Micrograms enough for LC on dyestuffs

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  • Published: Jan 16, 2018
  • Author: Ryan De Vooght-Johnson
  • Channels: HPLC
thumbnail image: Micrograms enough for LC on dyestuffs

A number of traditional dyestuffs are based on extracts of plant alkaloids. These include extracts from the bark of the Amur cork tree (Phellodendron amurense), which have been used in China and Japan for centuries. The Oregon grape (Mahonia aquifolium) was used as a source of dye by many of the first peoples of North America; the plant is now widely used in gardens and for landscaping purposes. The European barberry (Berberis vulgaris) has also long been used as a source of dye; this bush has become naturalised in many other countries outside its native European range, including parts of the USA. The detection of dyes from these plants in historical artefacts, such as textiles or prints, is of considerable interest but requires techniques that use the minimum amount of sample.

The Warsaw scientists used capillary HPLC with diode array detection (DAD or PDA) in combination with LC-MS/MS with a sensitive triple quadrupole instrument in order to separate coloured alkaloids from extracts of the three plants. For comparison, samples of wool and silk were dyed with the plant extracts and small sub-samples (0.15 to 0.20 mg) were then taken for analysis.

Lyophilized bark samples were extracted with aqueous methanol (70% MeOH) using ultrasound followed by heating in a 60 °C hot water bath. The resulting extracts were filtered through a PTFE syringe filter.

HPLC employed an Agilent 1200 instrument. Three columns were tested: an Agilent Zorbax SB-C18 150 × 0.5 mm, a Phenomenex Synergi Fusion-RP 150 × 0.3 mm (modified with polar groups) and a Phenomenex Luna PFP(2) 150 × 1 mm (containing pentafluorophenyl groups). Although the latter two columns gave good separation, they also gave low-intensity MS peaks, so the Zorbax column was used. The two eluting solvents were 0.15% aqueous formic acid and methanol, with the proportion of the latter increasing from 5 to 59% over 12 minutes, then going from 59 to 100% over the next 8 minutes, and finally being kept at 100% for 10 minutes. DAD detection was carried out at eight different wavelengths: 254, 270, 330, 400, 450, 500, 550 and 620 nm. An Agilent 6460 triple quadrupole mass spectrometer was used with electrospray ionisation (ESI).

The optimised HPLC method gave good separation of the coloured alkaloids, while careful examination of their mass spectra allowed nearly all the main compounds to be identified. Berberine was the most abundant alkaloid for all three plants; it had a limit of detection (LOD) of 3.2 ng/mL and a limit of quantification of 9.7 ng/mL, both lower than with previous methods.

HPLCs were also run with extracts from the dyed textiles, which gave fairly similar traces to the plant extracts. Specific alkaloids were associated with particular plants. For example, phellodendrine and menisperine were only found with the Amur cork tree, not with the other two plants. Berbamine and oxyacanthine were found in relatively high levels in Oregon grape extracts, in trace amounts in barberry extracts and not at all in Amur cork tree extracts.

Alkaloid HPLC method gives information on historic artefacts

The new HPLC method enables alkaloid profiles to be obtained from very small samples of textiles, thus ensuring minimal damage. Knowing the compounds present can aid preservation work on historic items. The plants giving rise to the dyes can also be identified, giving valuable information on an artefact’s geographical origin and on ancient dyeing practices. It would be useful to extend this method to other dyestuffs.

Related Links

Electrophoresis, 2017, Early View Paper. Dąbrowski et al. Capillary-HPLC with tandem mass spectrometry in analysis of alkaloid dyestuffs – a new approach.

Bioanalysis, 2015, 7, 1397-1411. Rainville et al. Integration of microfluidic LC with HRMS for the analysis of analytes in biofluids: past, present and future.

Studies in Conservation, 2010, 55, 177-185. Zhang et al. Preliminary studies toward identification of sources of protoberberine alkaloids used as yellow dyes in Asian objects of historical interest.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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