Determination of monoamine neurotransmitters and metabolites by high-performance liquid chromatography based on Ag(III) complex chemiluminescence detection

Journal Pre-proof Determination of monoamine neurotransmitters and metabolites by high-performance liquid chromatography based on Ag(III) complex chemiluminescence detection Li Ma, Tangjuan Zhao, PingPing Zhang, Mengying Liu, Hongmei Shi, Weijun Kang PII:

S0003-2697(19)31233-3

DOI:

https://doi.org/10.1016/j.ab.2020.113594

Reference:

YABIO 113594

To appear in:

Analytical Biochemistry

Received Date: 8 December 2019 Revised Date:

20 January 2020

Accepted Date: 20 January 2020

Please cite this article as: L. Ma, T. Zhao, P. Zhang, M. Liu, H. Shi, W. Kang, Determination of monoamine neurotransmitters and metabolites by high-performance liquid chromatography based on Ag(III) complex chemiluminescence detection, Analytical Biochemistry (2020), doi: https:// doi.org/10.1016/j.ab.2020.113594. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Author statment

I declare that all authors contribute to this article to varying degrees and assume different roles. Each of them contributes as follows: Li Ma: Methodology, Writing- Original draft preparation. Tangjuan Zhao: Formal analysis PingPing Zhang: Investigation Mengying Liu: Data curation, Validation. HongmeiShi: Writing - Review & Editing Weijun Kang: Funding acquisition

Determination of monoamine neurotransmitters and metabolites by high-performance liquid chromatography based on Ag(III) complex chemiluminescence detection Li Ma1,2 , Tangjuan Zhao1,2, PingPing Zhang3, Mengying Liu1,2 ,HongmeiShi1,2, Weijun Kang1,4

1. School of Public Health, Hebei Medical University, Shijiazhuang 050017, China 2. Key Laboratory of Environment and Human Health of Hebei Province, Shijiazhuang, 050017, China 3. Department of Reproductive Genetic Family, Hebei General Hospital, Shijiazhuang, 050011, China 4. Key Laboratory of Forensic Medicine of Hebei Province, Shijiazhuang 050017, China

Abstract A novel, simple and efficient chemiluminescence system has been developed for the determination of monoamine neurotransmitters and metabolites. By using the Ag (III)-luminol chemiluminescence system as a

detector,

a

high

performance

liquid

chromatography

chemiluminescence method (HPLC-CL) was established and used to detect seven monoamine neurotransmitters. Under the optimized conditions, the detection limits (3S/N) of epinephrine (E), levodopa (L-DOPA),

dopamine

(DA),

serotonin

(5-HT),

3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxypentylacetic acid (5-HIAA) were 20.0 µg dm-3,15.0 µg dm-3, 15.0 µg dm-3, 8.0 µg dm-3, 2.0 µg dm-3, 2.0 µg dm-3 and 3.0 µg dm-3, respectively. Moreover, they were well within the linear

range of 50-1000 µg dm-3, 50-1000 µg dm-3, 50-1000 µg dm-3, 25-1000 µg dm-3, 5-25 µg dm-3, 5-25 µg dm-3 and 10-30 µg dm-3, respectively. The average recovery varied between 84.82 % and 110.4%. The method has the attributes of simplicity, high sensitivity, and high efficiency. The sensitization and inhibition mechanisms for luminol-[Ag(HIO6)2]5–analytes CL system were proposed by CL spectra and free-radical capture experiment.

Keywords: Luminol-[Ag(HIO6)2]5-, Monoamine neurotransmitter, Metabolites，High-performance liquid chromatography， Chemiluminescence, Mechanism

1. Introduction Monoamine neurotransmitters (MNTs) and their metabolites primarily include norepinephrine (NE), epinephrine (E), dopamine (DA), 5-hydroxytryptamine

(5-HT),

levodopa

(L-DOPA),

3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxytryptamine (5-HIAA) (structures shown in Fig.1). L-DOPA is a precursor to the synthesis of DA. MHPG is the final metabolite of NE. DOPAC and 5-HIAA are respectively the metabolite of DA and 5-HT. MNTs and metabolites play an important role in the transmission of nerve signals via the central nervous system and have important physiological functions. The concentrations of the MNTs and their metabolites in blood are important for the diagnosis of diseases like schizophrenia [1], depression [2,3], Parkinson's disease [4] and Alzheimer's disease [5]. Therefore, accurate determination of these metabolites is essential for understanding the neurotoxic effects of substances and for the diagnosis of diseases as well as for evaluating the efficacy of drugs.

Fig.1. The molecular structures of seven MNTs and metabolite At present, the methods for determination of MNTs in diagnostic centers are based on enzyme-linked immune sorbent assay (ELISA) and electrochemical methods [6]. Although these methods are sensitive, they cannot be used for the simultaneous determination of multiple neurotransmitters. High-performance liquid chromatography with an electrochemical detector [7], fluorescence detector [8,9] and mass spectrometer detector [10-12] is considered one of the most powerful and popular techniques for MNTs analysis today. Although fluorescence and mass spectrometer detector are highly sensitive, the instruments are complex,

costly

or

difficult

to

operate.

However,

in

the

chemiluminescence (CL) method, the need for an excitation light source is eliminated. Thus, HPLC combining CL detection techniques have the advantages of sensitivity, accuracy, low interference, low cost and ease of operation [13,14].

The chemiluminescence system composed of luminol and hydrogen peroxide

or

luminol

and

potassium

ferricyanide

are

classical

chemiluminescence systems, which have been widely used in the analysis of environmental and biological samples. In a previous study, we found that, [Ag(HIO6)2]5- (short for Ag(III)), as a unusual oxidation metal complex, has strong oxidizability under alkaline conditions. The new system of Ag (III) - luminol chemiluminescence has the following advantages compared with known systems such as lumino-hydrogen peroxide, luminol - potassium ferricyanide: (1) The kinetic curve of chemiluminescence reaction shows that the chemiluminescence reaction is rapid. (2) It has a stronger luminescence efficiency at the same concentration of oxidant level. (3) It avoids the common bubble problem in the luminol - hydrogen peroxide system and the self-absorption problem produced by unreacted potassium permanganate. A variety of bioactive molecules can sensitize or inhibit the luminescence signal of this system [15-19]. Given its many advantages, it could be used as a new high performance liquid chromatography chemiluminescence detector, as a beneficial supplement to ultraviolet, mass spectrometry and other detectors, and will play an important role in many analytical fields. In this work, a new HPLC- CL detector was developed for the sensitive, accurate and rapid determination of seven monoamine neurotransmitters and metabolites in human serum based on the sensitizing or inhibiting

effects of seven analytes on Ag (III)-luminol CL system. Moreover, the possible mechanisms of signal sensitization and inhibition were proposed that could promote its application to a wider range of areas. 2. Experimental 2.1 Chemicals E, MHPG, DOPAC and 5-HIAA standard (purity ≥ 98%) were obtained from Sigma-Aldrich. DA, L-DOPA and 5-HT were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Luminol was purchased from TCI Company (purity ≥ 98%, Shanghai, China). 2.2 Preparation of solutions Stock solutions of

E, MHPG, L-DOPA, DA, 5-HT, DOPAC and

5-HIAA were prepared in 0.5% HCL containing 1mg dm-3 Na2S2O5 at a final concentration of 1 mg dm-3 each. The stocks were diluted serially with water and stored in the dark at 4 °C. A luminol stock solution was prepared by dissolving luminol (TCI, Japan) in 1 mol dm-3 NaOH solution. The synthesis of bis(hydrogenperiodato)argentate(III) or Ag(III) was carried out according to the procedure described previously.The concentrations were determined spectrophotometrically at 362 nm using Lambert-Beers law, and a molar absorbtivity (ε) = 1.26 × 104 (mol dm-3)–1cm–1 [22]. 2.3 Instrumentation and experimental conditions

The HPLC-CL detection system consist an HPLC system and a post-column CL detection system (illustrated in Fig.2). The HPLC (Shimadzu, Japan) was equipped with two pumps, a UV detection system, a manual sample valve injector with a 20 μL loop, and C18 columns (4.6mm × 250mm × 5 µm, DIKMA, China). The CL detection system used was Flow-Injection Analysis (FIA) CL System (Xi’an Remax Electronic Science-Tech Co. Ltd.). The fluorescence spectra were obtained using an F-7000 fluorescence spectrophotometer (Hitachi).

Fig. 2. Schematic diagram of the HPLC with Ag(III)-luminol chemiluminescence detection

The mobile phase A comprised of 15 mmol dm-3 KH2PO4 solution (containing 3% methanol), and B, which is methanol. The gradient elution conditions established were as follows: 0 ~ 15% B(0 ~ 9 min), 15 ~ 20% B (9 ~ 12 min), 20% B (12 ~ 20 min) and 0% B (20-24 min). The flow rate was kept at 1 mL min-1 and the column temperature was maintained at 30°C.

2.4 Sample preparation Serum samples of clinical patients were obtained and the protein was precipitated using 1mol dm-3 perchloric acid in the ratio of 2:1. The vortex was mixed for 30 s and centrifuged for 5 min at 15000 rpm at 4 °C. The samples were then filtered and the filtrate obtained was diluted with the mobile phase. 3. Results and Discussion 3.1 Optimization of Chromatographic conditions For the HPLC-CL method, selection of a proper mobile phase was important. The mobile phase should be suitable for the separation of analytes and compatible with the CL reaction. Methanol - acetic acid，methanol - acetic acid - sodium acetate and methanol - monopotassium phosphate were tested. Experimental results show the separation effect of methanol-acetic acid was poor, and the peak trailing was serious. Sodium acetate inhibits chemiluminescence signal. Monopotassium phosphate make the peak sharp and good symmetry. The reason may be that increasing the ionic strength of the solution can effectively inhibit the dissociation of analytes. Therefore, methanol monopotassium phosphate were chosen as the mobile phase solution for the experiment. If the phosphate concentration is too high, it is easy to precipitate in methanol. Finally, 15 mmol dm-3 KH2PO4 was selected

for all subsequent experiments. After optimization, the gradient elution procedure for sample separation was determined to be: 0 ~ 9 min, 0 ~ 15 % B; 9~12 min, 15 % ~ 20 % B; 12~30 min, 20% B; 0% B (20-24 min). Here, A is 15 mmol dm-3 KH2PO4 (containing 3% methanol, pH=4.61); and B is methanol. 3.2 Optimization of Chemiluminescence conditions The concentration, alkalinity and flow-rate of CL reagents, as well as the length of the pipeline has significant effects on the luminous intensity. By varying single factors, the optimal condition for detection was selected. The optimization results of the experiment are listed in Table 1. Table 1 Optimization of CL conditions for the determination MNTs and their metabolites. Variable [Luminol] ( µmol dm-3) [Ag(III)] (µmol dm-3) [OH-]in Ag(III)(mol dm-3)

Study Range 0.1-1.0 50-200 0.01-0.10

Flow rate (mL min-1) L1 lenth (cm) L2 lenth (cm)

1.5-4.5 1-5 2-10

Optimum Condition 0.1 80 0.03 for E, L-DOPA, MHPG and DA 0.04 for 5-HT, DOPAC and 5-HIAA 2.2 3.0 8.0

3.2.1 Optimization of luminol concentration The CL signal of the analytes was found to increase with increasing luminol concentrations. However, the baseline noise also increased

simultaneously. Based on our observations, an optimum signal to noise ratio was obtained at a luminol concentration of 1 × 10-7 mol dm-3. 3.2.2 Optimization of Ag(III) concentration The effect of Ag (III) complex concentration on the CL signal was investigated within the range of 0.5×10-4 mol dm-3 and 2×10-4 mol dm-3 (Fig. 3). The results showed that the CL signal of E and NE reached maximum when the Ag (III) concentration was 0.6 × 10-4 mol dm-3. The signal of DA and 5-HT reached maximum when Ag (III) concentration was 1.0 × 10-4 mol dm-3 and 0.8 × 10-4 mol dm-3. No significant changes in the luminous intensity of MHPG, L-DOPA, DOPAC and 5-HIAA were observed within the Ag (III) concentration range. Therefore, the most optimum concentration of Ag (III) for subsequent experiments was determined to be 0.8 × 10- 4 mol dm-3. 3000

CL intensity

2500 2000 1500

a

1000

b c d ef g

500 0

40

60

80

100

120

Ag (III) concentration(µmol dm-3) Fig. 3 Effect of [Ag(III)] concentration on the CL intensity.

Condition: MHPG(a): 20 µg dm-3; 5-HT (b): 40 µg dm-3; DOPAC (c): 20 µg dm-3; DA (d): 20 µg dm-3; 5-HIAA (e): 20 µg dm-3 ; E(f): 40 µg dm-3; L-DOPA (g): 20 µg dm-3 3.2.3 Optimization of NaOH in Ag(III) In the range of 0.01 ~ 0.1 mol dm-3, the concentration of NaOH in Ag(III) was optimized (Fig.4). The results show that the CL intensity increases at first and then decreases with the increase of NaOH concentration. The change of NaOH concentration has no obvious effect on the CL intensity for L-DOPA, DOPAC and 5-HIAA. Since DA has the largest signal at a alkalinity of 0.03 mol dm-3, hence, the concentration of NaOH in Ag(III) was identified as 0.03 mol dm-3 for detection of E, MHPG and DA . The [NaOH]= 0.04mol dm-3 was used for determination of 5-HT, DOPAC and 5-HIAA. 1600

a

CL intensity

1200

800

b 400

c d e f

0

g 0.02

0.03

0.04

0.05

-3 [OH-]in Ag(III)(mol dm )

0.06

Fig. 4 Effect of [OH– ] in [Ag(III)] and luminol solutions on the CL intensity. Condition: MHPG(a): 10 µg dm-3; L-DOPA(b):10 µg dm-3; E(c):20 µg dm-3; DOPAC(d): 20 µg dm-3; 5-HIAA(e):20 µg dm-3; 5-HT(f):20 µg dm-3; DA(g):20 µg dm-3 3.3 Detection limit, linearity, and precision Under the optimized condition, the standard curve equations, dynamic range and relative standard deviation and other variable were obtained and listed in table 2. The detection limits(LOD) and quantitation limits(LOQ）of the seven analytes were defined as the peak heights three times and ten times the baseline noise. LODs of 20.0 µg dm-3, 2.0 µg dm-3, 15.0 µg dm-3, 15.0 µg dm-3,8.0 µg dm-3, 2.0 µg dm-3 and 3.0 µg dm-3 were found for E, MHPG, L-DOPA, DA, 5-HT, DOPAC and 5-HIAA, respectively. RSD values in Table 1 correspond to 250µg dm-3 for E, DA, L-DOPA, 5-HT and 25μg dm-3 for MHPG, DOPAC and 5-HIAA. Table 2. Regression equation parameters of MNT and its metabolite r

LOD

LOQ

RSD

Regression equation

Linearity range

Analytes E

y = 10.79x + 239.7

50.0~1000.0

0.9999

20.0

50.0

2.8

MHPG

y = 41.92x + 207.7

5.0~25.0

0.9988

2.0

5.0

4.0

L-DOPA

y =12.02x + 316.5

50.0~1000.0

0.9996

15.0

50.0

3.5

DA

y =14.64x + 40.96

50.0~1000.0

0.9991

15.0

50.0

1.8

5-HT

y = 24.46x -79.2

25.0~1000.0

0.9999

8.0

25.0

3.0

DOPAC

y = 48.46x + 138.8

5.0~25.0

0.9998

2.0

5.0

2.0

5-HIAA

y =19.80x + 52.18

10.0~30.0

0.9990

3.0

10.0

0.5

（µg dm-3）

（µg dm-3） (µg dm-3 )

(n=5 ,% )

Under the conditions, the seven analytes were completely separated within 20 minutes. The CL chromatograms of blank serum and spilked serum were shown in Fig.5. The added standard level are 125 µg dm-3 for E, L-DOPA, DA, 5-HT, and 12.5 µg dm-3 for MHPG, DOPAC, 5-HIAA.

12000

CL intensity

10000 5-HT

8000 6000

E

L-DOPA DA

4000 DOPAC

MHPG

2000 200

5-HIAA

400

600

800

1000

1200

Time (s) Fig. 5. CL chromatograms of blank sample and spilked sample red line: blank sample; black line: spilked sample

3.4 Applications The current method was applied to determine seven MNTs and metabolites in human serum. Figure 6 shows the typical chromatograms obtained from human serum samples and human serum spiked with seven analytes. The recoveries of the seven MNTs and metabolites, investigated by

adding standard solutions to human serum in the ratio of 1:1 to examine the reliability of the method (Table 3). As can be seen from Table 3, the recovery rates of the seven analytes varied between 84.82 % to 110.4 %. RSD was 0.5% ~ 5.0% for three determinations. Table 3 Recovery of the seven analytes in serum sample

Average

Average

found

recovery

（µg dm-3）

（%）

50.00

42.41

84.82

3.1

100.0

104.9

104.9

2.9

200.0

195.6

97.79

2.8

5.000

5.490

109.8

4.9

10.00

9.810

98.15

5.0

20.00

19.07

95.37

4.4

50.00

45.10

90.20

3.6

100.0

101.5

101.5

3.5

200.0

198.0

99.01

3.2

50.00

43.16

86.32

2.4

100.0

103.0

103.0

2.1

200.0

196.0

98.01

1.8

50.00

42.85

85.69

2.8

100.0

101.6

101.6

3.1

200.0

201.8

100.9

3.0

5.000

5.010

100.2

4.0

10.00

11.04

110.4

2.6

20.00

19.65

98.27

2.1

5.000

4.640

92.87

2.2

10.00

10.00

100.0

1.1

Added Analytes

E

MHPG

L-DOPA

DA

5-HT

DOPAC

5-HIAA

（µg dm-3）

RSD (%, n=3)

20.00

20.00

100.0

0.5

14000

CL intensity

12000

5-HT

10000 8000 6000

L-DOPA

DA

E

4000 DOPAC

2000

5-HIAA

MHPG

0 200

400

600

800

1000

1200

Time/ s Fig.6. Chemiluminescence chromatogram of serum samples black line: human serum; red line: spilked human serum

3.5 CL mechanism study The mechanism of the CL reaction was investigated by studying the CL spectra obtained from the assembly of fluorescence spectrometer and FIA system. Figure 7 shows the maximum CL spectrum at about 425 nm, which is the characteristic wavelength of 3-aminophthalate. No new luminophores were generated in the Ag(III)-luminol-analytes system.

2500

B

CL intensity

2000 1500 A

1000 500

C

0 340 360 380 400 420 440 460 480 500 wavelength (nm) Fig. 7. CL spectra of Ag(III)-Luminol-analytes system A: Ag(III)-luminol;

B: Ag(III)-luminol-E;

C: Ag(III)-luminol-DOPAC

As observed in the free-radical capture experiment, some free radicals were produced when luminol reacted with Ag(III)as well as upon the action of certain reducing substances and Ag(III) [17]. Previous studies have shown that the free radicals produced from CL reaction were neither hydroxyl (·OH) nor superoxide radicals (·O2–) [21]. Based on our results, it may be inferred that luminol first was captured an electron by Ag (III) to form an anionic radical of luminol, and Ag(III) continues to oxidize the anionic radical to form 3-amino - phthalate of excited state (3-AP*) with an peak emission wavelength of 425nm when it returns to the ground state. DA/E /5-HT/L-DOPA is oxidized by Ag (III) to form DA/E/5-HT/L-DOPA free radical. Since the oxidation rate of

DA/E/5-HT/L-DOPA by Ag (III) is higher than that of luminol, a part of DA/ E/5-HT/L-DOPA radicals are transferred to luminol. As a result, the luminol radicals increase and CL signals enhanced. However, the reaction rates of MHPG/DOPAC/5-HIAA with Ag (III) is slower than luminol with Ag(III). Therefore, the luminol radical generated upon oxidization by Ag(III) is transferred to MHPG/DOPAC/5-HIAA partly, which reduces the number of luminophores, thereby demonstrating the inhibition of CL intensity. The mechanism were shown respectively in figure 8 and 9.

Fig.8. The sensitization mechanism inferred for CL system

Fig.9. The inhibition mechanism inferred for CL system 4. Conclusion A HPLC-CL method for the determination of seven monoamine neurotransmitters and metabolites was established and successfully applied for the detection of these analytes in human serum samples. The method had the advantages of being simple, rapid, selective, sensitive and accurate. Based on the CL spectra and previous studies, a possible mechanisms of sensitization and inhibition for CL system were proposed. Acknowledgment We gratefully acknowledge the financial support received from the National Science Foundation of China (81573202 and 81502846).

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Highlight ► We present a novel, sensitive and liable HPLC-CL method for the determination of seven monoamine neurotransmitters and metabolites in human serum. ► Luminol-[Ag(HIO6)2]5– chemiluminescence system was first used in the HPLC-CL method for monoamine neurotransmitters and metabolites analysis. ►The

sensitization

and

inhibition

mechanisms

Luminol-[Ag(HIO6)2]5–-analytes CL system were proposed.

for