 106), h2-modified serotonin (m/z = 205, rt = 10.91 min, y-axis intensity = 8.2 × 106), and d2-modified serotonin (m/z = 209, rt = 10.91 min, y-axis intensity = 1.6 × 107).
Figure 3. Signal enhancement of serotonin via reductive amination and a comparison of the peak areas of the unmodified, h2-modified, and d2-modified analogs: unmodified serotonin (m/z = 177, retention time [rt] = 10.50 min, y-axis intensity = 6.0 × 106), h2-modified serotonin (m/z = 205, rt = 10.91 min, y-axis intensity = 8.2 × 106), and d2-modified serotonin (m/z = 209, rt = 10.91 min, y-axis intensity = 1.6 × 107).
Molecules 28 04580 g003
Molecules 28 04580 g004 550
Figure 4. Demonstration of the applicability of serotonin, tryptophan, and 5-HTP derivatization with different ratios of h2-modified and d2-modified analogs. (A) Serotonin analogs blended with h2-modified vs. d2-modified at 1:1, (B) serotonin analogs blended with h2-modified vs. d2-modified at 1:20, (C) serotonin analogs blended with h2-modified vs. d2-modified at 20:1, (D) serotonin analogs blended with different ratios of h2:d2 analogs, including 20:1 (20), 10:1 (10), 5:1 (5), 1:1 (1), 1:5 (−5), 1:10 (−10), and 1:20 (−20), and illustrating the expected ratios vs. observed ratios. The coefficient of determination is 0.9831. (E) Tryptophan analogs illustrated the expected ratios vs. observed ratios with a coefficient of determination at 0.9998. (F) 5-HTP analogs illustrated the expected ratios vs. observed ratios with a coefficient of determination at 0.9927.
Figure 4. Demonstration of the applicability of serotonin, tryptophan, and 5-HTP derivatization with different ratios of h2-modified and d2-modified analogs. (A) Serotonin analogs blended with h2-modified vs. d2-modified at 1:1, (B) serotonin analogs blended with h2-modified vs. d2-modified at 1:20, (C) serotonin analogs blended with h2-modified vs. d2-modified at 20:1, (D) serotonin analogs blended with different ratios of h2:d2 analogs, including 20:1 (20), 10:1 (10), 5:1 (5), 1:1 (1), 1:5 (−5), 1:10 (−10), and 1:20 (−20), and illustrating the expected ratios vs. observed ratios. The coefficient of determination is 0.9831. (E) Tryptophan analogs illustrated the expected ratios vs. observed ratios with a coefficient of determination at 0.9998. (F) 5-HTP analogs illustrated the expected ratios vs. observed ratios with a coefficient of determination at 0.9927.
Molecules 28 04580 g004
Table
Table 1. Modified and unmodified molecules of serotonin, tryptophan and 5-HTP with molecular formula, molecular weight, MRM transitions with m/z of precursor ion and m/z of product ions, LOD and LOQ parameters are listed.
Table 1. Modified and unmodified molecules of serotonin, tryptophan and 5-HTP with molecular formula, molecular weight, MRM transitions with m/z of precursor ion and m/z of product ions, LOD and LOQ parameters are listed.
NameMolecular FormulaMolecular Weight (Da)MRM TransitionsLOD # (ng/mL)LOQ # (ng/mL)Coefficient of Determination (R2)
Precursor IonProduct Ions
SerotoninC10H12N2O176.22177115, 160---------
h2-modified serotoninC12H16N2O204.2720558, 1602.3514.540.9969
d2-modified serotoninC12H12D4N2O208.2920962, 160---------
TryptophanC11H12N2O2204.23205118, 146---------
h2-modified tryptophanC13H16N2O2232.28233102, 118, 1461.964.360.9959
d2-modified tryptophanC13H12D4N2O2236.30237106, 118, 146---------
5-HTPC11H12N2O3220.22221134, 162---------
h2-modified 5-HTPC13H16N2O3248.28249134, 1621.3915.360.9938
d2-modified 5-HTPC13H12D4N2O3252.30253134, 162---------
# Limits of detection (LOD) and limits of quantification (LOQ) values were ascertained following a statistical method, LOD: average minimum recognizable analytical signal of low-concentration samples + 3 × standard deviation of analytical signal of low-concentration samples; LOQ: average analytical signal of low-concentration samples + 10 × standard deviation of analytical signal of low-concentration samples (n ≥ 6).
Table
Table 2. The signal-enhancement statistical data of modified and unmodified serotonin, tryptophan and 5-HTP in MRM method.
Table 2. The signal-enhancement statistical data of modified and unmodified serotonin, tryptophan and 5-HTP in MRM method.
MRM/Average Peak Area (n = 4)
UnmodifiedRSD (%)d2-ModifiedRSD (%)Enhancement
Serotonin55,030,5864.265,536,4814.51.19-fold
Tryptophan6,765,2263.8127,646,7451.218.87-fold
5-HTP2,193,5036.35,257,5002.42.40-fold
Table
Table 3. Statistical intra-day and inter-day data for serotonin, tryptophan and 5-HTP analogs (n = 3).
Table 3. Statistical intra-day and inter-day data for serotonin, tryptophan and 5-HTP analogs (n = 3).
SerotoninTryptophan5-HTP
Concentration (ng/mL)Intra-DayInter-DayIntra-DayInter-DayIntra-DayInter-Day
5003.5–6.9%4.0%1.2–3.8%6.4%0.6–6.1%6.7%
1002.2–6.4%3.0%1.6–8.6%7.9%1.7–9.7%5.7%
503.4–3.9%9.7%2.0–10.8%3.7%4.1–16.3%6.1%
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Liang, S.-S.; Shen, P.-T.; Liang, Y.-Q.; Ke, Y.-W.; Cheng, C.-W.; Lin, Y.-R. Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry. Molecules 2023, 28, 4580. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28124580

AMA Style

Liang S-S, Shen P-T, Liang Y-Q, Ke Y-W, Cheng C-W, Lin Y-R. Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry. Molecules. 2023; 28(12):4580. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28124580

Chicago/Turabian Style

Liang, Shih-Shin, Po-Tsun Shen, Yu-Qing Liang, Yi-Wen Ke, Chieh-Wen Cheng, and Yi-Reng Lin. 2023. "Assisted Reductive Amination for Quantitation of Tryptophan, 5-Hydroxytryptophan, and Serotonin by Ultraperformance Liquid Chromatography Coupled with Tandem Mass Spectrometry" Molecules 28, no. 12: 4580. https://0-doi-org.brum.beds.ac.uk/10.3390/molecules28124580

Article Metrics

Back to TopTop