Method development for the

determination of thiols using

HPLC with fluorescence

detection

Anja Andersson

Student

Degree Thesis in Chemistry 30 ECTS

Bachelor’s Level

Report passed: 21 June 2012

Supervisors: Sylvain Bouchet, Erik Björn

I

Abstract

Hydrophilic thiols of low molecular weight are involved in a variety of physiological

and environmental processes. Of utmost importance are their strong binding

capacities towards class B, “soft” metals. To achieve a better understanding of the

speciation and fate of those metals, especially mercury species, the reliable analysis

of the different thiols present in the environment is first required.

Among the different methodologies reported in the literature, Reverse Phase High

Pressure Liquid Chromatography coupled with Fluorescence Detection (RP-HPLC-FD)

appears of relative easiness and fit the different requirements for thiol analyses in

relation with metal speciation studies.

The main objectives of this work consisted in the evaluation of the possibility to

analyze other thiols than the ones described in the literature and refining the

existing chromatographic methods to measure thiols.

Based on excitation-emission spectra, 11 out of 17 selected thiols were first found

to be suitable to be analyzed by RP – HPLC – FD. Different combinations of buffers,

such as ammonium acetate and citrate and organic modifiers, such as acetonitrile,

methanol, ethanol and tetrahydrofuran (THF) were tested for mobile phase. Three

different columns, two C18 and one phenyl were also tested for their retention

capacities. In the end, two chromatographic methods using an ammonium acetate

buffer and different proportions of THF were developed to analyze those

compounds.

II

Table of content

Abstract…………………………………………………………………………………………………………I

1. Introduction………………………………………………………………………………………………….3

1.1 Aim…………………………………………………………………………………………………………4

2. Method

2.1 Instrumentation……………………………………………………………………………………..5

2.2 Chemicals and reagents…………………………………………………………….……………6

2.3 Preparation…………………………………………………………………………………………….6

2.3.1 Derivatization…………………………………………………………………………………6

3. Results and Discussion

3.1 Excitation-Emission scans……………………………………………………………………….7

3.2 Test with different eluents and organic modifiers…………………………………..9

3.3 Columns……………………………………………………………………………………………….14

3.4 Calibration Curve………………………………………………………………………………….15

4. Conclusions…………………………………………………………………………………………………18

5. References………………………………………………………………………………………………….19

6. Appendix…………………………………………………………………………………………………….20

3

1. Introduction

Hydrophilic thiols of low-molecular-mass are involved in a variety of physiological

and environmental processes. Being able to determine different thiol compounds is

considered to be highly relevant as thiols play important roles in metabolism, cellular

homeostatis and antioxidant defense networks just to mention a few. Non-protein

thiols such as Cysteine (Cys) and Glutathione (GSH) have key functions in cells, as

they regulate the activity of trace metal ions. The determination of thiol compounds

in biological matrices is of interest since the trace metals such as Cu, Ag and Hg have

a high affinity for SH groups in these low molecular mass ligands [1,2]. Thiols are also

important in the transport and bioavailability of trace metals in aquatic ecosystems.

Recently, the importance of Hg complexation by thiols has been emphasized due to

the bioavailability of the complexes formed. It appeared that some Hg-thiolates

complexes are available for bacteria involved in Hg methylation and may therefore

promote the formation of monomethylmercury, a potent neurotoxic. However,

interactions between Hg and thiols are not well defined and partly rely on accurate

and sensitive thiol measurements. Many techniques have been reported for thiols

analyses but liquid chromatography coupled with fluorescence detection appears as

one of the most widely used due sensitivity and relative easiness of the

instrumentation and samples preparation.

Figure 1. The first step shows how Cysteine spontaneously oxidizes. TCEP is used to cleave the

disulfide bond and convert the oxidized forms to the reduced thiol. After reduction, SBD-F is

attached by an elimination reaction and the new molecule formed is fluorescent.

4

As thiols compounds are not themselves fluorescent, a previous derivatization step

with a fluorescent probe (SBD-F) is required before separation by reverse phase

liquid chromatography (RP-HPLC).

Figure 2. Thiol structures and acronyms

5

1.2 Aim

The aim of this project was to further develop previously published analytical

protocol for thiols [3,4]. Indeed, those two different methodologies already allow

the analysis of 6 thiols (Cys, GSH, MAC, 3-MPA, NacCys, GluCys). However, others

appeared equally important in the biogeochemistry of different trace elements.

The main objectives were:

- To evaluate the possibility of analyzing additional thiols;

- To improve the chromatographic resolution for the most hydrophilic

thiols, i.e. Cysteine (Cys), Cysteamine (Cyst) and N-Cysteineglycine

(CysGly) which are usually co-eluting.

In order to do so, excitation-emission spectra were acquired to refine the

fluorescence detector wavelength setup and ESI-MS analyses were carried out to

check for sub-products causing unknown peaks. Selected compounds were then

analyzed with the instrumental setup described below.

2. Method

2.1 Instrumentation

The instrumentation setup included:

a Perkin Elmer (PE) high performance liquid chromatography (HPLC).

a PE quaternary 200 series pump;

a PE autosampler 200 Series;

a Jasco FP-920 fluorescence detector.

A Perkin Elmer Diode Array Detector 200 EP was also used to interface the system

with TotalChrom 6.3.1 Workstation software. Emission-Excitation scans were

performed on a Waters HPLC, Acquity and ESI-MS data were also acquired on a

Perkin Elmer sciex API 2000 LC/MS/MS with a Perkin Elmer micro pump.

6

Listed columns were tested during development of the analytical protocol:

Phenomenex C18 5 μm column C18, 2.0 x 150 mm

HAMILTON C18 HxSil 5 μm column 4.6 x 150mm

Intersil Ph-3 5 μm column 2.1 x 150 mm

2.2 Chemicals and reagents

Organic solvents used were of HPLC grade. Acetonitrile was purchased from Sigma,

tetrahydrofuran from VWR, methanol and ethanol from Baker. Trifluoroacetic acid

(TFA) 99+% spectrophotometric grade, n,n-dimethyl-formamide (DMF) and thiols

were purchased from Sigma. 7-Fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid

amomonium (SBD-F), methanesulfonic acid (MSA), tris(2-carboxyethyl)phosphine

hydrochloride salt (TCEP) from Fluka. Ammonium acetate was reagent grade quality

from Scharlau and Acetic acid from Merck.

2.3 Preparation

10 mM stock solutions of thiols were prepared in a deoxygenated ammonium

acetate solution (0.1 M) in a Nitrogen-filled glove box and stored in the freezer at -

20°C until used. 3 µM standard solution was then prepared weekly from the 10 mM

thiol stock solution. Subsequent dilutions were then performed to match the mobile

phase composition and carried out from the 3 µM standard solutions to a final

volume of 1.5 ml in suitable vials.

2.3.1 Derivatization

The SBD-F was dissolved in DMF and then diluted to a ratio 1:4 mg/ml. The thiols

standards were reduced in 10 min with the addition of 10 µl of a 10 % (w/v) TCEP

solution. After the reduction step, 200 µl of 0.1 M potassium borate buffer with 2.0

mM EDTA (pH 9.5), 40 µl SBD-F and 20 µl 1 M NaOH were added. The vials were then

incubated for 60 min at 60°C. The reaction was stopped after incubation time by the

addition of 100 µl of 1M MSA.

7

3. Results and Discussion

3.1 Excitation-Emission scans

Excitation-emission scans were acquired with scan wavelength for excitation from 300

to 400 nm and emission from 430 to 700 nm. All thiol derivatives were 30 mM and

showed an excitation wavelength centered around 365nm with a quite sharp peak

(Table 1 and Appendix).

The emission wavelengths scan on the other side showed a much broader peak in the

500 nm region, demonstrating that they are less sensitive to one specific emission

wavelength. This was found to be an advantage later on as this broad peak for each

thiol allowed their detection with the same emission wavelength. As illustrated in the

table below, the majority of the emission wavelengths are located in the spectra

between 470-520 nm. By selecting a wavelength in the middle, around 510 nm, all

thiols should be detected with high sensitivity. Cysteamine, Cysteinylglycine and

Mercaptoacetic acid were also tested with different organic modifiers to check if it

had any impact on the wavelength. The results show clearly that similar Excitation-

Emission wavelengths were obtained for all organic solvents. For the compounds that

were not detected with this instrument, tests were done with ESI-MS to try to see if

they were attached to the probe or not. Unfortunately, no conclusions could be made

from it; it seems that the probe was polymerized in the ESI source, preventing the

detection of the compounds.

As Cys, CysGly, GSH, MAC, Hcys, Cyst, Glyc, ETH, SULF, 2MPA and 3MPA gave clear

excitation-emission spectra, they were therefore selected for analysis with HPLC.

GluCys, NacPEN, NacCys, PYR, SUC and PEN were also tested but could not be

detected by RP-HPLC-FD. This can be explained by lower intensity (EU) on the

excitation-emission spectra. However, the intensity of 3MPA on excitation-emission

spectra was quiet low and could only be detected on the HPLC at 1000nM.

8

Table 1 Excitation-Emission wavelengths

Thiol Abbreviated Excitation (nm) Emission (nm)

Cysteine Cys 365 494

Glutathione GSH 365 512

Penicillamine* PEN 365 455

N-Acetyl-Penicillamine* NacPEN 365 455

Mercaptoacetic acid MAC 365 527

2-mercaptopropionic acid 2MPA 365 520

3-mercaptopropionic acid 3MPA 365 520

Cysteinylglycine CysGly 365 498

Cysteamine Cyst 365 508

γ-glutamylcysteine* GluCys 365 470

N-acetyl-L-cysteine* NacCys 365 470

Mercaptopyruvate* PYR 365 455

Homocysteine HCys 365 470

Mercaptosuccinic acid* SUC 365 455

Mercaptoethanol ETH 365 520

Mercaptosulfonate SULF 365 520

Monothioglycerol Glyc 365 521

Cysteamine 5% MeOH 365 505

Cysteamine 5% THF 365 505

CysGly 5%MeOH 365 598

CysGly 5% THF 365 598

MAC 5% MeOH 365 526

MAC 5% THF 365 526

*low intensity obtained in the Excitation-Emission scans carried out with the

Waters HPLC Acquity system.

9

3.2 Test with different eluents and organic modifiers

The Phenomenex C18 column was used throughout the following tests. The first

tests regarding mobile phase composition were carried out with 0.1 % (v/v) TFA (pH

2) and acetonitrile, according to Tang’s protocol [3] but problems were observed

such as co-elution of Cysteine, Cysteamine and N-Cysteineglycine and unstable

baseline. Alternative mobile phase compositions were tried as 30 mM ammonium

acetate (pH 5.8) and 30 mM ammonium citrate (pH 6.3) which showed clearly that

ammonium acetate buffer was more stable than the ammonium citrate buffer

(Figure 4) and was therefore mostly used later on. Different organic modifiers were

also tested according to different criteria: system stability including baseline drift

and bubbles formation perturbing the detector, column backpressure and signal

intensity. Ethanol and methanol were first tested with the citrate buffer. Ethanol is

supposed to enhance the signal intensity [5] but showed several drawbacks, such as

high column backpressure, unstable baseline and caused bubbles in the system,

whereas methanol on the other side was much more stable and there were no

problems with bubbles (figure 5 and 6). Why this happened is not clear, but one fact

could be that ethanol mixed with water has a reducing impact on the volume [6]. A

gradient with acetonitrile was also tested, but the unstable baseline made it

impossible to identify peaks (figure 7). Overall, a good gradient was hard to obtain

even with methanol where a problem with ghost peak appeared (figure 8 & 9). In the

end, THF was found out to be the best organic modifier in most aspects, i.e. good

peak shapes were obtained and the system was really stable. However, it was too

efficient for the more hydrophilic thiols and they eluted almost at the same time, at

the minimal mixing amount allowed by the pump (0.1% v/v). Therefore, a small

amount of THF (0.4 ‰ v/v) was instead added in the acetate buffer in order to

improve peak shape.

10

Figure 4. Baseline stability of isocratic run with 30 mM ammonium acetate and 30

mM ammonium citrate

Figure 5. Baseline stability of 30 mM ammonium acetate buffer with 5% (v/v)

methanol

11

Figure 6. Baseline stability of 30 mM ammonium acetate buffer and 5% (v/v) ethanol

Figure 7. A typical chromatogram obtained with the ammonium acetate buffer and

acetonitrile with a gradient from 5-20% (v/v), which shows the unstable baseline.

12

Figure 8. Chromatogram of ETH with gradient from 5-20% (v/v) methanol. The ghost

peak appears (around 10-11 min) in all the chromatogram due to the gradient

Figure 9. Chromatogram of Glyc, the same conditions as Figure 8 with the same ghost

peak.

13

Figure 10. MIX standard 250 nM of 1.Cys, 2.MAC, 3.Hcys, 4 Cysteamine/CysGly,

5.GSH (Isocratic 30 mM ammonium acetate buffer, 0.4 ‰ (v/v) THF and 0.1% (v/v)

methanol).

Figure 11. Isocratic run with 30 mM ammonium acetate buffer and 7% (v/v) THF of

MIX 250 nM 1. SULF, 2.Glyc, 3.ETH/2MPA.

14

3.3 Columns

An HAMILTON C18 and an Inertsil Phenyl columns were then tested to see if better

separation could be obtained for the co-elution between Cyst/CysGly and

ETH/2MPA. No significant improvement could be observed for the Hamilton column

and as can be seen in figure 11, the thiols eluted earlier with the phenyl column and

caused co-elution of several peaks. Up to now, the Phenomenex C18 gave the best

results.

Figure 12. Phenyl column (30 mM ammonium acetate buffer and 0.1% (v/v)

methanol)

MIX ISOCRATIC NEWC.raw

20

30

40

50

60

70

80

90

100

110

0 2 4 6 8 10 12 14 16 18 20 22 24

15

3.4 Calibration Curve

The data below have been collected from three different runs with Ex 365 - Em 510.

Table 2 and figure 13 present data from isocratic runs with 30 mM acetate buffer

and 0.1% (v/v) methanol, while Table 3 and figure 14 present data from an isocratic

run with 30 mM acetate buffer and 3% THF (v/v). The calibration curves for each

compound were drawn using the averaged peak area values from these three runs.

Standard deviations (SD) are always smaller than the markers.

In a similar way, data were processed to calculate slopes for peak heights versus

concentrations for each compound. The standard deviation of the baseline was also

calculated for different periods of the relevant chromatograms and the detection

limits (DL in nM) were calculated as:

The relative standard deviation (RSD) and detection limits (DL) are quite good for

most of the compounds, however Cys and MAC which give small peaks have much

higher DLs (20 nM respectively 24 nM) than the rest of the compounds. Detection

limits for Cyst/CysGly and ETH/2MPA are not accurate because of the co-elution, but

it gives a rough estimate. All these four compounds have high peaks, especially Cyst

and will probably be around the lowest detection limit of the compounds listed in

Table 2.

16

Table 2: Averaged peak areas (AV) standard deviation (SD) and relative standard

deviation (RSD) for different compounds.

100 250 500 1000 Slope R2 DL (nM)

Cys AV 50,5 111,3 210,2 411,5

SD 0,1 3,5 6,2 11,4 0,4155 0,99788 20

RSD 0,15 3,11 2,94 2,76

MAC AV 43,3 65,7 166,9 302,3

SD 1,5 2,9 1 1,9 0,3074 0,98848 24

RSD 3,42 4,39 0,57 0,64

Hcys AV 185,6 421,2 902,1 1808,3

SD 10,7 17 9,6 9,9 1,8021 0,9994 4

RSD 5,78 4,03 1,06 0,55

Cyst/CysGly AV 1849,4 3365,5 7367,1 19942

SD 18,9 64,3 21,2 45,4 18,64 0,96448 1

RSD 1,02 1,91 0,29 0,23

GSH AV 173,3 336,2 722,7 1579

SD 10,6 1,5 17,9 11,8 1,5439 0,99451 7

RSD 6,12 0,44 2,47 0,75

Figure 13. Calibration curve (peak area) for Cys, MAC, HCys (left y axis), Cyst/CysGly and GSH (right y axis

17

Table 3: Averaged peak areas (AV) standard deviation (SD) and relative standard

deviation (RSD) for different compounds.

100 250 500 1000 Slope R2 DL (nM)

SULF AV 50,5 111,3 210,2 411,5

SD 0,1 3,5 6,2 11,4 0,238 0,98846 2

RSD 0,15 3,11 2,94 2,76

Glyc AV 43,3 65,7 166,9 302,3

SD 1,5 2,9 1 1,9 0,1415 0,99073 3

RSD 3,42 4,39 0,57 0,64

ETH/2MPA AV 185,6 421,2 902,1 1808,3

SD 10,7 17 9,6 9,9 0,1949 0,98505 2

RSD 5,78 4,03 1,06 0,55

Figure 15. Calibration curve (peak area) of SULF, Glyc, and ETH/2MPA.

18

4. Conclusion

Excitation-emission scans were conducted to investigate excitation-emission

wavelength of the different thiols. The optimum wavelength was found out to be Ex

365 - Em 510. For better separation different buffers phases (TFA, ammonium

acetate, ammonium citrate) and organic modifiers (methanol, ethanol, THF,

acetonitrile) were tested. The most stable mobile phase was ammonium acetate

and the best organic modifier THF due to good peak shape. However the elution

strength of THF was too high for the more hydrophilic thiols so instead only a small

amount of THF were added to the ammonium acetate and methanol was used as

organic modifier. Another attempt to achieve better separation was to investigate

HAMILTON C18 and Intersil Phenyl columns instead of Phenomenex C18. No

significant improvement was observed with these two columns. 11 thiols

compounds can now be analyzed compared to only 6 with the original method. The

detection limits of the different thiols range from 2-25 nM. However, these 11 thiols

will have to be determined in 2 separate analyses because the mobile phase

conditions differed too much between the most hydrophilic and the most

hydrophobic ones. Attempts to develop a common gradient method remained

unsuccessful during this work.

19

5. Reference

[1] Damian. Shea, and William A. MacCrehan (1988) Determination of hydrophilic

thiols in sediment porewater using ion-pair liquid chromatography coupled to

electrochemical detection. Analytical Chemistry, 60 (14), 1449-1454.

[2] J. W. Rijstenbil and J. A. Wijnholds (1996) HPLC analysis of nonprotein thiols in

planktonic diatoms: pool size, redox state and response to copper and cadmium

exposure. Marine Biology, 127, 45-54.

[3] Degui Tang, Liang-Saw Wen, Peter H. Santschi (2000) Analysis of biogenic thiols

in natural water samples by high-performance liquid chromatographic separation

and fluorescence detection with ammonium 7-fluorobenzo-2-oxa-1.3-diazole-4-

sulfonate (SBD-F). Analytica Chimica Acta, 299-307.

[4] Jinzhong Zhang, Feiyue Wang, James D. House, and Bryan Page (2004) Thiols in

wetland interstitial waters and their role in mercury and methylmercury speciation.

Limnol. Oceanogr., 49 (6), 2276-2286.

[5] Toshimasa Toyo’oka and Kazuhiro Imai (1983) High-performance liquid

chromatography and fluorometric detection of biologically important thiols,

derivatized with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F)

[6] Lide, D R., ed (2000). CRC Handbook of Chemistry and Physics 81st edition. ISBN

0-8493-0481-4

20

6. Appendix

6.1 Excitation-Emission Spectra

Cysteamine (Cyst)

36.489 Extracted

508.1

EU

0.0

1000.0

2000.0

3000.0

4000.0

nm

450.00 500.00 550.00 600.00 650.00 700.00

21

Cysteine (Cys)

36.489 Extracted

493.9

EU

0.00

100.00

200.00

300.00

nm

450.00 500.00 550.00 600.00 650.00 700.00

22

Glutathione (GSH)

36.505 Extracted

512.4519.5

EU

0.00

200.00

400.00

600.00

800.00

nm

450.00 500.00 550.00 600.00 650.00 700.00

23

γ-glutamylcysteine (GluCys)

36.505 Extracted

469.7

EU

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

nm

450.00 500.00 550.00 600.00 650.00 700.00

24

3-mercaptopropionic acid (3MPA)

36.505 Extracted

519.5526.5

EU

0.00

50.00

100.00

150.00

200.00

250.00

nm

450.00 500.00 550.00 600.00 650.00

25

Cysteinylglycine (CysGly)

36.505 Extracted

498.2

EU

0.00

500.00

1000.00

1500.00

2000.00

nm

450.00 500.00 550.00 600.00 650.00

26

Homocysteine (HCys)

36.489 Extracted

469.7

EU

0.00

200.00

400.00

600.00

nm

450.00 500.00 550.00 600.00 650.00

27

Mercaptoacetic acid (MAC)

36.505 Extracted

526.5

EU

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

nm

450.00 500.00 550.00 600.00 650.00

28

N-acetyl-L-cysteine (NacCys)

36.489 Extracted

469.7

EU

0.00

20.00

40.00

60.00

80.00

100.00

120.00

nm

450.00 500.00 550.00 600.00 650.00

29

2-mercaptopropionic acid (2MPA)

36.489 Extracted

519.5526.5

EU

0.0

1000.0

2000.0

3000.0

4000.0

nm

450.00 500.00 550.00 600.00 650.00

30

N-Acetyl-Penicillamine (NacPEN)

36.489 Extracted

455.4462.6

EU

0.00

50.00

100.00

150.00

nm

450.00 500.00 550.00 600.00 650.00

31

Mercaptosulfonate (SULF)

36.489 Extracted

519.5

EU

0.00

500.00

1000.00

1500.00

nm

450.00 500.00 550.00 600.00 650.00

32

Mercaptoethanol (ETH)

36.505 Extracted

519.5526.5

EU

0.0

1000.0

2000.0

3000.0

nm

450.00 500.00 550.00 600.00 650.00

33

Mercaptopyruvate (PYR)

36.505 Extracted

455.4

EU

50.00

100.00

150.00

200.00

nm

450.00 500.00 550.00 600.00 650.00

34

Monothioglycerol (Glyc)

36.489 Extracted

519.5526.5

EU

0.00

500.00

1000.00

1500.00

nm

450.00 500.00 550.00 600.00 650.00

35

Penicillamine (PEN)

36.489 Extracted

455.4462.6

EU

0.00

50.00

100.00

150.00

nm

450.00 500.00 550.00 600.00 650.00

36

Mercaptosuccinic acid (SUC)

36.439 Extracted

455.4462.6

EU

0.00

50.00

100.00

150.00

nm

450.00 500.00 550.00 600.00 650.00

37

Cysteamine (Cyst) with 5% MeOH

36.489 Extracted

505.3

EU

0.0

1000.0

2000.0

3000.0

4000.0

5000.0

nm

450.00 500.00 550.00 600.00 650.00

38

Cysteamine (Cyst) with 5% THF

36.489 Extracted

505.3

EU

0.0

1000.0

2000.0

3000.0

4000.0

nm

450.00 500.00 550.00 600.00 650.00

39

Cysteinylglycine (CysGly) with 5% MeOH

36.505 Extracted

498.2

EU

0.00

500.00

1000.00

1500.00

nm

450.00 500.00 550.00 600.00 650.00

40

Cysteinylglycine (CysGly) with 5% THF

36.424 Extracted

498.2

EU

0.00

500.00

1000.00

1500.00

nm

450.00 500.00 550.00 600.00 650.00

41

Mercaptoacetic acid (MAC) with 5% MeOH

36.472 Extracted

526.5

EU

0.00

200.00

400.00

600.00

800.00

1000.00

1200.00

1400.00

nm

450.00 500.00 550.00 600.00 650.00

42

Mercaptoacetic acid (MAC) with 5% THF

36.489 Extracted

526.5

EU

0.00

200.00

400.00

600.00

800.00

1000.00

nm

450.00 500.00 550.00 600.00 650.00

43

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