Towards a more accurate future for chlorophyll a and b determinations: the inaccuracies of Daniel Arnon's assay.

A recent publication (Esteban in New Phytol 217:341-342, 2018) describes how the use and citation of the assay of chlorophylls a and b extracted in aqueous 80% acetone by Arnon (Plant Physiol 2:1-15, 1949) is increasing, even in journals with high impact factors. This is a very disconcerting situation: the assay is outdated because it relies on the seriously under-estimated extinction coefficients of Mackinney (J Biol Chem 140:315-322, 1941), and the assay of chlorophylls is one of the most important, and much reported, procedures in studies of photosynthesis and related plant biological fields. Using the assay has led to the accumulation of masses of inaccurate data and confusion during the resolution of some plant biological problems.

Triacylglycerol "hand-shape profile" of Argan oil. Rapid and simple UHPLC-PDA-ESI-TOF/MS and HPTLC methods to detect counterfeit Argan oil and Argan-oil-based products.

The marketing of new argan-based products is greatly increased in the last few years and consequently, it has enhanced the number of control analysis aimed at detecting counterfeit products claiming argan oil as a major ingredient. Argan oil is produced in Morocco and it is quite expensive. Two simple methods for the rapid screening of pure oil and argan-oil based products, focused on the analysis of the triacylglycerol profile, have been developed.

Screening of preservatives by HPLC-PDA-ESI/MS: A focus on both allowed and recently forbidden compounds in the new EU cosmetics regulation.

Commission regulation (EU) No 358/2014 amending the new regulation (EC) No 1223/2009 on cosmetics has prohibited the use of isopropyl-, isobutyl-, phenyl-, benzyl- and pentylparaben. Furthermore, Commission regulation (EU) No 1004/2014 has lowered the maximum permitted concentration of butyl- and propylparaben in cosmetics and it has also banned them in leave-on products designed for application on the nappy area of children under three years of age. A HPLC-PDA-ESI/MS method has been developed herein for the detection of seventeen preservatives, both the most utilised and the recently forbidden by the new EU regulations.

Photostability of bacteriochlorophyll a and derivatives: potential sensitizers for photodynamic tumor therapy.

The photostabilities of bacteriochlorophyll a and several of its derivatives, which are of interest as potential sensitizers in photodynamic tumor therapy, were investigated. The pigments were irradiated with light >630 nm in organic solvents (acetone, tetrahydrofuran, pyridine, methanol, ethanol, n-propanol, 2-propanol and toluene) and in aqueous detergent solutions (cetyl-trimethyl-ammonium bromide [CTAB], lauryldimethyl-aminoxide [LDAO] or sodium dodecyl-sulfate [SDS] and Triton X-100 [TX100]). Their stabilities in these different solvents were determined in the presence and absence of an external sensitizer (pyromethyl-pheophorbide a), oxygen, sodium ascorbate and inert gas (Ar) or vacuum.

The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b.

Over the last half century, the most frequently used assay for chlorophylls in higher plants and green algae, the Arnon assay [Arnon DI (1949) Plant Physiol 24: 1-15], employed simultaneous equations for determining the concentrations of chlorophylls a and b in aqueous 80% acetone extracts of chlorophyllous plant and algal materials. These equations, however, were developed using extinction coefficients for chlorophylls a and b derived from early inaccurate spectrophotometric data. Thus, Arnon's equations give inaccurate chlorophyll a and b determinations and, therefore, inaccurate chlorophyll a/b ratios, which are always low.

(18)O and mass spectrometry in chlorophyll research: Derivation and loss of oxygen atoms at the periphery of the chlorophyll macrocycle during biosynthesis, degradation and adaptation.

Chlorophylls, magnesium-containing tetrapyrrolic pigments of photosynthesis, are widely-distributed in Nature and participate in both light harvesting and in the transduction of light energy to chemical energy for the photosynthetic fixation of carbon dioxide. We briefly discuss the extensive role of various isotopic labelling techniques in elucidating the pathway of tetrapyrrole-pigment biosynthesis and we acknowledge the classic and meticulous research of David Shemin who, approximately 50 years ago, introduced isotopic tracer techniques with (15)N and (14)C isotopes to study the biosynthesis of the carbon/nitrogen macrocycle of haem, an iron tetrapyrrole. The main focus of this review is the application of mass spectrometry and (18)O labelling to the study of the incorporation of oxygen atoms from molecular oxygen or water into the periphery of the chlorophyll macrocycle during biosynthesis and their loss during degradation and light acclimation.

Development of an HPLC method for the identification and dosage of non-allowed substances in cosmetic products. Part I: local anaesthetics and antihistaminics.

An HPLC method with ultraviolet detection coupled with a solid-phase extraction sample clean up was developed for the analysis of five local anaesthetics and four antihistaminics in cosmetic products. The presence of these compounds in commercial cosmetic samples is fordbidden. Extracts from real samples were applied to a solid-phase extraction C18 cartridge, and the analytes were eluted with 8:2 (v/v) acetonitrile/water containing 1% trifluoroacetic acid.

HPLC determination of imidazole antimycotis in antidandruff cosmetic products.

A simple HPLC method for the determination of imidazole antimycotics in cosmetic antidandruff formulations has been developed. HPLC was carried out on a Discovery RP-Amide C16 column and spectrophotometric detection was performed at 220 nm. The initial mobile phase was a mixture of acetonitrile and aqueous 10(-3) M NaClO4 (pH 3.

LTB4 as marker of 5-LO inhibitory activity of two new N-omega-ethoxycarbonyl-4-quinolones.

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.

Stability of reconstituted solutions of ceftazidime for injections: an HPLC and CE approach.

The stability of aqueous reconstituted ceftazidime injection vials containing ceftazidime pentahydrate blended with anhydrous sodium carbonate was investigated in different storage conditions (4 degrees C and 10 degrees C for 7 days in a refrigerator, 20 and 30 degrees C for 24 h) with validated HPLC and (micellar) CE methods. Stability indicating data were obtained for ceftazidime and two degradation products: pyridine and the delta2-ceftazidime isomer. Other degradation products were also identified (the complementarity of the two used experimental procedures was useful in such exercise) and characterized by their UV spectra and retention times.

Analysis of ceftazidime and related compounds by micellar electrokinetic chromatography.

A micellar electrokinetic chromatographic method for the separation and quantification of ceftazidime, its delta2-isomer and pyridine (two ceftazidime related impurities) was developed and validated. Optimised conditions were obtained using an electrolyte system consisting of 25 mM sodium tetraborate, at pH 9.2, and 75 mM sodium dodecylsulphate.

Biosynthesis of the 3-acetyl and 13(1)-oxo groups of bacteriochlorophyll a in the facultative aerobic bacterium, Rhodovulum sulfidophilum--the presence of both oxygenase and hydratase pathways for isocyclic ring formation.

Using (18)O-labelling and mass spectrometry, we have examined bacteriochlorophyll a formation in Rhodovulum sulfidophilum, formerly known as Rhodobacter sulfidophilus, which forms large amounts of BCh1 a both aerobically in the dark and anaerobically in the light. R. sulfidophilum, growing under strict anaerobiosis in the light, possesses hydratases which incorporate (18)O label from H2(18)O into both the 13(1)-oxo and 3-acetyl oxygens; in addition, the four carboxyl oxygens at C13(3) and C17(3) were labelled by H2(18)O.

Analysis of venlafaxine by capillary zone electrophoresis.

Capillary electrophoresis has been used for the separation of venlafaxine and two of its impurities deriving from the synthesis process. The electrophoretic experiments were performed using background electrolytes at different pHs in the 2.5-9.

Origin of the two carbonyl oxygens of bacteriochlorophyll a. Demonstration of two different pathways for the formation of ring E in Rhodobacter sphaeroides and Roseobacter denitrificans, and a common hydratase mechanism for 3-acetyl group formation.

A respiring culture of Rhodobacter sphaeroides, grown in the dark under defined aerobic conditions, produced cells capable of immediately commencing adaptation to photosynthetic growth on exposure to light and further reduction of oxygen tension. Adaptation was complete after 12 h and the bacteriochlorophyll a content increased 10-20-fold. This adaptation was performed in the presence of either H2(18)O or 18O2.

The derivation of the oxygen atoms of the 13(1)-oxo and 3-acetyl groups of bacteriochlorophyll a from water in Rhodobacter sphaeroides cells adapting from respiratory to photosynthetic conditions: evidence for an anaerobic pathway for the formation of isocyclic ring E.

Using mass spectrometry, we have demonstrated 18O-labelling of both the 13(1)-oxo and 3-acetyl groups of newly-formed bacteriochlorophyll a synthesized by Rhodobacter sphaeroides cells during adaptation from respiratory to photosynthetic conditions in the presence of H218O. This derivation of the 13(1)-oxo group of bacteriochlorophyll a from water provides a stark contrast with that of chlorophylls in higher plants where ring E formation is an aerobic process in which the 13(1)-oxo group arises from molecular oxygen via an oxygenase activity. The formation of the 3-acetyl group of bacteriochlorophyll a, however, is consistent with the enzymic hydration of the 3-vinyl group of a derivative of chlorophyll a.

The derivation of the formyl-group oxygen of chlorophyll b in higher plants from molecular oxygen. Achievement of high enrichment of the 7-formyl-group oxygen from 18O2 in greening maize leaves.

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7-formyl-group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark-grown plants, by illumination under white light in the presence of either H2(18)O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7-formyl substituent by reversible formation of chlorophyll b-7(1)-gem-diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process. Mass spectra of chlorophyll a and [7-hydroxymethyl]-chlorophyll b extracted from leaves greened in the presence of either H2(18)O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7-formyl group of chlorophyll b but also for the well-documented incorporation into the 13(1)-oxo group of both chlorophylls a and b [see Walker, C.

Derivation of the formyl-group oxygen of chlorophyll b from molecular oxygen in greening leaves of a higher plant (Zea mays).

Using mass spectroscopy, we demonstrate as much as 93% enrichment of the 7-formyl group oxygen of chlorophyll b when dark-grown, etiolated maize leaves are greened under white light in the presence of 18O2. This suggests that a mono-oxygenase is involved in the oxidation of its methyl group precursor. The concomitant enrichment of about 75% of the 13(1)-oxygen confirms the well-documented finding that this oxo group, in both chlorophyll a and b, also arises from O2.

The identification of related substances in 9 alpha-fluoroprednisolone-21 acetate by means of high-performance liquid chromatography with diode array detector and mass spectrometry.

A method for the analysis and identification of the principal related substances in 9 alpha-fluoroprednisolone acetate is described. This compound has been chosen as a model for the investigation of related substances which can be originated in the general procedure for introducing a fluorine substituent at position 9 of a corticosteroid molecule. HPLC procedures, both in reversed and in normal phase were used; a rapid scanning UV detector which allows direct spectrophotometric data to be obtained on chromatographic peaks, proved to be a tool of great importance.

Analytical and regulatory considerations for ferritin-containing pharmaceutical products.

Ferritin, an iron-containing protein widely diffused in nature, has the important biochemical function of being the principal reserve and regulator for Fe3+ levels in blood and tissue. Due to its natural, effectively non-toxic iron content, ferritin has been the object of strong interest in the development of pharmaceutical products for use in iron deficiency treatment. Therefore, the need has arisen for the analytical characterization of this industrial product, be it in the hydroglyceric solution or dry powder form.

Carotenoids, retinoids and alpha-tocopherol in human serum: Identification and determination by reversed-phase HPLC.

A rapid, simple and specific high performance liquid chromatographic procedure for assaying alpha- and beta-carotene is described. The method also enables the simultaneous determination of retinol and dl-alpha-tocopherol in human serum. The same chromatographic procedure can be used to assay the major carotenoids in human serum, provided analyses are replicated and the effluent is monitored at 450 nm.

Labelling of chlorophylls and precursors by [2-14C]glycine and 2-[1-14C]oxoglutarate in Rhodopseudomonas spheroides and Zea mays. Resolution of the C5 and Shemin pathways of 5-aminolaevulinate biosynthesis by thin-layer radiochromatography.

The C-5 of 5-aminolaevulinate, a tetrapyrrole precursor which accumulates when inhibitory laevulinate is present, is derived from either the C-2 of glycine by the 5-aminolaevulinate-synthase-mediated Shemin pathway or the C-1 of 2-oxoglutarate by the C5 pathway. Thin-layer-radiochromatographic procedures are described for determining whether [2-14C]glycine or 2-[1-14C]oxoglutarate labelled the macrocycle of bacteriochlorophyll a, in addition to or rather than the methyl ester or phytyl ester moieties of the side-chains. The method was also used for detecting whether the same substrates label the formaldehyde (C-5) or the succinate (C-1 to C-4) fragments, obtained by periodate cleavage of 5-aminolaevulinate.

[Oral load test with beta-carotene in normal and dysthyroid subjects].

An oral load of beta-carotene (500 mg) was administered to four normal, four hypo and four hyperthyroid subjects. Plasma beta-carotene content was determined at the 2nd, 4th, 6th, 8th, 10th, 12th and 24th hour after administration and at every 24th hour thereafter for 5 consecutive days. Plasma assays were performed by HPLC.

[Effect of renal and liver failure on blood levels of vitamin A, its precursor (beta-carotene) and its carrier proteins (prealbumin and retinol binding protein)].

Plasma beta-carotene and retinol assay was performed by high pressure liquid chromatography (HPLC) in subjects with chronic renal failure or liver cirrhosis. In the same subjects blood prealbumin (PA) and retinol binding protein (RBP) were determined by immunological technique. A considerable increase of retinol and in a lesser extent of beta-carotene was noted in the blood of patients with renal insufficiency.

[Behavior of vitamin A, beta-carotene, retinol binding protein and prealbumin in the plasma of hypo- and hyperthyroid subjects].

Plasma concentrations of beta-carotene and retinol, determined by HPLC, and of transport proteins, ascertained by immunodiffusion technique, in hypo and hyperthyroid subjects are reported. In hypothyroid subject a considerable increase in carotene was noted. This was not the case for retinol.