High-performance Liquid Chromatography Methodology Research Articles

Chemiluminescence detectors for liquid chromatography

In this tutorial we describe the construction of chemiluminescence detectors for high performance liquid chromatography (HPLC), comprising the components required to deliver the chemiluminescence reagent, a coiled-tubing flow cell, photomultiplier tube and detector housing, and various options for data acquisition. We also discuss two state-of-the-art commercially available chemiluminescence detectors for HPLC and other flow analysis methodology.

Development of the radiosynthesis of high-specific-activity 123I-NKJ64

(123)I-NKJ64, a reboxetine analogue, is currently under development as a potential novel single photon emission computed tomography radiotracer for imaging the noradrenaline transporter in brain. This study describes the development of the radiosynthesis of (123)I-NKJ64, highlighting the advantages and disadvantages, pitfalls and solutions encountered while developing the final radiolabelling methodology. The synthesis of (123)I-NKJ64 was evaluated using an electrophilic iododestannylation method, where a Boc-protected trimethylstannyl precursor was radioiodinated using peracetic acid as an oxidant and deprotection was investigated using either trifluoroacetic acid (TFA) or 2 M hydrochloric acid (HCl). Radioiodination of the Boc-protected trimethylstannyl precursor was achieved with an incorporation yield of 92±6%. Deprotection with 2 M HCl produced (123)I-NKJ64 with the highest radiochemical yield of 98.05±1.63% compared with 83.95±13.24% with TFA. However, the specific activity of the obtained (123)I-NKJ64 was lower when measured after using 2 M HCl (0.15±0.23 Ci/μmol) as the deprotecting agent in comparison to TFA (1.76±0.60 Ci/μmol). Further investigation of the 2 M HCl methodology found a by-product, identified as the deprotected proto-destannylated precursor, which co-eluted with (123)I-NKJ64 during the high-performance liquid chromatography (HPLC) purification. The radiosynthesis of (123)I-NKJ64 was achieved with good isolated radiochemical yield of 68% and a high specific activity of 1.8 Ci/μmol. TFA was found to be the most suitable deprotecting agent, since 2 M HCl generated a by-product that could not be fully separated from (123)I-NKJ64 using the HPLC methodology investigated. This study highlights the importance of HPLC purification and accurate measurement of specific activity while developing new radiosynthesis methodologies.

A RP-HPLC Method Using -Hydroxy Isobutyric Acid for Preconcentration and Determination of Uranium in Seawater

A high-performance liquid chromatographic (HPLC) method is developed for the determination of uranium (U), present at ppb levels, in seawater. α-Hydroxy isobutyric acid is used as the complexing agent for preconcentration as well as separation of U from other interfering metal ions (Mo, V, Ti, Ni etc.). A C 18 reversed-phase monolithic column and α-HIBA of pH 6-7 are employed for the selective preconcentration of U. Mobile phase consisting of α-HIBA of pH 2 and MeOH is used for elution of adsorbed U which is monitored after post-column derivatisation with Arsenazo(III), using a spectrophotometric detector. The methodology is validated by using simulated as well as seawater samples. Quantitative recovery for U is possible with linear response over a concentration range of 1 to 30 ppb of U. Metal ions which have been previously reported to give interferences do not give any problem in the present approach. The developed HPLC methodology is validated by comparing the results with those of isotope dilution-thermal ionization mass spectrometry.

Improvement of an HPLC method to determine urinary δ-aminolevulinic acid

OBJECTIVE: The purpose of this study was to evaluate and improve a high performance liquid chromatography (HPLC) methodology to determine urinary δ-aminolevulinic acid (ALA-U) with small volumes of sample. METHOD: The method was based on the formation of a fluorescent compound and subsequent 15-minute chromatographic run. RESULTS: The method shows suitable linearity, precision and recovery. Urine samples showed 1.2 ± 0.9 mg/l (media ± standard deviation) of ALA-U. CONCLUSION: The method was considered suitable for the routine analysis of ALA-U.

β-Diketones: Peak shape challenges and the use of mixed-mode high-performance liquid chromatography methodology to obtain fast, high resolution chromatography

Historically, indirect methods have been used for the HPLC analysis of β-diketone compounds because of the very poor peak shapes and resolution obtained on conventional HPLC stationary phases. In this paper we demonstrate that it is possible to obtain good peak shapes for underivatised β-diketone compounds, in a simulated reaction mixture, using only conventional mobile phases with mixed-mode stationary phase HPLC columns. Optimum conditions were obtained using the mixed-mode reversed-phase strong anion exchange column Primesep B, supplied by SIELC Technologies, with a 0.1% aq. TFA/MeOH gradient method and a column temperature of 55 °C.

Measurement of plasma 5-hydroxyindole acetic acid by liquid chromatography tandem mass spectrometry—Comparison with HPLC methodology

In patients with carcinoid disease, urinary concentration of the serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) is currently used to monitor disease progression or response to treatment as it is the metabolic end-product resulting from free and stored serotonin turnover. However, due to the undignified, cumbersome and error-prone nature of 24-h urine collections, there is constant pressure to replace them. It has been demonstrated using high performance liquid chromatography (HPLC) with fluorescence detection technology that plasma can achieve this, with the added advantage that it can be used for diagnostic purposes also. Here we describe a much simpler method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) that is twice as fast as a HPLC method currently in routine use. The sample preparation protocol requires 50 μL of plasma and a simple protein precipitation step facilitated by acetonitrile. Chromatography was performed on a Phenomenex C18 Security Guard™ column coupled to a SIELC Primesep B reversed-phase, anion-exchange dual chemistry column and methanolic mobile phase gradient elution. Eluant was directly connected to a Waters ® Quattro Premier™ XE tandem mass spectrometer operating in positive ion mode. We detected multiple reaction monitoring transitions m/ z 191.9 > 145.6 and 193.9 > 147.6 for 5-HIAA and d2-5-HIAA respectively, which co-eluted at 2.1 min. Ion suppression was negligible, recovery from spiked plasma was 103% (range 97–113%) and the method showed good linearity to 10,000 nmol/L ( r 2 = 0.999). Within-batch and between-batch imprecision was <10% and bias <15% at 3 concentrations, the limit of detection was 5 nmol/L and lower limit of quantitation 15 nmol/L. No interference was observed with l-tryptophan or 5-hydroxytryptamine. Comparison of LC–MS/MS and HPLC showed good agreement between the two methods but this LC–MS/MS assay displays several advantages; it requires 10-fold less sample, has a simpler extraction procedure and extended linearity, thus increasing laboratory throughput, lowering reagent costs and removing the need to dilute samples in patients with established carcinoid disease being monitored for therapeutic efficacy.

Advances in PAHs/nitro-PAHs fractioning

The aim of this work was to develop an efficient methodology for the reliable fractioning of nitrated-polycyclic aromatic hydrocarbons (nitro-PAHs) and polycyclic aromatic hydrocarbons (PAHs). Unlike what usually occurs under pressures developed by HPLC (high performance liquid chromatography) systems (above 11 bar) we observed that when normal phase chromatographic fractioning procedures are accomplished under very low pressures (about 1 bar), dipole molecules (nitro-PAHs) elute much faster than non-polar organic molecules (PAHs). This finding allowed developing an original and very efficient methodology for fractioning nitro-PAHs and PAHs. This method is based on normal-phase liquid chromatography through a home-made phenyl column by using hexane as mobile phase at very low speed flow (0.05 ml min−1). Unlike typical HPLC methodology, the fractioning of nitro-PAHs and PAHs was accomplished as a function of their polarity (first the polar compounds as a unique peak and further, the non-polar compounds, PAHs) rather than as a function of their medium polarizability.

Clinical Validation of a Noninvasive, Raman Spectroscopic Method to Assess Carotenoid Nutritional Status in Humans

Background: Carotenoids are an important group of phytonutrients that are abundant in fruits and vegetables. Epidemiological and clinical intervention studies have implied the presence of protective qualities of these nutrients against the development of a variety of chronic diseases. Previously, human carotenoid status has been assessed in serum and tissue using high-performance liquid chromatography (HPLC) methodology. Recently, a Raman spectroscopy (RS)–based photonic method has been developed to accurately and noninvasively measure the carotenoid concentration in human skin.Objectives: (1) To validate skin RS methodology against standard serum carotenoid measurements by HPLC and (2) to establish and compare the reliability of the 2 methods.Design: This study included 372 healthy adults who provided 3 blood samples and 3 RS skin carotenoid measurements within an 8-day period; each day-matched blood sample and RS determination was spaced by ≥48 hours.Results: Consistent positive correlations were observed for each of 3 separate same-day correlation plots of total serum versus RS skin carotenoids. Overall estimate of the line of best fit from analysis of covariance, using all 3 samples (n = 1116), yielded a Pearson correlation of R = 0.81 (r2 = 0.66; p < 0.001). Based on analysis of variance, RS skin carotenoid methodology exhibited 0.9% less variance over the 3 tests than serum carotenoids by the HPLC method (p < 0.03).Conclusions: RS accurately measures total carotenoids in human skin with less intra-individual variability than measurement of serum carotenoids by HPLC analysis. RS technology is a valid and reliable noninvasive method to rapidly assess carotenoid nutritional status in humans.

Evaluation of Kahweol and Cafestol in Coffee Tissues and Roasted Coffee by a New High-Performance Liquid Chromatography Methodology

A reverse phase high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantification of kahweol and cafestol in tissues of fresh fruits, leaves, and roasted coffee beans. The best resolution was obtained with isocratic elution of acetonitrile/water (55/45% v/v) and UV detection. A single sample preparation method carried out by direct saponification and extraction with organic solvent was standardized for all matrices. Good recovery (average of 99% for kahweol and 94% for cafestol), repeatability, and linearity were obtained. Detection limits of 2.3 and 3.0 mg/100 g were observed for kahweol and cafestol. The HPLC method was effective in quantifying these diterpenes in the different coffee matrices. The endosperm and perisperm of Coffea arabica cv. IAPAR 59 showed elevated amounts of kahweol as compared to the pericarp and leaves. On the other hand, cafestol was detected in all samples except in leaves from Coffea canephora cv. Apoatā.

Measurement of serotonin in platelet depleted plasma by liquid chromatography tandem mass spectrometry

5-Hydroxytryptamine (5-HT) in human platelet depleted plasma (PDP) is a biomarker in functional gastrointestinal disorders (FGID), with levels reflecting acute changes in circulating 5-HT concentration. PDP 5-HT is currently measured by reversed phase high performance liquid chromatography (HPLC) fluorimetric detection. We have developed a simple and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method that is two times more rapid than the current HPLC methodology. Our method employs a simple protein precipitation requiring no further downstream sample preparation. 10μL of extract was injected directly onto a SecurityGuard SCX cation exchange column followed by isocratic elution onto an Onyx Monolithic C18 analytical column and methanolic gradient elution. Eluant was connected directly to a Quattro Premier XE tandem mass spectrometer operating in ES+ mode. We detected multiple reaction monitoring transitions m/z 160>114.9 and m/z 164.1>118.9 for 5-HT and d4-5-HT, respectively. 5-HT and d4-5-HT co-eluted at 2.79min and cycle time between injections was 6min. Mean recovery was 98%, limit of detection 1.5nmol/L, lower limit of quantification 5nmol/L, linearity to 1000nmol/L (r2=0.999), imprecision <10% and bias <13.4%. 5-HT eluted with no ion suppression. No interference was found with l-tryptophan or 5-hydroxyindoleacetic acid (5-HIAA). This assay was compared to a previously published HPLC method. Passing-Bablok regression analysis showed LC–MS/MS=0.91 (HPLC)–0.83, r2=0.97, n=80. Bland Altman analysis showed general agreement, with a mean bias of 3.3nmol/L. We have developed a simple and robust assay for PDP 5-HT that will increase throughput for clinical trials.

Application of HPLC and MALDI-TOF MS for Studying As-Synthesized Ligand-Protected Gold Nanoclusters Products

Samples of polydisperse gold nanoclusters (AuNCs) protected with monolayers of N-acetyl-L-cysteine (NAC) have been chromatographically separated by a C18 column (4.6 mm x 250 mm) using a gradient elution program with a mobile phase of methanol (MeOH)/water containing tetrabutylammonium fluoride (Bu(4)N(+)F(-)) and sodium chloride. The effects of Bu(4)N(+)F(-) and MeOH on the separation have been investigated in detail. In conjunction with absorbance-based, fluorescence, and electrochemical detectors, the elution order of these water-soluble AuNCs is confirmed according to their core size in an ascending order. The onset oxidation potential closely follows the core size of AuNC. The separated fractions from high-performance liquid chromatography (HPLC) were collected and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the number of Au atoms in the fractions. The sizes of AuNCs in some selected HPLC fractions were also assessed by transmission electron microscopy and high-resolution transmission electron microscopy. Photoluminescence spectra of the fractions show that the luminescent shift in the visible/near-infrared region does not follow with the core size of AuNC. More importantly, the proposed HPLC methodology has been successfully applied to analyze various polydisperse AuNC products synthesized from different gold-to-ligand mole ratios (Au/NAC) and reaction temperatures. The results confirm that larger Au/NAC and higher temperature will produce larger core size AuNCs products with narrower dispersity. In addition, AuNC samples obtained from various synthesis reaction times were analyzed by our HPLC methodology, demonstrating that the reaction's behavior follows the nucleation-growth-disintegration process.

Improved HPLC methodology for food control – furfurals and patulin as markers of quality

This work describes an improved, simple high performance liquid chromatography (HPLC) method for the simultaneous determination of furfuraldeydes (2-furfural (2-F), 5-methylfurfural (5-MF) and 5-hydroxymethylfurfural (5-HMF)) and patulin in food products. The compounds were simultaneously determined by reversed-phase HPLC using a C 18 column and photodiode array (DAD) detection. The method, simple and accurate, showed recoveries of furfuraldeydes from 6% to 100% and the recovery of patulin is about 90%. The method was applied to different commercial food matrices: honey, white and demerara table sugars, white and red balsamic vinegars, caramel, nutritional supplement, sugar substitute, apple juices (clear and cloudy) and also to a mould infected apple. The one-step, sensitive (the limits of detection (LODs) for patulin, 5-HMF, 5-MF and 2-F were 0.09, 0.06, 0.23 and 0.73 ppb, respectively), no time consuming methodology will contribute to characterize and quantify the presence of these compounds in human diet and to determine the quality of foods.

In vitro Inhibition of CYP3A4 Metabolism and P‐Glycoprotein‐Mediated Transport by Trade Herbal Products

Extracts of six commonly used commercially available herbal products, St. John's wort, common valerian, common sage, Ginkgo biloba, Echinacea purpurea and horse chestnut were investigated for their in vitro inhibitory potential of CYP3A4 mediated metabolism and P-glycoprotein efflux transport activity. C-DNA baculovirus expressed CYP3A4 and Caco-2 cells were used. Ketoconazole and verapamil were applied as positive control inhibitors, respectively. A validated high-performance liquid chromatography methodology was used to quantify the formation of 6-OH-testosterone and scintillation counting was used to quantify the transport of (3)H-digoxin. All the investigated herbs inhibited CYP3A4 activity. St. John's wort was the strongest inhibiting herb with an IC(50) value of 15.4 microg/ml, followed by common sage, Ginkgo biloba, common valerian, horse chestnut and Echinacea purpurea. All herbs also inhibited P-glycoprotein activity. Ginkgo biloba was the strongest inhibiting herb, inhibiting the net digoxin flux with an IC(50) value of 23.6 microg/ml, followed by St. John's wort, horse chestnut, common sage, common valerian and Echinacea purpurea. No correlation was found between the herbs inhibitory potentials towards CYP3A4 and P-glycoprotein activities. Ginkgo biloba, horse chestnut and common sage, besides St. John's wort, are suggested candidates for in vivo intestinal herb-drug pharmacokinetic interactions.

Improved Methodology for Monitoring Poly(amidoamine) Dendrimers Surface Transformations and Product Quality by Ultra Performance Liquid Chromatography

Ultra performance liquid chromatography (UPLC) analysis was utilized for the first time as a methodology for monitoring poly(amidoamine) (PAMAM) dendrimer surface transformations and product quality. Results were compared to high‐performance liquid chromatography (HPLC) and were found to provide a vastly improved analytical method for the characterization of dendrimer polydispersity and variance in a typical surface modification. The application of UPLC increased the average number of theoretical plates by a factor of 7 and reduced retention times of analytes by 36%, while improving the resolution capability to discriminate surface variances in dendrimers. The new UPLC procedures were used to monitor surface modification of [core: ethylenediamine]; (G = 4); dendri‐poly(amidoamine)‐ (i.e., [EDA]; (G4); dendri‐PAMAM‐) to produce biotinylated dendrimer conjugates. The enhanced sensitivity and efficiency of the UPLC analyses allowed resolution of biotin substituent levels and a better characterization of the targeted dendrimer conjugates compared to traditional HPLC methodology.

The in vitro Inhibitory Potential of Trade Herbal Products on Human CYP2D6‐Mediated Metabolism and the Influence of Ethanol

The six commonly used trade herbal products, St. John's wort, common valerian, common sage, Ginkgo biloba, Echinacea purpurea and horse chestnut, and ethanol, were investigated for their in vitro inhibitory potential of cytochrome P450 2D6 (CYP2D6)-mediated metabolism. Herbal components were extracted from commercially available products in a way that ensured the same composition of constituents in the extract as in the original trade products. c-DNA baculovirus expressed CYP2D6 was used with dextromethorphan as substrate. Quinidine was included as a positive control inhibitor. A validated high performance liquid chromatography methodology was used to quantify the formation of dextrorphan (product of dextromethorphan O-demethylation). Ethanol showed a biphasic effect on CYP2D6 metabolism, increasing initially the CYP2D6 activity with 175% of control up to a concentration of 1.1%, where after ethanol linearly inhibited the CYP2D6 activity. All the investigated herbs inhibited CYP2D6 activity to some extent, but only St. John's wort, common sage and common valerian were considered possible candidates for in vivo clinically significant effects. They showed IC50 values of 0.07 +/- 7 x 10(-3) mg/ml, 0.8 +/- 0.05 mg/ml and 1.6 +/- 0.2 mg/ml, respectively. St. John's wort inhibited CYP2D6-mediated metabolism in an uncompetitive manner, while common valerian and common sage in a non-competitive manner demonstrated interherb differences in inhibition patterns and differences when compared to the more homogenous competitive inhibitor quinidine. Common valerian was the only herb that showed a mechanistic inhibition of CYP2D6 activity and attention should be paid to a possible toxicity of this herb.

Rapid determination of Papaver somniferum alkaloids in process streams using monolithic column high-performance liquid chromatography with chemiluminescence detection

We have combined high-performance liquid chromatography (HPLC) separations using a monolithic column with acidic potassium permanganate and tris(2,2′-bipyridyl)ruthenium(II) chemiluminescence detection in a rapid and highly sensitive method to monitor the process of extracting opiate alkaloids from Papaver somniferum. Due to the high flow rates allowed with the monolithic column and the inherent selectivity of the chemiluminescence reactions, the four predominant alkaloids – morphine, codeine, oripavine and thebaine – were determined in less than 2 min. The results obtained with numerous process samples compared favourable with those of the standard HPLC methodology. Limits of detection were 1 × 10 −10 M, 5 × 10 −10 M, 5 × 10 −10 M and 1 × 10 −9 M, for morphine, codeine, oripavine and thebaine, respectively.

Pharmacokinetics of Cefprozil in Plasma and Middle Ear Fluid

Despite the wide-scale use of cefprozil for acute otitis media (AOM), there are only limited data available regarding the pharmacokinetic profile of this agent in the pediatric population. To characterize the plasma and middle ear fluid (MEF) pharmacokinetic profile of cefprozil in pediatric patients with AOM. Pharmacokinetic sampling was obtained as part of a phase IV, multicenter, open-label study of children with AOM receiving cefprozil suspension 15 mg/kg twice daily. A single blood sample was obtained 4-6 days after the initiation of cefprozil therapy and a simultaneous MEF sample was obtained by tympanocentesis when clinically indicated. Cefprozil concentrations in both matrices were determined using a validated high-performance liquid chromatography methodology. A composite profile of cefprozil concentration data in each matrix was constructed and values for the pharmacokinetic parameters were obtained using conventional modeling techniques. Plasma concentrations were obtained in 53 children aged 6-48 months. In this population the maximum concentration (C(max)) in plasma was 9.18 microg/mL, the time to C(max) (t(max)) was 1.5 hours, and the terminal elimination half-life (t((1/2))(beta)) was 0.98 hours. Simultaneous plasma and MEF concentration data were available in 22 children. In this subset the C(max) in plasma was 8.2 microg/mL, the t(max) was 1.9 hours, and the t((1/2))(beta) was 1.02 hours; the corresponding MEF C(max) was 2.4 microg/mL, the t(max) was 3.5 hours, and the t((1/2))(beta) was 1.23 hours. Cefprozil MEF penetration as assessed using the ratio of the area under the concentration-time curves from the two matrices was 28%. Moreover, concentrations in MEF approximated 1 microg/mL 6 hours' post-dose. The plasma profile of cefprozil in the current analysis is consistent with previously reported values in children receiving the 15 mg/kg twice daily dose. MEF penetration and the duration of drug exposure at the site of infection support the clinical utility of this agent for organisms with minimum inhibitory concentrations (MIC) of < or =1 microg/mL. However, these results also predict higher clinical failure when using this dose of cefprozil against penicillin-non-susceptible Streptococcus pneumoniae or Haemophilus influenzae because of typically higher MIC values for these organisms.

Assessment of the quantitative determination of paralytic shellfish poisoning toxins by pre-column derivatization and elimination of interfering compounds by solid-phase extraction

Monitoring programmes for paralytic shellfish poisoning toxins in bivalve molluscs still rely heavily on the use of mouse bioassays (MBA) for consumer protection. A high-performance liquid chromatography (HPLC) methodology (Lawrence method) was implemented in 1996 in the Portuguese monitoring programme as a complementary means of analysis. Comparison between MBA and HPLC was done at the time only by a qualitative approach due to the scarce number of positive samples tested. More quantitative data were obtained recently when studying toxin profiles in Moroccan shellfish, and the correlation found between these two methodologies is reported here for the first time. Two different matrices were studied: blue mussel and the giant cockle Acanthocardia tuberculatum. A good linear correlation was obtained for both matrices. However, a second-degree polynomial best fitted the data at both low and high extremes of toxicity. According to the HPLC quantitative results, 13% of false-negatives could be obtained by MBA due to an underestimation of toxicity near the limit of detection of the MBA. Difficulties on relying solely on HPLC for consumer protection have been aroused with uncommon matrices, such as imported clams or crustaceans, due to the presence of high concentrations of interfering compounds. The solid-phase extraction step of the Lawrence method was implemented to eliminate an unknown compound that could be mistaken for saxitoxin, and an 80% reduction of another common unknown compound eluting close to decarbamoylsaxitoxin. The implementation of the HPLC methodology achieved so far allows a high degree of consumer protection without the need to resource to animal sacrifice.

Iso-coenzyme A

Iso-coenzyme A is an isomer of coenzyme A in which the monophosphate is attached to the 2'-carbon of the ribose ring. Although iso-CoA was first reported in 1959 (Moffatt, J. G., and Khorana, H. G. (1959) J. Am. Chem. Soc. 81, 1265-1265) to be a by-product of the chemical synthesis of CoA, relatively little attention has been focused on iso-CoA or on acyl-iso-CoA compounds in the literature. We now report structural characterizations of iso-CoA, acetyl-iso-CoA, acetoacetyl-iso-CoA, and beta-hydroxybutyryl-iso-CoA using mass spectrometry (MS), tandem MS, and homonuclear and heteronuclear NMR analyses. Although the 2'-phosphate isomer of malonyl-CoA was recently identified in commercial samples, previous characterizations of iso-CoA itself have been based on chromatographic analyses, which ultimately rest on comparisons with the degradation products of CoA and NADPH or have been based on assumptions regarding enzyme specificity. We describe a high performance liquid chromatography methodology for separating the isomers of several CoA-containing compounds. We also report here the first examples of iso-CoA-containing compounds acting as substrates in enzymatic acyl transfer reactions. Finally, we describe a simple synthesis of iso-CoA from CoA, which utilizes beta-cyclodextrin to produce iso-CoA with high regioselectivity, and we demonstrate a plausible mechanism that accounts for the existence of iso-CoA isomers in commercial preparations of CoA-containing compounds. We anticipate that these results will provide methodology and impetus for investigating iso-CoA compounds as potential pseudo-substrates or inhibitors of the >350 known CoA-utilizing enzymes.

Identification of Red Natural Dyes in Post‐Byzantine Icons by HPLC

A high performance liquid chromatography (HPLC) methodology, combined with UV‐Vis Diode Array Detection, is developed for the separation and identification of five reddish natural dyestuffs: cochineal, madder, lac dye, dragon blood, and brazilwood. The method is used for the identification of organic dyes in extracts originating from five icons, four of which are representative for the post Byzantine era (15th to 19th century) and one is typical for the Byzantine coloring technology (created at the 14th century). The origin and the nature of the coloring content of Mediterranean art objects, created in these historical periods are not well known. Carminic acid, the main active, coloring, ingredient of cochineal was identified in four icons, including the one of the 14th century. Brazilwood was found to be the only organic colorant in one icon of the post Byzantine era. It was also present, in addition to cochineal, in the icon of the Byzantine period. Further discussion, associated with the dyestuff origin, is provided based on the analytical results and the available historical data.