Dyestuff Analyses on Historic Objects by LC-ESI-MS/MS Using a Superspher RP-18 HPLC Column

By: Prof. Dr. René Csuk and Dr. Annemarie E. Kramell
Department of Organic Chemistry, Martin-Luther-University, Halle-Wittenberg, Germany

Introduction

Prior to the development of synthetic dyestuffs starting in the middle of the 19th century, organic dyes have only been gained from a variety of plants, fungi, lichens and insects. These colorants have been used to color skin, hair, foods, textiles and other artifacts. For instance, various ancient cultures dyed textiles with a brilliant red hue utilizing scale insects of the superfamily Coccoidea.

Dyestuff analyses on historic objects give some clues concerning applied dyeing technologies, available resources and questions of cultural exchange. Furthermore, material analyses are essential for the conservation and restauration of these objects. However, historic colorations are often based on complex mixtures of different compounds. Thus, for the separation and characterization of these dyestuffs, liquid chromatographic techniques are commonly considered as methods of choice. In this study, LC-ESI-MS/MS under reversed phase conditions using a Superspher RP-18 column is applied to analyze dyestuffs of a reddish historic textile from South America.

 

Detected compounds - anthraquinone dyes gained from scale insects of the genus Dactylopius
Substance R1 R2 Molecular formula Nominal mass (Da)
Carminic acid C-α-D-Glucopyranosyl OH C22H20O13 492
Flavokermesic acid H H C16H10O7 314
Kermesic acid H OH C16H10O8 330

 

Detected compounds - anthraquinone dyes gained from scale insects of the genus Dactylopius

 

Experimental Conditions
Column LiChroCART® Superspher RP-18 (4 µm) 125x2 mm + LiChroCART® LiChrospher RP-18 (5 µm) 4x4 mm Injection volume: 5 µL
Detection API 2000 mass spectrometer from Applied Biosystem (ion source: ESI, MRM mode) Flow Rate: 0.3 mL/min
Mobile phase A Acetonitrile + 0.1 % formic acid (v/v) Temperature: 25 °C
Mobile Phase B Water + 0.1 % formic acid (v/v)
Time (min) A (%) B (%)
0 20 80
2 20 80
3 40 60
10 75 25
11 100 0
12 100 0
Gradient See table
Sample material Reddish wool fibers of a historic fabric fragment (Ichma culture, 1100 – 1440 AD)
Sample preparation The sample (5 mg) was extracted with MeOH/H2O (200 µL, 1:1 v/v) in the presence of formic acid (200 µL, 5 M) for 5 min at 100 °C under reflux. An aqueous solution of EDTA (200 µL, 0.5 mM) was added, and heating under reflux was continued for another 5 min at 100 °C. After cooling to room temperature, the supernatant was removed, and the extraction process was repeated twice. The remnants of the textile were rinsed with MeOH; this solution and the supernatants were combined, filtered (0.2 µm, PTFE-filter), and the solvents were evaporated under a gentle flow of argon at 65 °C. The residue was suspended in a mixture of MeOH (100 µL) and HCl (conc., 20 µL) and stirred at room temperature for 3 min. The mixture was extracted with ethyl acetate (6 x 100 µL), the organic phases were combined, and the solvent was evaporated at 65 °C using a gentle flow of argon. The remaining residue was dissolved in ACN/MeOH 1:1 (v/v), and an aliquot of this solution was directly injected into the LC-MS/MS-system.

 

MRMs of an extract of a historic sample (anion sensitive detection)

MRMs of an extract of a historic sample (anion sensitive detection)

Chromatographic Data

No. Compound MRM transition (m/z) Retention Time (min) S/N Tailing Factor
1 Carminic aicd 491 → 357
491 → 327
10.20
10.20
24
28
1.32
1.48
2 Kermesic acid 329 → 285 12.77 5 1.50
3 Flavokermesic acid 313 → 269 12.66 27 2.12