Comparison of extraction methods for the analysis of natural dyes in historical textiles by high-performance liquid chromatography

Different methods for the extraction of Dactylopius coccus Costa, Rubia tinctorum L., Isatis tinctoria L., Reseda luteola L., Curcuma longa L. and Cotinus coggygria Scop. from wool fibres are investigated using high-performance liquid chromatography with diode array detector (HPLC-DAD). The efficiencies of five extraction methods which include the use of HCl (widely used extraction method), citric acid, oxalic acid, TFA and a combination of HCOOH and EDTA are compared on the basis of the (a) number, (b) relative quantities, measured as HPLC peak areas and (c) signal-to-noise ratios (S/N) of the compounds extracted from the wool substrates. Flavonoid glycosides and curcuminoids contained in R. luteola L. and C. longa L., respectively, according to liquid chromatography with mass spectrometry (LC-MS) identifications, are not detected after treating the fibres with HCl. All the other milder methods are successful in extracting these compounds. Experiments are performed using HPLC-DAD to compare the HPLC peak areas and the S/N of the following extracted compounds: indigotin, indirubin, curcumin, demethoxycurcumin, bisdemethoxycurcumin, fisetin, sulfuretin, luteolin, luteolin-7-O-glucoside, apigenin, carminic acid, alizarin, puruprin and rubiadin. It is shown that the TFA method provides overall the best results as it gives elevated extraction yields except for fisetin, luteolin, apigenin and luteolin-7-O-glucoside and highest S/N except for fisetin and luteolin-7-O-glucoside. It is noteworthy that treatment of the fibres with the typical HCl extraction method results overall in very low S/N. The TFA method is selected for further studies, as follows. First, it is applied on silk dyed samples and compared with the HCl method. The same relative differences of the TFA and HCl methods observed for the wool dyed samples are reported for the silk dyed samples too, except for rubiadin, luteolin and apigenin. Thus, in most cases, the nature of the substrate (wool or silk) appears to have negligible effects on the relative difference of the two extraction methods. Second, the selected TFA method is applied to treat wool and silk historical samples extracted from textiles of the Mamluk period, resulting in the identification of several colouring compounds. In all extraction methods mentioned above, DMSO is used to dissolve the dyes, after acid treatment.     

LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles

Identification of dyes in historic textiles was until recently only based on reversed phase liquid chromatography and diode-array detection (RPLC–DAD). Although in the last years mass spectrometry (MS) is increasingly used as a detection system for liquid chromatography, most applications in the field are directed to identification of the molecular ions or in studies dedicated to degradation products which may be used as markers in RPLC–DAD. In the present work, an analytical protocol for the identification of dyes using RPLC/ESI/MS is presented. Atmospheric pressure electrospray ionization (ESI) was applied, in the negative ion monitoring mode. Both single stage and tandem MS (MS/MS) approaches were considered. An ion trap was used as mass analyzer. Experiments are based on the characterization of standards (natural dyes and/or dyed fibers) with the mass spectrometer sequentially working in the following modes: single MS/full scan, followed by plotting chromatograms through ion extraction (IEC) according to mass/charge ratios corresponding to molecular ions; single MS/selected ion monitoring (SIM) mode; tandem MS/single reaction monitoring (SRM) mode; tandem MS/multiple reactions monitoring (MRM) or product ion scanning modes. A faster chromatographic separation could be applied as MS detection readily balanced the selectivity of the analytical process. In a case study, 11 dyes from 3 biological sources were detected in a 0.5 mg historic sample.     

Liquid chromatography determination of natural dyes in extracts from historical Scottish textiles excavated from peat bogs

Textiles excavated from Scottish sites belonging now to the collections of the National Museums of Scotland, including seventeenth century textiles from peat bogs in the Scottish Highlands and Islands, were selected for analysis by high performance liquid chromatography with photodiode array detection (PDA HPLC) to detect whether any dyes remained and, if so, to identify their biological sources. Dye components were identified in 36 of the 81 samples analysed. Although it was not possible to identify the exact sources of the dyestuffs because of the wide-spread occurrence of these natural dyes components, the study has shown that textiles previously not thought to have been coloured had detectable traces of dye. Before the historical textiles were analyzed, an improved extraction procedure that combined the routine acid hydrolysis method with one using dimethylformamide (DMF) was applied. The DMF method enabled increased recovery of major flavonoid and anthraquinoid compounds, and very high efficiency of recovery of indigotin even in textiles with no colour visible, thereby complementing the acid hydrolysis method already in use. Extracts from historical thread samples were analysed by PDA HPLC using a reversed-phase gradient system comprising of a C18 column (150 mm x 4.6 mm i.d., 25 +/- 1 degrees C) with water, methanol and o-phosphoric acid at an eluent flow rate of 1.2 ml/min. A preliminary investigation to improve the detection limits further for a selection of natural dyes was made by comparing results from the 4.6mm internal diameter (i.d.) column with a narrow bore C18 column (2.1 mm i.d.). An increase in the detector response was observed for narrow-bore column proving its possibility of enhancement of sensitivity.     

Investigation of natural dyes occurring in historical Coptic textiles by high-performance liquid chromatography with UV-Vis and mass spectrometric detection

Liquid chromatography (LC) combined with ultraviolet-visible (UV-Vis) and mass spectrometric (MS) detection was utilized to study the chemical components present in extracts of natural dyes originating from fiber samples obtained from Coptic textiles from Early Christian Art Collection of National Museum in Warsaw. Chromatographic retention, ionization, UV-Vis and mass spectra of twenty selected dye compounds of flavanoid-, anthraquinone- and indigo-types were studied. Most of the investigated compounds could be ionized by positive and negative ion electrospray ionization. Difficulties with the ionization by electrospray were experienced for indigotin and brominated indigotins, but these were ionized by atmospheric pressure chemical ionization. Mass spectrometric detection, utilizing different scanning modes of a triple quadrupole mass spectrometer, combined with the UV-Vis detection was demonstrated to be a powerful approach to detection and identification of dyes in the extracts of archeological textiles. Using this approach the following compounds were identified in the extracts of Coptic textiles: luteolin, apigenin, rhamnetin, kaempferol, alizarin, purpurin, xanthopurpurin, monochloroalizarin, indirubin, and so the type of dye that was utilized to dye the textiles could be identified. Detection capabilities for several dye-type analytes were compared for the UV-Vis and mass spectrometric detection. The signal-to-noise ratios obtained for luteolin, apigenin, and rhamnetin were higher for the MS detection for most of the examined sample extracts. Purpurin, alizarin, and indirubin showed similar signal-to-noise ratios for UV-Vis and mass spectrometric detection.     

Colour fading in textiles: A model study on the decomposition of natural dyes

This paper describes an analytical procedure based on GC-MS to identify in textiles the most common flavonoid yellow dyes used in Europe since ancient times, extracted from weld, young fustic, dyer's broom, sawwort and the berries of some species of Rhamnus. Later on, old fustic and quercitron bark were introduced as sources of yellow colours.The method is based on the solvent extraction of flavonoids from raw plant materials (weld, dyer's broom and old fustic), aged and not aged alum-mordanted wool dyed specimens; subsequently, flavonoids are derivatised with N,O-bis(trimethylsilyl)trifluoroacetamide and analysed by GC-MS. The method easily allows the identification of a dyestuff by the detection of the molecular markers apigenin, luteolin, genistein, morin, maclurin, together with 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,4,6- trihydroxybenzoic acid, which survive in aged textiles. Two photo-oxidative degradation pathways for colour fading, one involving the mordant metallic ion and the other the light as a catalyst, are suggested.     

LC/MS/MS study for identification of entacapone degradation product obtained by photodegradation kinetics

Entacapone is indicated for clinical use as an adjunct to levodopalcarbidopa to treat patients with idiopathic Parkinson's Disease who experience the signs and symptoms of end-of-dose wearing-off. The aim of this study was to determine the photodegradation kinetics and to elucidate the structure of the main degradation product. The stability of entacapone was studied in order to investigate the degradation kinetics of this drug using LC as a stability indicator. Entacapone was subjected to accelerated photodegradation. This study was carried out with methanolic solutions, prepared from coated tablets, in quartz cells under UV light at 254 nm. The degradation process of entacapone in solutions can be described by second-order kinetics under the experimental conditions used in this study. The LC/MS/MS determinations revealed that in the above conditions the photodegraded product formed the geometric isomer of entacapone (Z-entacapone). The obtained results show the importance of appropriate light protection during the drug development process, storage, and handling.     

Sampling and identification of natural dyes in historical maps and drawings by liquid chromatography with diode-array detection

A simple and rapid liquid chromatographic with diode-array UV-vis spectrophotometric detection (HPLC-DAD) method for identification of natural dyes has been developed. Chromatographic retention of carminic acid, indigotin, crocetin, gambogic acid, alizarin and purpurin has been studied. The mobile phase consisted of 40 mM SDS-10 mM phosphate buffer solution (pH 2.3)-0.1% TFA (eluent A) and acetonitrile (eluent B) using a programmed gradient (5% B to 95% B). Analyses were carried out on a Phenomenex, Luna 5u NH2 100(a) column (250 mm x 4.60 mm i.d., 5 microm particle) and the operating conditions were: 0.6 ml min(-1) flow rate, 20 microl volume injection and 35 degrees C column temperature. Extracts of samples of natural dyes taken from historical maps belonging to The Royal Chancellery Archives in Granada were successfully analyzed using the proposed method including a new technique for sampling.     

Simultaneous identification of natural dyes in the collection of drawings and maps from The Royal Chancellery Archives in Granada (Spain) by CE

A simple and rapid capillary electrophoretic method with UV detection (CE-UV) has been developed for the identification of five natural dyes namely, carmine, indigo, saffron, gamboge and Rubia tinctoria root. The separation was performed in a fused-silica capillary of 64.5 cm length and 50 microm id. The running buffer was 40 mM sodium tetraborate buffer solution (pH 9.25). The applied potential was 30 kV, the temperature was 25 degrees C and detections were performed at 196, 232, 252, 300 and 356 nm. The injections were under pressure of 50 mbar during 13 s. The method was applied to the identification of carminic acid, gambogic acid, crocetin, indigotin, alizarin and purpurin in the collection of drawings and maps at the Royal Chancellery Archives in Granada (Spain). The method was validated by using HPLC as a reference method.     

Analysis of natural red dyes (cochineal) in textiles of historical importance using HPLC and multivariate data analysis

A new analytical approach based on high-performance liquid chromatography with diode array detector (HPLC-DAD) and multivariate data analysis was applied and assessed for analyzing the red dye extracted from cochineal insects, used in precious historical textiles. The most widely used method of analysis involves quantification of specific minor compounds (markers), using HPLC-DAD. However, variation in the cochineal markers concentration, use of aggressive dye extraction methods and poor resolution of HPLC chromatograms can compromise the identification of the precise insect species used in the textiles. In this study, a soft extraction method combined with a new dye recovery treatment was developed, capable of yielding HPLC chromatograms with good resolution, for the first time, for historical cochineal-dyed textiles. After principal components analysis (PCA) and mass spectrometry (MS), it was possible to identify the cochineal species used in these textiles, in contrast to the accepted method of analysis. In order to compare both methodologies, 7 cochineal species and 63 historical cochineal insect specimens were analyzed using the two approaches, and then compared with the results for 15 historical textiles in order to assess their applicability to real complex samples. The methodology developed here was shown to provide more accurate and consistent information than the traditional method. Almost all of the historical textiles were dyed with Porphyrophora sp. insects. These results emphasize the importance of adopting the proposed methodology for future research on cochineal (and related red dyes). Mild extraction methods and HPLC-DAD/MSⁿ analysis yield distinctive profiles, which, in combination with a PCA reference database, are a powerful tool for identifying red insect dyes.     

Validation of multivariate screening methodology. Case study: Detection of food fraud

Multivariate screening methods are increasingly being implemented but there is no worldwide harmonized criterion for their validation. This study contributes to establish protocols for validating these methodologies. We propose the following strategy: (1) Establish the multivariate classification model and use receiver operating characteristic (ROC) curves to optimize the significance level (α) for setting the model’s boundaries. (2) Evaluate the performance parameter from the contingency table results and performance characteristic curves (PCC curves). The adulteration of hazelnut paste with almond paste and chickpea flour has been used as a case study. Samples were analyzed by infrared (IR) spectroscopy and the multivariate classification technique used was soft independent modeling of class analogies (SIMCA). The ROC study showed that the optimal α value for setting the SIMCA boundaries was 0.03 in both cases. The sensitivity value was 93%, specificity 100% for almond and 98% for chickpea, and efficiency 97% for almond and 93% for chickpea.     

Simple methodology coupling microwave-assisted extraction to SPE/GC/MS for the analysis of natural steroids in biological tissues: Application to the monitoring of endogenous steroids in marine mussels Mytilus sp.

A simple analytical procedure for the simultaneous determination of eight endogenous steroids (testosterone, androstenedione, 17β-estradiol, estrone, pregnenolone, progesterone, dihydroandrostenedione, and dihydrotestosterone) in aquatic molluscs by solid-phase extraction (SPE) and gas chromatography/mass spectrometry (GC-MS) has been developed. After a microwave-assisted extraction, samples were further extracted and purified using two successive SPE (EnviChrom-P and NH₂) cartridges. Steroids were derivatized with a mixture of N-methyl-N(trimethylsilyl)trifluoroacetamide (MSTFA)/mercaptoethanol/ammonium iodide (NH₄I) and determined by GC-MS in selective ion monitoring mode. Recoveries were in the range 85-114%, although slightly lower for dihydrotestosterone, and the repeatability of the procedure, expressed as the coefficient of variation, was lower than 16%. The limits of detection determined in digestive glands of mussels were in the range 0.1-0.4 ng g⁻¹ wet weight for all the steroids. The developed procedure was then applied to the monitoring of steroid profiles in the digestive glands of mussels from the Arcachon Bay (France) during two reproductive cycles. In parallel, two physiological parameters (lipid content and the condition index of mussels) were also monitored, as well as the seawater temperature and salinity. Only progesterone and pregnenolone were detected in the digestive glands of mussels, and the seasonal variations of progesterone levels seemed to be related to the spawning periods of Mytilus sp. in the Bay. The current challenge for the determination of natural steroids in aquatic invertebrates is also briefly discussed.     

Phytochemical analysis of young fustic (Cotinus coggygria heartwood) and identification of isolated colourants in historical textiles

Young fustic (Cotinus coggygria Scop.; Anacardiaceae) has been used as a dyestuff since antiquity. Phytochemical investigation of the methanol extract of the heartwood has led to the isolation and structure elucidation by nuclear magnetic resonance and mass spectrometry (MS) of 3′,4′,6-trihydroxyaurone (sulfuretin) and 3′,4′,7-trihydroxyflavonol (fisetin) as well as 3′,4′,7-trihydroxyflavanol (fustin), 3′,4′,5,7-tetrahydroxyflavonol (quercetin), 3′,4′,5,7-tetrahydroxyflavanol (taxifolin), 4′,7-dihydroxyflavanol, 3′,4′,7-trihydroxyflavanone (butin), 4′,7-dihydroxyflavanone (liquiritigenin), trans-2′,3,4,4′-tetrahydroxychalcone (butein), 4′,5,7-trihydroxyflavanone and trans-2′,4,4′-trihydroxychalcone (isoliquiritigenin). The isolated compounds were used as reference materials for the development of a high-performance liquid chromatography–diode array detector–MS method, which was then applied to analyse (1) fresh silk samples dyed with young fustic, (2) dyed silk subjected to artificially accelerated light ageing and (3) historical silk micro-samples, extracted from ecclesiastical post-Byzantine garments (fifteenth to eighteenth century), which belong to monasteries of Mount Athos. Sulfuretin and fisetin, which are usually used as markers for the identification of the yellow dye and, for the first time, some of the aforementioned flavonoid components of young fustic were identified in the historical extracts. Furthermore, preliminary experiments suggested that although the amounts of the dye components decrease with light ageing, the relative ratio of fisetin and sulfuretin, after a first step of ageing, seems to be almost unaffected by such degradation processes raised by light. The effect of the latter on the morphology of the dyed silk fibres is briefly investigated by scanning electron microscopy.     

Trace-level determination of pharmaceutical residues by LC-MS/MS in natural and treated waters. A pilot-survey study

A pilot-survey study was performed by collecting samples (influent and effluent wastewaters, rivers and tap waters) from different locations in Europe (Spain, Belgium, Germany and Slovenia). A solid-phase extraction (SPE) followed by liquid chromatography–tandem mass spectrometry method was applied for the determination of pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). Method detection limits and method quantification limits were at the parts-per-trillion level (7.5–75 ng/L). The recovery rates of the SPE from deionized water and effluent wastewater samples spiked at 100- and 1,000-ng/L levels ranged from 87 to 95%. Identification criteria in compliance with the EU regulation for confirmatory methods of organic residues were applied. A detailed study of signal suppression evaluation for analysis of pharmaceutical residues in effluent wastewaters is presented.     

Response surface methodology for the microwave-assisted extraction of insecticides from soil samples

The extraction of two pyrethroid insecticides (deltamethrin and α-cypermethrin) together with three organophosphorus insecticides (dimethoate, diazinon and malathion) from soil samples was carried out with microwave-assisted technology. Experimental designs showed that extraction temperature, addition of water to the extractant and solvent/soil ratio were the variables that affected the recoveries of the pesticide the most. Response surface methodology was applied to find the optimum values of the variables involved in the extractions of the analytes. In addition, in order to achieve near-optimal extraction conditions, a desirability function was used to optimize the five pesticides simultaneously. The optimized conditions were applied to different types of soils.     

Optimization of the solid phase extraction method for determination of Cu(II) in natural waters by using response surface methodology

A solid-phase extraction method was proposed for the preconcentration of Cu(II) in different samples in a mini-column packed with functionalized multi-walled carbon nanotubes (MWCNTs-COOH) as an effective sorbent, without using any complexing reagent, prior to its determination by flame atomic absorption spectrometry using response surface methodology. The experimental optimization step was performed by both a two-level full factorial design, with a center point, and a Box-Behnken design combined with response surface methodology. Three variables (pH, amount of Cu(II), and sample volume) were regarded as factors in the optimization. It was found that pH is the most significant factor affecting the preconcentration of Cu(II). The preconcentration factor was obtained as 100. The linear range was 1-5 mg L(-1) (R(2) = 0.999). Under the optimized experimental conditions, the detection limit (3s) of the proposed method followed by FAAS was found to be 0.27 μg L(-1). The relative standard deviation for 10 replicate measurements of 50 and 100 μg L(-1) Cu(II) was 2.39% and 0.98%, respectively. The response surface methodology was successfully applied to the determination of Cu(II) in water samples and mussel samples, and in a certified standard reference material (BCR-320R, Channel sediment).     

The methodology of knowledge acquisition from the collection of IR and UV spectra

An expert system for identification of parts of chemical molecules from IR and UV spectra is described. To develop the system, the intelligent knowledge engineering program environment SCANKEE¹ (elaborated at the Department of Computer Chemistry) was applied. The expert systems for identification of a chemical compound described in the literature use hand-crafted knowledge bases. To eliminate the time-consuming writing of the rules, a module for the automatic creation of rule knowledge bases was developed. The knowledge derives from the collection of molecular spectra of organic compounds and their structural formulae. The algorithm for the automatic generation of the production rules (in the rule knowledge base) is realized in two steps: (i) the creation of the correlation tables and (ii) the generation of the production rules base.     

Optimizing the extraction of anti-tumor polysaccharides from the fruit of Capparis spionosa L. by response surface methodology

Capparis spionosa L. is a traditional medicinal plant in China and central Asia. In this study, an experiment was designed to investigate the optimization of the extraction of anti-tumor polysaccharides from the fruit of Capparis spionosa L. (CSPS) by response surface methodology (RSM). Four independent variables (extraction temperature, extraction time, ratio of water to sample and extraction cycles) were explored. Meanwhile, the in vivo anti-tumor activity of CSPS was investigated. The results showed that the experimental data could be fitted to a second-order polynomial equation using multiple regression analysis. The optimum extraction conditions were as follows: extraction temperature 92 °C, extraction time 140 min, ratio of water to sample 26 mL/g, and three extraction cycle. Under these conditions, the yield of polysaccharides reached 13.01%, which was comparable to the predicted yield (12.94%, p > 0.05). This indicated that the model was adequate for the extraction process. Additionally, CSPS could prolong the survival time of H22 bearing mice in vivo. The anti-tumor activities of CSPS were dose-dependent.     

Multifunctional sorbents for the extraction of pesticide multiresidues from natural waters

In this work multifunctional sorbents, based on surfactant-coated mineral oxides, are assessed for the simultaneous extraction/preconcentration of pesticide multiresidues from aqueous environmental samples. Seventeen pesticides, representative of all the common groups (triazines, phenylureas, carbamates, azols, anilides, chloroacetanilides, organophosphorous, phenoxyacids, aryloxy acids and phenols), are selected for this study. The sorbents assessed are pure sodium dodecyl sulphate (SDS) and mixed tetrabutylammonium (TBA)-SDS hemimicelles and/or admicelles adsorbed onto alumina. Because of their multifunctional character, these sorbents provide different retention mechanisms (i.e. hydrophobic, ionic and/or π-cation interactions), which highly contribute to the efficient retention of pesticides with different polarities and acidities (bases, neutrals and acids). In addition, the low volume of eluent required for complete elution of analytes (typically 1-2 mL) avoided the need of using time-consuming and tedious evaporation steps that generally are needed when cross-linked polymeric resins or carbon materials are used as sorbents. The performance of two sorbents, i.e. SDS and TBA-SDS mixed hemimicelles/admicelles, for the admicellar solid-phase extraction (ASPE) of pesticide multiresidue was comparatively investigated. The latter was selected on the basis of the higher breakthrough volumes permitted, the lower volume of eluent required and the higher sample and eluent flow rates allowed. The proposed ASPE/LC/UV approach provided detection limits lower than 100 ng L⁻¹ for the determination of the 17 pesticides tested. Recoveries from spiked (at the ng L⁻¹ level) river and underground water samples was quantitative for most of the pesticides tested.