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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

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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.

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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

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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.

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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).

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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/MSwith a Perkin Elmer micro pump.

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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 Nitrogenfilled 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.

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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.

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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.

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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.

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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

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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.

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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.

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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.

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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

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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.

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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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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43 Department of Chemistry S-901 87 Umeå, Sweden Telephone +46 90 786 50 00 Text telephone +46 90 786 59 00 www.umu.se

References

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