Development and Validation of HPLC Method for Determination of Sodium Copper Chlorophyllin - A Food Colorant and Its Application in Pharmacokinetic Study.

A simple, accurate, and precise bioanalytical method was developed and validated for the determination of pharmacokinetic parameters of sodium copper chlorophyllin, a USFDA approved food additive and colorant in rat plasma. The column used was Luna® C18 250×4.6 mm, 100 Å, having particle size 4.5 µm, and the mobile phase used was methanol (MeOH), and 10 mM ammonium acetate buffer in the ratio of 90 : 10, the flow rate was 1 ml/min, and the injection volume of 20 µL. The retention time of sodium copper chlorophyllin was obtained at 9 min. The method was found to be linear at the range of 0.50-8.00 µg mL-1 .

HPLC-hrTOF-MS study of copper chlorophylls: Composition of food colorants and biochemistry after ingestion.

Despite the daily consumption of copper chlorophylls (E-141i), the green food colorants in foods high in fats, there is a general need for knowledge regarding their exact composition. Consequently, we have analyzed by HPLC-ESI(+)/APCI(+)-hrTOF-MS2 the accurate composition of different commercial copper chlorophyll colorants for the first time. Data showed a favored yield of copper pheophytins from a series, while pheophytins from b series are preferentially no complexed with copper. The copper pheophytins present in the food colorants consisted mainly of three structural rearrangements. New fragmentation patterns and structural assignments have been described for several copper pheophytins. During the ingestion of copper chlorophylls, no chlorophyll derivative was present in serum nor urine except a new copper-pyroporphyrin a accumulated in a few livers. In any case, this green additive could represent the ideal food colorant, as most of the copper pheophytins are excreted in the feces showing almost no absorption of copper-chlorophylls compounds.

Synthesis of different metallochlorophyllins and quantification in food samples by reversed phase - high performance liquid chromatography.

The complex formation between metals (Ni, Co, Zn, Fe, Pb, Sn and Ag) and natural chlorophyll extracted from green leaves was monitored in the present study. The respective metallochlorophyllin was prepared by the reaction of metal chloride or nitrate (1M) to chlorophyll extracted from Ficus leaves extract. All synthesized metallochlorophyllins were stable and Na-Cu-chlorohyllin (E141) which is permitted to add in food and are listed in European Directive 94/36/EC on food colouring materials, was identified in commercially available food commodities (candies). In this study different synthesized metallochlorophyllins were characterised by using UV-Vis, FT-IR, HPLC, AAS and HR-MS techniques. Many food commodities (i.e. candy, chips, drink, and cream biscuits) were monitored for metallochlorophyllins and Na-Cu-Chlorophyllin was detected only in real candy samples.

Simultaneous determination of sodium iron chlorophyllin and sodium copper chlorophyllin in food using high-performance liquid chromatography and ultra-performance liquid chromatography-mass spectrometry.

A simultaneous method for analyzing sodium iron chlorophyllin (SIC) and sodium copper chlorophyllin (SCC) using high-performance liquid chromatography was developed. This method employed an Inertsil ODS-2 column and diode array detection at 395 nm, using methanol-water (97:3 and 80:20, v/v) containing 1% acetic acid as the mobile phase. Liquid chromatography-tandem mass spectrometry was used to identify the main components of SIC and SCC as Fe-isochlorine e4 and Cu-isochlorine e4, respectively. The limits of detection and quantitation of SIC were 1.2 and 4.1 mg/kg, respectively, while those of SCC were 1.4 and 4.8 mg/kg, respectively. For intraday and interday tests, the SIC recoveries from candy ranged from 81% to 101%, while SCC recoveries ranged from 100% to 109%. The developed method can be applied to the rapid determination of SIC and SCC in candy.

Determination of synthetic and natural colorants in selected green colored foodstuffs through reverse phase-high performance liquid chromatography.

The current study performed simultaneous identification of semisynthetic chlorophyllin (Cu-Chl) and synthetic food colorants in food matrices. The quantitative assessment of these synthetic and semisynthetic colorants was determined by using analytical method based on reverse-phase high-performance liquid chromatography (RP-HPLC) equipped with UV-visible detection. The concentration of Cu-Chl in hard candy was 3.334 mg/kg and 4.489 mg/kg in the soft candy, while contents of other colorants i.e. Tartrazine and Brilliant Blue were within 4.74-140.284 mg/kg. The extraction of Cu-Chl and synthetic colorants from different food matrixes was performed with a recovery range of 90-97% and RSD of 1-9%. Overall, the current developed RP-HPLC method could detect both green coloured synthetic and semisynthetic food colorants by a single run and could be used for monitoring different colorants in other complex matrices. This is the first report in India to determine Cu-Chl in foodstuffs and beverages by using RP-HPLC with UV-vis detector.

Direct Photometric Assay for Copper Chlorophyll Adulterants in Edible Oil by the Aid of an Ultraviolet-Photobleaching Pretreatment.

Adulterating edible oil with copper chlorophyll derivatives (E141i) has made a substantial impact on the edible oil industry and food safety. This study demonstrates an efficient and reliable screening method to directly identify the color adulteration by the aid of a simple photobleaching pretreatment using a 365 nm ultraviolet-light-emitting diode working at a photon flux density of 480 mmol m-2 s-1 for 24 min. The content of copper chlorophyll [predominantly Cu-pyropheophytin a (Cu-py a)] can be calculated by A600, A650, and A700 with satisfactory spike recovery [97.9-103.6%; six kinds of edible oils spiked with 1 ppm of Cu-py a; n = 3 for each kind of oil; relative standard deviation (RSD) < 5%], linearity ( R2 = 0.9961 when spiking 0.1-10 ppm of Cu-py a into soybean oil standard; n = 3 for each concentration; RSD < 5%), and reproducibility (RSD < 5% for spiking 1 ppm of Cu-py a into soybean oil standard; n = 3 over 3 days). The detection limit (S/N > 5) was 0.05 ppm. The analytical results of 50 commercially available oil samples were verified by the official high-performance liquid chromatography method.

Identification and quantification of Cu-chlorophyll adulteration of edible oils.

Cu-pyropheophytin a, the major Cu-pigment of Cu-chlorophyll, was determined in edible oil by high-resolution mass spectrometry with a high-performance liquid chromatography-quadrupole (HPLC-Q)-Orbitrap system and by HPLC coupled with a photodiode-array detector. Respective limit of detection and limit of quantification levels of 0.02 µg/g and 0.05 µg/g were obtained. Twenty-nine commercial oil products marked as olive oil, grapeseed oil and blended oil, all sourced directly from a food company that committed adulteration with Cu-chlorophyll, were investigated. In this company, four green dyes illegally used in oils were seized during factory investigation by the health authorities. The food additive Cu-pyropheophytin a was found in all confiscated samples in concentrations between 0.02 and 0.39 µg/g. Survey results of another 235 commercial oil samples manufactured from other companies, including olive pomace oil, extra virgin olive oil, olive oil, grapeseed oil and blended oil, indicated high positive incidences of 63%, 39%, 44%, 97% and 8%, respectively, with a concentration range between 0.02 and 0.54 µg/g. High Cu-chlorophyll concentrations are indications for fraudulent adulteration of oils.

Rapid detection of copper chlorophyll in vegetable oils based on surface-enhanced Raman spectroscopy.

The addition of copper chlorophyll and its derivatives (Cu-Chl) to vegetable oils to disguise them as more expensive oils, such as virgin olive oils, would not only create public confusion, but also disturb the olive oil market. Given that existing detection methods of Ch-Chl in oils, such as LC-MS are costly and time consuming, it is imperative to develop economical and fast analytical techniques to provide information quickly. This paper demonstrates a rapid analytical method based on surface-enhanced Raman spectroscopy (SERS) to detect Cu-Chl in vegetable oils; the spectroscopic markers of Cu-Chl are presented and a detection limit of 5 mg kg(-1) is demonstrated. The analysis of a series of commercial vegetable oils is undertaken with this method and the results verified by a government agency. This study shows that a SERS-based assessment method holds high potential for quickly pinpointing the addition of minute amounts of Cu-Chl in vegetable oils.

Retrieval chlorophyll-a concentration from coastal waters: three-band semi-analytical algorithms comparison and development.

The objectives of this study are to validate the applicability of a three-band algorithm in determining chlorophyll-a in eutrophic coastal waters, and to improve the model using improved three-band algorithm. Evaluated using two independent data sets collected from the West Florida Shelf, the variation three-band model was found to have a superior performance to both the three-band and modified three-band model. Using the variation three-band algorithm decreased 18% and 56% uncertainty, respectively, from the three-band and modified three-band algorithms. The significantly reduced uncertainty in chlorophyll-a estimations is attributed to effective removal of absorption of gelbstoff and suspended solids and backscattering of water molecules.

Comparative study of biological activity of three commercial products of Sasa senanensis Rehder leaf extract.

BACKGROUND: We have previously reported that alkaline extract of Sasa senanensis leaves (SE) has several biological activities characteristic of lignin-carbohydrate complex (LCC). In the present study, we compared the biological activity of three commercially available products of SE (products A, B and C). MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected, UV-irradiated cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Radical intensity was determined by electron spin resonance spectroscopy. Cytochrome P-450 (CYP)3A4 activity was measured by ß-hydroxylation of testosterone in human recombinant CYP3A4. RESULTS: Product A is a pure SE that contains Fe(II)-chlorophyllin, whereas products B and C contain Cu(II)-chlorophyllin and less LCC. Product C is supplemented with ginseng and pine (Pinus densiflora) leaf extracts. Product A exhibited 5-fold higher anti-HIV, 4-fold higher anti-UV, 5-fold higher hydroxyl radical-scavenging, and 3-fold lower CYP3A4 inhibitory activities as compared to those of product B, and 5-fold higher, 1.5-fold higher, comparable, and 7-fold lower activities, respectively, as compared to those of product C. CONCLUSION: The present study demonstrates for the first time the superiority of product A over products B and C, suggesting the beneficial role of LCC and Fe(II)-chlorophyllin.

Role of chlorophyllase in chlorophyll homeostasis and post-harvest breakdown in Piper betle L. leaf.

Piper betle L., a dioecious shade-loving perennial climber is one of the important Pan-Asiatic plants. More than hundred landraces having marked variation in leaf chlorophyll (Chl) content are in cultivation in India. In this study, role of chlorophyllase (Chlase) in Chl homeostasis and post-harvest breakdown was investigated in two contrasting P. betle landraces Kapoori Vellaikodi (KV) with light green and Khasi Shillong (KS) with dark green leaves. The two landraces showed negative correlation between Chl content and Chlase activity in fresh as well as stored leaves. Accumulation of chlorophyllide a (Chlid a) was correlated with the level of Chlase activity, which was higher in KV than KS. The overall response of abscisic acid (ABA) and benzylaminopurine (BAP) was similar in KV and KS, however, the time-course was different. ABA-induced Chl loss was accompanied by rise in Chlase activity in KV and KS and the delay in Chl loss by BAP was accompanied by reduction in Chlase activity. While there were significant differences in Chlase activity in KV and KS, only minor differences were observed in the enzyme properties like pH and temperature optima, Km and Vmax. No landrace-related differences were observed on the effect of metal ions and functional group reagents/amino acid effectors on Chlase activity. These results showed that despite significant differences in Chl content and Chlase activity between landraces KV and KS, the properties of Chlase were similar. The findings show that in P. betle Chlase is involved in Chl homeostasis and also in Chl degradation during post-harvest storage and responds to hormonal regulations. These findings might be useful in predicting the stability of Chl during post-harvest storage and also the shelf-life in other P. betle landraces.

Methods for analysis of photosynthetic pigments and steady-state levels of intermediates of tetrapyrrole biosynthesis.

Tetrapyrroles and carotenoids are required for many indispensable functions in photosynthesis. Tetrapyrroles are essential metabolites for photosynthesis, redox reaction, and detoxification of reactive oxygen species and xenobiotics, while carotenoids function as accessory pigments, in photoprotection and in attraction to animals. Their branched metabolic pathways of synthesis and degradation are tightly controlled to provide adequate amounts of each metabolite (carotenoids/tetrapyrroles) and to prevent accumulation of photoreactive intermediates (tetrapyrroles). Many Arabidopsis mutants and transgenic plants have been reported to show variations in steady-state levels of tetrapyrrole intermediates and contents of different carotenoid species. It is a challenging task to determine the minute amounts of these metabolites to assess the metabolic flow and the activities of both pigment-synthesising and degrading pathways, to unravel limiting enzymatic steps of these biosynthetic pathways, and to characterise mutants with accumulating intermediates. In this chapter, we present a series of methods to qualify and quantify anabolic and catabolic intermediates of Arabidopsis tetrapyrrole metabolism, and describe a common method for quantification of different plant carotenoid species. Additionally, we introduce two methods for quantification of non-covalently bound haem. The approach of analysing steady-state levels of tetrapyrrole intermediates in plants, when applied in combination with analyses of transcripts, proteins, and enzyme activities, enables the biochemical and genetic elucidation of the tetrapyrrole pathway in wild-type plants, varieties, and mutants. Steady-state levels of tetrapyrrole intermediates are only up to 1/1,000 of the amounts of the accumulating end-products, chlorophyll, and haem. Although present in very low amounts, the accumulation and availability of tetrapyrrole intermediates have major consequences on the physiology and activity of chloroplasts due to their additional photoreactive and possible signalling functions. Although adjusted for Arabidopsis tetrapyrrole metabolites, the presented methods can also be applied for analysis of cyanobacterial and other plant tetrapyrroles.

[Light absorption by suspended particulate matter in Chagan Lake, Jilin].

Spectral characteristics and the magnitudes of light absorption by suspended particulate matter were determined by spectrophotometry in this optically complex Lake Chagan waters for the purpose of surveying the natural variability of the absorption coefficients to parameterize the bio-optical models for converting satellite or in-situ water reflectance signatures into water quality information. Experiments were carried out on seasonal frozen Lake Chagan, one representative inland case-2 water body in Northeast of China. Particulate absorption properties analyzed using the field data on July 15th and October 12th 2009 were measured using the quantitative filter technique to produce absorption spectra containing several fractions that could be attributed to two main optical active constituents (OACs) phytoplankton pigments and non-algal particulates (mineral sediments, and organic detritus). Results suggested that the suspended particulate matter (SPM) concentration was higher while phytoplankton biomass (chlorophyll-a concentration) was lower in July and that in October. The spectral shape of total suspended particulate matter resembled that of non-algal particulates which contributed greater than phytoplankton in total particulate absorption during both periods. An obvious absorption peak occurring at around 440 nm exhibited an increase in phytoplankton contribution in October. Non-algal particulate absorption at 440 nm (a(NAP) (440)) had better correlation with total suspended particulate matter concentration than that with chlorophyll-a over the two periods. Light absorption by phytoplankton pigments in the Chagan lake region was generally lower than that of non-algal components. Chl. a dominating phytoplankton pigment composition functioned exponentially with its absorption coefficients at 440 and 675 nm specifically, the average values of which in July were 0.146 8 m2 x mg(-1) and 0.050 3 respectively while in October they were 0.153 3 and 0.013 2 m2 x mg(-1) varying regionally and seasonally due to the changes in specific composition, light and nutrient conditions.

[Content of biologically active substances--selenium, flavonoids, ascorbic acid and chlorophyllin of Allium ursinum L and Allium victorialis L].

Accumulation of selenium, flavonoids, chlorophyll and vitamin C by Allium ursinum L. and Allium victorialis L. are studied. Allium victorialis L. is shown to accumulate twice more selenium and flavonoids compared to Allium ursinum L. Increase of insolation elevates the concentration of the element, flavonoids and ascorbic acid in plants. Selenium concentration in Allium victorialis L. after selenium fertilization is higher compared to Allium ursinum L. The results indicate the significance of Allium victorialis L. as a source of natural antioxidants: selenium, flavonoids and ascorbic acid.

Behavioural responses of the mangrove fiddler crabs (Uca annulipes and U. inversa) to urban sewage loadings: results of a mesocosm approach.

The study aimed at investigating the effects of sewage loadings on the behaviour of two fiddler crabs species maintained in a system of experimental mesocosms, built in a mangrove area in Tanzania and inundated with different seawater/sewage mixtures. Our results show that sewage loads led to a modification of the overall activity budget of the crab community as a result of increased hypertrophic conditions (high COD, increased chlorophyll-a concentrations). During their activity period, crabs inside contaminated mesocosms seemed to satisfy their feeding demand faster than those of the control cells, spending a significant longer time in other activities like courtship and territorial defence. Apart from being a good biological indication of ecosystem eutrophication, such a reduced foraging activity by fiddler crabs also depresses their sediment bioturbation activity, important factor for the health of mangrove systems, suggesting practical implications regarding the efficiency of mangrove-based wetlands for treatment of domestic sewage.

Method development and HPLC analysis of retail foods and beverages for copper chlorophyll (E141[i]) and chlorophyllin (E141[ii]) food colouring materials.

An analytical method using high performance liquid chromatography with photodiode array and fluorescence detection has been developed and applied to the determination of the food colour additives copper chlorophylls and copper chlorophyllins (E141[i] and [ii]) in foods and beverages. The analytical procedures from previously reported methods have been refined to cover a range of food colour formulations and retail foods. The method was single-laboratory validated. Recoveries of the polar copper chlorophyllins from spiked samples (at 14.5 mg/kg in all but one case) were in the range 79-109%, except for jelly sweets (49%). Recoveries of relatively non-polar copper chlorophylls were in the range 77-107% (except for 'made' jelly at 50%). The %RSD for recoveries was generally below 12%. Quantitative estimates of the total copper chlorophyll/chlorophyllin content of a small range of food commodities are reported, based on the use of trisodium copper chlorophyllin as a surrogate standard. The majority of E141-containing foods and colour formulations analysed exhibited a multiplicity of components due to the various extraction and purification processes that are used to obtain these colour additives. This was confounded by the presence of overwhelming amounts of native chlorophylls in certain samples (e.g. mint sauce). Food commodities containing significant amounts of emulsifiers (i.e. ice cream), gelatine or fats were problematic during extraction hence further development of extraction regimes is desirable for such products. All of the samples analysed with added E141, had estimated total copper chlorophyllin contents of below 15 mg/kg (range 0.7-13.0).

Carotenoid, chlorophyll, and chlorophyll-derived compounds in grapes and port wines.

Carotenoids and chlorophyll-derived compounds in grapes and Port wines were investigated by HPLC-DAD and HPLC-DAD-MS (ESP+) analysis. A total of 13 carotenoid and chlorophyll-derived compounds are formally reported in grapes, 3 are identified for the first time, pheophytins a and b and (13Z)-beta-carotene, and 3 others remain unknown. In Port wines 19 compounds with carotenoid or chlorophyll-like structures are present, 8 still unidentified. The young wines showed higher total carotenoid content and chlorophyll-like compounds compared to aged Ports, with lutein and beta-carotene as major carotenoids. Among samples analyzed of monovarietal Vitis vinifera L. cultivar wines produced with the five most important Douro varieties, Tinta Roriz contained the highest levels of carotenoids and Touriga Franca the lowest. The forced-aging study indicated that lutein was more sensitive to temperature than beta-carotene. Additionally, aged wines showed higher ratios of beta-carotene/lutein concentrations compared to new Ports. Rates of degradation of chlorophyll derivative compounds were higher than those for carotene and lutein.

A chemotaxonomic method to quantify phytoplankton groups in freshwater lentic mesocosms: an approach including chlorophyll a breakdown products.

The use of HPLC methods to determine and quantify phytoplankton population composition, is sometimes less time-consuming than microscopic identification. However, its general application poses problems since high discrepancies between chlorophyll a calculated using chemotaxonomic methods and direct measurements were noticed. For instance, chemotaxonomic protocols generally employed can lead to a poor estimation of total and relative abundance when high amounts of chlorophyll a breakdown products are present. Therefore, we propose a new approach to calculate relative abundance of algal groups in a phytoplankton population, based on integration of these degradation products in the chemotaxonomic assessment in lentic and shallow freshwater ecosystems.

[Studies on the analysis of copper chlorophyll and sodium copper chlorophyllin in imported foods].

The analysis of copper chlorophyll (CuCh) and sodium copper chlorophyllin (CuCh-Na) in imported foods was carried out. Standards of CuCh and CuCh-Na were examined by ultraviolet and visible spectrophotometer. CuCh gave absorption maximum around 650 nm, while CuCh-Na gave around 630 nm. Imported sweets and liqueurs were homogenized and extracted with ethyl acetate. These extracts also gave absorption maxima absorbance around 650 nm or 630 nm. The results suggested that a distinction between CuCh and CuCh-Na in foods was possible by spectrophotometry. Visible spectrophotometric quantitation was also tried. However the estimated levels of CuCh or CuCh-Na in the samples by this method did not meet with those by atomic absorption spectrometry.

Chlorophyll precursors in the plasma membrane of a cyanobacterium, Anacystis nidulans. Characterization of protochlorophyllide and chlorophyllide by spectrophotometry, spectrofluorimetry, solvent partition, and high performance liquid chromatography.

Plasma membranes were isolated and separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation of crude membranes prepared by French pressure cell extrusion of lysozyme-treated Anacystis nidulans. Two distinct populations of chlorophyll-free plasma membrane vesicles were obtained exhibiting buoyant densities of 1.087 and 1.100 g/cm3 as opposed to a uniform density of 1.192 g/cm3 for thylakoid membranes. Plasma and thylakoid membranes were characteristically different also with respect to fatty acid and protein composition, cytochrome oxidase activity, and pigment content as analyzed by spectrophotometry, spectrofluorimetry, and high performance liquid chromatography. Apart from carotenoids, chlorophyll a was the only major photosynthetic pigment detected in thylakoid membranes while plasma membranes contained virtually no chlorophyll a but (besides large amounts of carotenoids) protochlorophyllide a and chlorophyllide a as revealed by solvent partition (between n-hexane and acetone or methanol), room and low temperature fluorescence emission and excitation spectra, and analytical separation and identification by high performance liquid chromatography and comparison with authentic standards. The protochlorophyllide in the plasma membrane could be transformed into chlorophyllide in the dark in vitro by incubating the membrane preparation with NADPH; NADP+ effected the reverse transition.