HPLC Peak Areas Research Articles

A Method for Converting HPLC Peak Area from Online Reaction Monitoring to Concentration Using Nonlinear Regression

Online HPLC reaction progress monitoring provides detailed data-rich profiles; however, extracting kinetic information requires ultraviolet-visible response factors to determine concentrations from peak areas. If the reaction's overall mass balance is known and some analytical trend for all relevant species can be recorded, it is possible to estimate the absolute response factors of all species using a system of linear equations. We delineate a method using the Microsoft Solver plug-in to convert time course profiles to reagent concentrations without analytical standards.

Determination of optical density (OD) of oligodeoxynucleotide from HPLC peak area.

Oligodeoxynucleotides (ODNs) are typically purified and analysed with HPLC equipped with a UV-Vis detector. Quantities of ODNs are usually determined using a UV-Vis spectrometer separately after HPLC, and are reported as optical density at 260 nm (OD260). Here, we describe a method for direct determination of OD260 of ODNs using the area of the peaks in HPLC profiles. It is expected that the method will save significant time for researchers in the area of nucleic acid research, and minimize the loss of oligonucleotide samples.

Identification of Dyes in Coptic Textiles from the Museum of Faculty of Archaeology, Cairo University

High Performance Liquid Chromatography coupled to a Diode-Array-Detector (HPLC-DAD) is used to investigate samples which were extracted from ancient Egyptian textiles (4th–5th c. AD) of the Museum of Faculty of Archaeology, Cairo University. Madder is identified in several samples. According to semi-quantitative results, which are obtained from HPLC peak areas measured at 254 nm, madder that is rich in purpurin and poor in alizarin is identified in samples which were treated (i) only with madder and (ii) with madder and either indigo/woad (Indigofera species and other/Isatis tinctoria L.) or weld (Reseda luteola L.). The madder dye used in these samples could have been originated from Rubia peregrina L. However, the possible use of Rubia tinctorum L. (or other plants of the Rubiaceae family) by the Egyptian dyers cannot be ruled out, particularly if methods were developed by the ancient dyers to affect and control the relative composition of madder dye. The HPLC peak area ratio of alizarin versus purpurin is very high (>2.2) for samples which were treated with madder (probably originated from R. tinctorum) and a tannin source. Finally, in some samples, only indigoid dyes (indigo/woad) are identified.

Purity determination of pyributicarb by internal standard correction-high-performance liquid chromatography-quantitative nuclear magnetic resonance.

An internal standard correction-high-performance liquid chromatography-quantitative nuclear magnetic resonance (ISC-HPLC-qNMR) procedure was established as a reliable quantitative method for complex organic compounds with low purity in order to solve the risk of qNMR inaccuracy because of insufficient resolution of impurity peaks from the selected quantitative peak. This method collects a small quantity of target analyte from low-purity organics by LC. After drying and re-dissolving in deuterated solvent containing internal standard, the solution was analyzed by 1H NMR and HPLC. Another solution prepared by accurately weighing unpurified low-purity substance and internal standard was analyzed by HPLC. Based on the theoretical derivation derived from the Beer-Lambert law, using the ratio of the HPLC peak areas of two solutions as correction, the purity was then calculated without the same reference as target analyte. Compared to previous methods with similar selectivity and accuracy, it has advantages such as a less purified sample is required, time for lyophilization is reduced by half, and sample preparation is more controllable. The proposed method was verified by analysis of a suite of six commercially available, high-purity compounds, and the difference of results between it and direct qNMR was within 0.1%. The result of pyributicarb using ISC-HPLC-qNMR was 97.6% (U = 0.5%; k = 2), and the reference value was 97.61% (U = 0.22%; k = 2). The results demonstrate that the proposed method provides a new way for reference material producers to calibrate lower-purity organics and has the potential advantage of accurate quantification of lower-purity organics.

Dataset of certified food dye levels in over the counter medicines and vitamins intended for consumption by children and pregnant women.

Food, Drug, & Cosmetic (FD&C) dyes can be found in various products outside of food that are consumed by children. The amount of FD&C dyes used in commercial products is proprietary. Determining the contribution of dye intake from commercial products requires direct assessment of FD&C dyes in the products. This dataset contains the raw data of HPLC peak areas, absolute values, averages, SDs and % RSD for FD&C dyes in children's gummy vitamins, children's tablet vitamins, prenatal vitamins, children's cough/cold/allergy tablets & syrups, and children's pain reliever tablets & syrups obtained using high performance liquid chromatography with a photometric diode array detector (HPLC-PDA). The data can be used for further interpretations of dye intake in children, based upon dose levels suggested for distinct age groups, to evaluate the consumption of the FD&C dyes and accepted daily intake (ADIs) suggested for each FD&C dye by the United States Food & Drug Administration (US FDA). The variability associated within each category is critical for understanding how products on the market can differ between lot especially with large gaps between expiration dates. The interpretation of the data is described in “Certified Food Dyes in Over the Counter Medicines and Supplements Marketed for Children and Pregnant Women” in the Journal of Food and Chemical Toxicology [1].

A new composite based on graphene oxide-poly 3-aminophenol for solid-phase microextraction of four triazole fungicides in water and fruit juices prior to high-performance liquid chromatography analysis

An environmentally friendly method which is based on preparation and use of a new composite consisting of poly 3-aminophenol and graphene oxide for solid-phase microextraction of triazole fungicides from natural water and juices is introduced. The composite was synthesized by in-situ polymerization of 3-aminophenol and graphene oxide in weak alkaline media. By using the obtained results from the characterization, a suitable mechanism for polymerization and a proper structure for the polymer were proposed. Under optimal conditions, there are linear relationships between concentration of triazoles and their HPLC peak areas in the range of 0.5–100 µg L−1. The recovery percentages, pre-concentration factors and LOQ for all triazoles were 95.2–98.0%, about 200 and 0.2–0.4 µg L−1, respectively. The offered method was applied for extraction and determination of triazoles from natural water, some juices and it represents a promising alternative method for analysis of triazoles.

Identification of dyes in Egyptian textiles of the first millennium ad from the collection Fill-Trevisiol

High-performance liquid chromatography coupled to a diode-array detector (HPLC-DAD) is used to investigate 30 samples which were removed from 27 ancient Egyptian fabrics of the Fill-Trevisiol collection. Attention is focused in this paper on fabrics of the Roman and Byzantine periods, with red and deep violet–blue wool weft threads which are Z-spun. The following dyes are identified in fabrics which date to the Roman period (first–fifth c. ad): mollusc purple, madder, and indigo/woad (Indigofera species and other, Isatis tinctoria L.). The results for the Byzantine (fifth–seventh c. ad) fabrics are richer in terms of identified dyes: apart from the three aforementioned dyes, the use of kermes (Kermes vermilio Planchon) and cochineal is revealed as the two coccid dyes have been detected in six Egyptian–Byzantine fabrics. Moreover, a yellow dye (probably Reseda luteola L.) is identified in one sample. Finally, samples taken from two fabrics, which date to the Islamic period, were dyed using indigo/woad and lac (Kerria lacca, Kerr). Mixing madder and indigo/woad to imitate true purple was a common practice in ancient Egypt and this is confirmed by the HPLC results in several samples. Semi-quantitative results are obtained from the HPLC peak areas and lead to the following conclusions. Madder dyes which were rich or poor in alizarin, compared to purpurin, and could have been therefore obtained from Rubia tinctorum L. and Rubia peregrina L., respectively are detected in Byzantine fabrics. Only alizarin-rich madder dyes are identified in some samples from Roman fabrics, but in some other samples the relative alizarin-to-purpurin ratio cannot be measured with confidence. Cochineal is found in two samples. The biological source of cochineal (Porphyrophora hamelii Brandt) is possible to be chemically identified only in one sample. The HPLC results of the molluscan dye detected in a Roman fabric are compared with the relative compositions of extracts of the three Mediterranean molluscs leading to the speculation that the Roman purple sample was probably dyed using Hexaplex trunculus L. The molluscan dye was furthermore detected in a Byzantine sample where it was combined with madder. Finally, it is reported that the standard (hydrochloric) acid hydrolysis method which is commonly applied to extract dyes from archaeological samples does not have any noticeable effect on the relative composition of the molluscan dye.

Identification of the molecular species of acylglycerols containing hydroxy fatty acids in wild edible mushroom Ganoderma lucidum

Abstract Edible Philippine mushrooms including Ganoderma lucidum have many health benefits. Seventy-two molecular species of triacylglycerols and five molecular species of diacylglycerols containing hydroxy fatty acids (FA) in the lipid extract of this mushroom were identified by HPLC and MS. The mono-, di- and tri-hydroxy FA constituents of the molecular species of acylglycerols were identified by various degrees of dehydration of [FA + Li] + . Many odd carbon numbered hydroxy FA constituents of acylglycerols were also identified. The contents of the 77 molecular species of acylglyerols and their constituent hydroxy FA in the lipid extract were estimated by the relative ion signal intensities of the molecular species of acylglycerols and the HPLC peak area % (ELSD). The contents (%) of the molecular species of acylglycerols containing hydroxy FA in the lipid extract were as: P-19:0-2OH18:2, 0.69%; 2OH18:2-2OH18:2-2OH18:2, 0.60%; P-OH19:1-OH19:1, 0.48%; S-OH19:1-OH19:1, 0.43%; 19:0-19:0-2OH18:2, 0.27% and the total of about 5.8%. “2OH18:2” stands for dihydroxy FA with 18 carbon atoms and two double bonds. The mushroom lipid extract was 1.67% of freeze-dried mycelium. The contents (%) of individual hydroxy FA in the mushroom lipid extract in decreasing order were: 2OH18:2, 1.4%; OH19:1, 1.1%; OH18:1, 0.38%; 2OH19:0, 0.19%; 2OH18:0, 0.13% and the total of about 3.5%. The odd carbon numbered hydroxy FA was about 1.44% of the mushroom lipid extract. The high contents of acylglycerols containing hydroxy FA, odd carbon numbered FA and odd carbon numbered hydroxy FA in edible mushroom might be related to one or more of its health benefits. As far as we are aware of, this mushroom contains the highest amounts of hydroxy FA and odd carbon numbered FA among the human foods.

Authentication of Schisandra chinensis and Schisandra sphenanthera in Chinese patent medicines

Authentication of species is crucial for ensuring the safety and efficacy of herbal medicines. The fruits of Schisandra chinensis and S. sphenanthera have been used for the same traditional Chinese drug, Wuweizi, but are found to be quite different according to their constituents, pharmacological effects, and qualities. These two fruits have been recorded as Schisandrae Chinensis Fructus (Wuweizi) and Schisandrae Sphenantherae Fructus (Nan-wuweizi), respectively, by Chinese Pharmacopoeia, 2000 edition. However, Nan-wuweizi is often found to be taken as Wuweizi in some Chinese patent drugs intentionally or by mistake because of its lower price and similar characteristics to Wuweizi. In this study, the selection and validation of special chemical markers for the identification of Schisandra species were established by HPLC-DAD-MS profiling analysis. Simple TLC and HPLC methods were proposed for the accurate determination of Nan-wuweizi from Wuweizi in Chinese patent medicines, using schisandrin and anwulignan as the identifying markers for Wuweizi and Nan-wuweizi, respectively. Through the establishment of a statistical model, adulterated or misused ratios of Nan-wuweizi in Wuweizi (w/w), as well as in Fenghan Kesou pills, can be determined. The limit of detection of Nan-wuweizi in a mixture (w/w) using both TLC and HPLC methods is 5% (mixed crude drugs of 50mg and 5g in a 1000g prescribed amount). The constructed statistical model relating the HPLC peak area ratio (anwulignan/schisandrin) and adulteration ratio is suitable for mixed crude drugs and Fenghan Kesou pills, and the two fitting equations have a good correlation (r=0.9979). Furthermore, 36 commercial Chinese patent medicines containing Wuweizi or Nan-wuweizi according to their labels were checked by these methods, and Nan-wuweizi was detected in Renshen Wuweizi Granules and Fenghan Kesou Pills. The ratios of Nan-wuweizi in these mixtures (w/w) were 100:0 for both, which does not comply with the statutory prescription. This study provided a simple and reliable method to prevent the adulteration or misuse of Nan-wuweizi in crude drugs and patent medicines of Wuweizi.

Combined effect of Bacillus cereus CPOU13 and B. subtilis SPC14 on polycyclic aromatic hydrocarbons degradation in vitro

Biodegradation of polycyclic aromatic hydrocarbons (PAHs) with suitable bacterial strains conveys much interest in recent years. We studied biodegradation of PAHs namely phenanthrene, anthracene and pyrene using two efficient PAHs degrading strains, B. cereus CPOU13 and B. subtilis SPC14 in vitro experiments. Standard HPLC chromatograms for phenanthrene, anthracene and pyrene were plotted separately based on HPLC peak area values and Retension time of known concentrations of the test PAHs and using software, ‘Origin 6.0’. Biodegradation of PAHs mixture containing phenanthrene, anthracene and pyrene was studied in for 13 days. We found that the combination of bacterial strains, B. cereus CPOU13 and B. subtilis SPC14 degraded high amounts of phenanthrene, anthracene and pyrene in 13 days of incubation. The recorded degradation percentages of phenanthrene, anthracene and pyrene were 85.31, 92.82 and 85.70 respectively. Concentration of phenanthrene was degraded from 217µg/5ml to 31.9µg/5ml. Concentration of anthracene was degraded from 211µg/5ml to 16µg/5ml. Concentration of pyrene was degraded from 233µg/5ml to 33µg/5ml in 13 days of incubation. We also observed biodegradation of phenanthrene, anthracene and pyrene from 1st day to 13th day.

Determination of equilibrium constant of amino carbamate adduct formation in sisomicin by a high pH based high performance liquid chromatography

Amino carbamate adduct formation from the amino group of an aminoglycoside and carbon dioxide has been postulated as a mechanism for reducing nephrotoxicity in the aminoglycoside class compounds. In this study, sisomicin was used as a model compound for amino carbamate analysis. A high pH based reversed-phase high performance liquid chromatography (RP-HPLC) method is used to separate the amino carbamate from sisomicin. The carbamate is stable as the breakdown is inhibited at high pH and any reactive carbon dioxide is removed as the carbonate. The amino carbamate was quantified and the molar fraction of amine as the carbamate of sisomicin was obtained from the HPLC peak areas. The equilibrium constant of carbamate formation, Kc, was determined to be 3.3×10−6 and it was used to predict the fraction of carbamate over the pH range in a typical biological systems. Based on these results, the fraction of amino carbamate at physiological pH values is less than 13%, and the postulated mechanism for nephrotoxicity protection is not valid. The same methodology is applicable for other aminoglycosides.

N-Acetyl-S-(N,N-diethylcarbamoyl) cysteine in rat nucleus accumbens, medial prefrontal cortex, and in rat and human plasma after disulfiram administration

Disulfiram (DSF), a treatment for alcohol use disorders, has shown some clinical effectiveness in treating addiction to cocaine, nicotine, and pathological gambling. The mechanism of action of DSF for treating these addictions is unclear but it is unlikely to involve the inhibition of liver aldehyde dehydrogenase (ALDH2). DSF is a pro-drug and forms a number of metabolites, one of which is N-acetyl-S-(N,N-diethylcarbamoyl) cysteine (DETC-NAC). Here we describe a LCMS/MS method on a QQQ type instrument to quantify DETC-NAC in plasma and intracellular fluid from mammalian brain. An internal standard, the N,N-di-isopropylcarbamoyl homolog (MIM: 291>128) is easily separable from DETC-NAC (MIM: 263>100) on C18 RP media with a methanol gradient. The method's linear range is 0.5–500nM from plasma and dialysate salt solution with all precisions better than 10% RSD. DETC-NAC and internal standards were recovered at better than 95% from all matrices, perchloric acid precipitation (plasma) or formic acid addition (salt) and is stable in plasma or salt at low pH for up to 24h. Stability is observed through three freeze-thaw cycles per day for 7 days. No HPLC peak area matrix effect was greater than 10%. A human plasma sample from a prior analysis for S-(N,N-diethylcarbamoyl) glutathione (CARB) was found to have DETC NAC as well. In other human plasma samples from 62.5mg/d and 250mg/d dosing, CARB concentration peaks at 0.3 and 4nM at 3h followed by DETC-NAC peaks of 11 and 70nM 2h later. Employing microdialysis sampling, DETC-NAC levels in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and plasma of rats treated with DSF reached 1.1, 2.5 and 80nM at 6h. The correlation between the appearance and long duration of DETC-NAC concentration in rat brain and the persistence of DSF-induced changes in neurotransmitters observed by Faiman et al. (Neuropharmacology, 2013, 75C, 95–105) is discussed.

Comparison of extraction methods for the analysis of Indigofera tinctoria and Carthamus tinctorius in textiles by high performance liquid chromatography

The extractions of indigo (Indigofera tinctoria L.) and safflower (Carthamus tinctorius L.) red and yellow from wool fibres are investigated using high performance liquid chromatography with photo-diode-array detection (HPLC–PDA) which is frequently coupled to Mass Spectrometry (HPLC–PDA–MS) for identification purposes. The efficiencies of dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) to extract indigo and safflower from wool, are compared for a wide range of temperature (from 40 to 120°C) and treatment time (from 1 to 30min). Extraction procedures are investigated by measuring integrated HPLC peak areas of selected dyestuff components, evaluating simultaneously the effects of solvent (DMSO and DMF), treatment temperature (T) and time (t). Thus, cross-influence of the different extraction parameters (solvent, T and t) is taken into account. Indigotin, isatin and indirubin are monitored to evaluate the extraction of indigo. Carthamin, a decomposition product of carthamin, apigenin, safflomin A, 6-hydroxykaempferol-3-O-β-D-glucoside, anhydrosafflor yellow Ct1 and Ct4 are monitored to investigate the extractions of safflower red and yellow. Overall, it is reported that DMSO gives better extraction yields than DMF.The best conditions for the extraction of the marker compounds of the three dyes are summarized as follows. For indigotin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for t=1min give the best yield. Longer treatment (t>1min) at 120°C results to decomposition of indigotin. For carthamin, treatments of the wool with DMSO at 80°C for t>5min or at 120°C min for short t (<5min) give the best yield, considering that longer (>5min) treatment at 120°C results to decomposition of the marker compound. Finally, for safflomin A treatments of the wool with DMSO at 100°C for t>10min or at 120°C min for t=5min give the best result.

Estrogen receptor affinity chromatography: A new method for characterization of novel estrogenic disinfection by-products

To identify the unknown estrogenic disinfection by-products (DBPs) from the chlorination extract, an effective method based on affinity chromatography with immobilized human recombinant estrogen receptor α (ERα) was developed, which has an advantage in targeting different potential estrogenic compounds from mixed sample simultaneously by comparing their relative binding activities to ER. The new method worked well for six known environmental estrogens. To further test the validity of this method for unknown chemicals, six DBPs of diethylstilbestrol (DES) with relatively strong ER binding affinity after chlorination were isolated and identified. It was found that except for 2-chloro-DES which showed 1.36 times stronger binding affinity than DES, most of the by-products bound to ER much more weakly than DES. All these seven by-products induced a dose-dependent transcriptional activation in two-hybrid-yeast assays. Z,Z-dienestrol (DE) and 2-chloro-DES, which exhibiting the weakest and the strongest binding affinity, were further tested for their transcriptional potential as 0.00243 and 0.014 compared to DES, respectively. However, they were still potential harmful environmental estrogenic disruptors as their estrogenic activities were much stronger than that of bisphenol A (BPA). These results demonstrated that the new method can help to screen unknown estrogenic compounds from mixture more efficiently.

Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2%), bi-(23.9%), tri-(9.0%), tetra-(2.7%), and penta-(2.8%) antennary oligosaccharides. However, OM contained mostly tri-(33.5%) and penta-(31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.

Preparative separation and purification of flavonoids and stilbenoids from Parthenocissus tricuspidata stems by dual-mode centrifugal partition chromatography

We successfully used dual-mode centrifugal partition chromatography (CPC) to separate and purify major components from an ethyl acetate extract of Parthenocissus tricuspidata stems. Dual-mode CPC with a solvent system composed of ethyl acetate–acetonitrile–water (3:3:4, v/v) was performed in reverse-phase mode followed by normal-phase mode, through the use of a switching valve from descending to ascending mode. In reverse-phase mode (descending mode), more polar components such as aromadendrin-3-O-β-d-glucopyranoside (1), trans-piceid (2), and catechin (3) were isolated. Switching to normal-phase mode (ascending mode) yielded the less polar compounds, resveratrol (4) and engeletin (5). As a result, five major compounds, aromadendrin-3-O-β-d-glucopyranoside (46.6mg), trans-piceid (13.5mg), catechin (42.0mg), resveratrol (13.9mg), and engeletin (17.6mg), were isolated from 500mg of P. tricuspidata ethyl acetate extract. The purity of each isolated compound was determined to be 95% (1), 85% (2), 90% (3), 90% (4) and 95% (5) based on the HPLC peak areas.

Rapid Determination of Residual Quinolones in Honey Samples by Fast HPLC with an On-Line Sample Pretreatment System

A method of on-line pretreatment coupled to HPLC with fluorescence detection was developed and validated for the determination of nine quinolones in honey samples. This method simplified the complicated process of sample pretreatment and reduced sample treatment time. Recovery of the quinolones was between 92% - 101% for spiked honey samples. The limit of detection was 0.22 - 3.78 ng/mL (signal/noise ratio = 3). There was good linear correlation between HPLC peak area and concentration of the quinolones, with a linear range of 1.0 - 100.0 ng/mL and correlation coefficients >0.9997. The method proposed was validated for detecting quinolones in honey samples.

Biotransformation of Meloxicam by Cunninghamella blakesleeana: Significance of Carbon and Nitrogen Source

Influence of carbon and nitrogen source, on biotransformation of meloxicam was studied by employing Cunninghamella blakesleeana NCIM 687 with an aim to achieve maximum transformation of meloxicam and in search of new metabolites. The transformation was confirmed by HPLC and based on LC-MS-MS data and previous reports the metabolites were predicted as 5-hydroxymethyl meloxicam, 5-carboxy meloxicam and a novel metabolite. The quantification of metabolites was performed using HPLC peak areas. The results obtained indicate that glucose as carbon source, ammonium nitrate as nitrogen source, were found to be optimum for maximum transformation of meloxicam. The study suggests the significance of these factors in biotransformation of meloxicam using microbial cultures. The fermentation was scaled up to 1l level.

Enlargement of the variances in amount and composition of anthocyanin pigments in sweetpotato storage roots and their effect on the differences in DPPH radical-scavenging activity

To investigate the effect of differences in anthocyanin composition on radical-scavenging activity in the purple-fleshed sweetpotato storage root, 124 sweetpotato clones with various compositions of anthocyanin were produced by crosses among superior sweetpotato genetic resources, and were analyzed for anthocyanin composition and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity (RSA). The highest content of anthocyanin among the clones was twofold higher than the content of cv. Ayamurasaki, which has been released for use in industrial pigment production in Japan. The range of variance in anthocyanin composition was very wide, as the ratio of peak areas of cyanidin-based anthocyanins to total anthocyanin peak areas ranged from 85.7% to 6.0%. The relationship between anthocyanin content and RSA was positively correlated. Correlation coefficients between RSA and the HPLC peak areas of cyanidin-based anthocyanins ranged from 0.606 to 0.687, whereas those of peonidin-based anthocyanins were 0.124–0.271. These results clearly demonstrated that cyanidin-based anthocyanins are closely related to DPPH RSA in sweetpotato storage roots.

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.