SUPPLEMENTARY MATERIAL

Use of a Core-shell Column for the Development of a Green LC Method for

Thiol Determination in Fresh Fruits Following Derivatization with Methyl Propiolate

Journal: Food Analytical Methods

Alexandra Samara, Anastasia Zotou*, Paraskevas Tzanavaras

Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki,

GR 54124 Thessaloniki, Greece

*Corresponding author: Prof.Anastasia Zotou

E-mail:

Fax: +30 2310 997719

Phone: +302310 997746

SUPPLEMENTARY MATERIAL

Use of a Core-shell Column for the Development of a Green LC Method for

Thiol Determination in Fresh Fruits Following Derivatization with Methyl Propiolate

Journal: Food Analytical Methods

Precision and Accuracy

Table S1. Within-day and between-day precision of assay for the determination of thiols in standard solutions

Thiol / Injected
concentration
(μmol L-1) / Mean measured concentrationa ± SD
(μmol L-1) / RSD
(%)
WD / BD / WD / BD
CYS / 3.0
10.0
50.0 / 2.95± 0.01
10.03±0.05
50.15± 0.06 / 2.93±0.06
9.93±0.04
50.10±0.12 / 0.3
0.5
0.1 / 2.0
0.4
0.2
GSH / 3.0
10.0
50.0 / 3.08± 0.02
9.78± 0.01
50.11± 0.08 / 3.10±0.03
9.52±0.05
50.14±0.17 / 0.6
0.1
0.2 / 1.0
0.5
0.3
NAC / 3.0
10.0
50.0 / 3.05± 0.03
9.33± 0.05
49.91± 0.04 / 3.06±0.04
9.52±0.02
49.94±0.05 / 1.0
0.5
0.08 / 1.3
0.2
0.1

a Means of values calculated from the regression line equations for 8 derivatizations within a day (WD) and 3 derivatizations per day over 6 consecutive days (BD)

Method Robustness

A ± 2% change in methanol volume fraction (i.e. values of 6% and 10% v/v) in the mobile phase was studied and compared with the 8% (v/v) used in the established method. These changes had rather slight effects on the retention times of theelutedpeaks. The resolution (Rs) of the GSH - NAC pair, however, seems to be most affected in thiscase, as shown in Figure S1.

Flow rate changes of ± 0.02 mL min-1, with respect to the original method, i.e. flow rates of 0.18 and 0.22 mL min-1 were also studied, and as shown in Figure S2 had negligible effect on the resolution.

The effect of mobile phase-pH on the elution behavior of the thioacrylates was studied at pH-values of 3.0, 4.0 and 5.0. As shown in Figure S3, atpH = 5.0CYS- and GSH derivatives co-eluted, whileatpH = 4.0the resolution (Rs) of the CYS-GSHpair is marginally ≈1. Good separationis achievedat pHvaluesof 3.0 and 2.2, the latter beingselectedasoptimumin order to achieve improved separation ofthiolsfromsample interferences. The elution order of the thioacrylates is affected by the ionization of the carboxylic acid moiety. The carboxylic acid groups are ionized when the pH of the mobile phase exceeds their pKa values (CYS: pKa(COOH) = 2.0; GSH: pKa(COOH)1 = 2.1, pKa (COOH)2 = 3.5; NAC: pKa(COOH) = 3.2) , resulting in shorter retention times, due to the more polar nature of the ionized analytes. At pH <3, the ionization of carboxylic acids is suppressed, minimizing potential ionic interactions with the surface of the silica stationary phase and generally increasing retention, due to the less polar nature of the non-ionized analytes.

According to the above findings, the method proved to be adequately robust.

Fig. S1 Effect of variations in mobile phase composition (volume fractionof methanol) on resolution; Cthiols=100 μmol L-1, Cbuffer=20 mmol L-1, CMP =20 mmol L-1 (final concentrations), pH=8.0, t=10 min, room temperature

Fig. S2 Effect of flow rate changes on resolution; Cthiols=100 μmol L-1, Cbuffer=20 mmol L-1, CMP =20 mmol L-1 (final concentrations), pH=8.0, t=10 min, room temperature

Fig. S3 Effect of mobile phase-pH on the separation of thioacrylates; Cthiols=100 μmol L-1,Cbuffer=20 mmol L-1, CMP =20 mmol L-1 (final concentrations), pH=8.0, t=10 min, room temperature; peak numbers 1, 2 and 3 are assigned to CYS, GSH and NAC respectively

Complience with Ethics Requirements

Conflict of Interest: Alexandra Samara declares that she has no conflict of interest. Anastasia Zotou declares that she has no conflict of interest. Paraskevas Tzanavaras declares that he has no conflict of interest.

This article does not contain any studies with human or animal subjects.

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