Comparative Metabolite Profiling of Three Savannic Species of Banisteriopsis (Malpighiaceae) via UPLC-MS/MS and Chemometric Tools.

Banisteriopsis (Malpighiaceae) is an important genus of neotropical savannas with related biological and medicinal activities but under-explored metabolomic profiles. We present a chemometric analysis for discriminating secondary metabolites of three species of Banisteriopsis (B. laevifolia, B. malifolia, and B. stellaris) leaves. Initially, each species was separately extracted with ethanol:water (4 : 1, v/v) and analysed by Ultra Performance Liquid Chromatography coupled with Mass Spectrometry (UPLC-MS/MS). The chromatographic profiles were subjected to Global Natural Product Social (GNPS) and Partial Least Squares Discriminant Analysis (PLS-DA). Eighty-nine compounds (cosine≥0.90) were annotated, including flavonoids, phenolics, and acids. The chemometric analysis (VIP Score) showed each species' relative concentration of the more relevant compounds. In addition, four compounds that discriminate the metabolomic profiles of B. laevifolia, B. malifolia, and B. stellaris were identified by PLS-DA.

Identification of volatile organic compounds in purple and white soybean flowers by HS-SPME/GC-MS.

This study aimed to establish a method for the extraction, enrichment, and identification of volatile organic compounds (VOCs) released by the flowers of purple (BRS 399) and white (DONMARIO 6563) soybean varieties. We tested the Static Headspace (HS) and Solid Phase Microextraction (SPME) methods using various fibre types: PDMS (Polydimethylsiloxane), PDMS/DVB (Divinylbenzene), and PDMS/DVB/CAR (Carboxen). We employed gas chromatography-mass spectrometry (GC-MS) to identify the VOCs. The SPME method with PDMS/DVB and PDMS/DVB/CAR fibres yielded the highest number of extracted compounds for both soybean cultivars. Notably, 67 compounds were detected in Glycine max. L for the first time. Using the developed method, we were able to detect 52 and 57 VOCs in the purple and white soybean varieties, respectively, including ketones, alcohols, aldehydes and benzenoids. In conclusion, the method we developed effectively identified VOCs in soybean flowers, thus enriching our understanding of the interactions between soybean flowers and their pollinators.

In vitro antiglycation and antioxidant properties of Eugenia pyriformis leaves and fruits.

Eight phenolic compounds were isolated from Eugenia pyriformis leaves fraction by semi-preparative HPLC and characterized by Nuclear Magnetic Resonance (NMR) and mass spectrometry (ESI-MS). Five compounds were isolated and identified for the first time in E. pyriformis species, while this is the first report of the accumulation of isoquercitrin, quercitrin, and the aglycone quercetin in its leaves. E. pyriformis leaves and fruits extracts, as well as the compounds isolated from the leaves most active fraction, were evaluated for their antiglycation and antioxidant activities. The mixture of myricetin-3-O-(2″-O-galloyl)-α-L-rhamnoside and myricetin-3-O-(4″-O-galloyl)-α-L-rhamnoside showed the highest antiglycation activity. These results suggest that this species is a promising source of bioactive compounds. Further studies to investigate the inhibition of the glycation process in vivo are necessary to evaluate its use in the treatment and/or prevention of advanced glycation end-products (AGEs)-associated diseases.

Chemical composition and chromatographic fingerprint of three strains of Agaricus subrufescens cultivated with handmade and commercial supplements.

Exploratory factor analysis was applied to determine the chemical differences between fruitbodies of three Agaricus subrufescens mushroom strains [from Japan (JP), Brazil (ABZ), and Belgium (T2)] grown with handmade and commercial supplements. The composition of the ABZ strain cultivated with agro-industrial waste supplement presented a high nutritional composition regarding the amounts of fibre and protein, similar to mushrooms cultivated with the commercial supplement. The chromatographic fingerprints obtained for T2 and JP strains grown with commercial supplements presented similar profiles compared to those cultivated with the supplement based on peanut and the mix of supplements. The chromatographic analysis also showed that the similarities are correlated with the relative abundance of antioxidant compounds annotated by HPLC-MS, such as vanillic acid deoxyhexoside, caffeic acid hexoside, catechin hexosemalonate, digallic acid, cinnamic acid derivative, and p-coumaroylmalic acid. This study showed that handmade supplements based on agro-industrial waste could be viable alternatives for replacing high-cost supplements.

Identification of a Prenyl Chalcone as a Competitive Lipoxygenase Inhibitor: Screening, Biochemical Evaluation and Molecular Modeling Studies.

Cyclooxygenase (COX) and lipoxygenase (LOX) are key targets for the development of new anti-inflammatory agents. LOX, which is involved in the biosynthesis of mediators in inflammation and allergic reactions, was selected for a biochemical screening campaign to identify LOX inhibitors by employing the main natural product library of Brazilian biodiversity. Two prenyl chalcones were identified as potent inhibitors of LOX-1 in the screening. The most active compound, (E)-2-O-farnesyl chalcone, decreased the rate of oxygen consumption to an extent similar to that of the positive control, nordihydroguaiaretic acid. Additionally, studies on the mechanism of the action indicated that (E)-2-O-farnesyl chalcone is a competitive LOX-1 inhibitor. Molecular modeling studies indicated the importance of the prenyl moieties for the binding of the inhibitors to the LOX binding site, which is related to their pharmacological properties.

Chalcones and their B-aryl analogues as myeloperoxidase inhibitors: In silico, in vitro and ex vivo investigations.

In the present study, a series of chalcones and their B-aryl analogues were prepared and evaluate as inhibitors of myeloperoxidase (MPO) chlorinating activity, using in vitro and ex vivo assays. Among these, B-thiophenyl chalcone (analogue 9) demonstrated inhibition of in vitro and ex vivo MPO chlorinating activity, exhibiting IC50 value of 0.53 and 19.2 µM, respectively. Potent ex vivo MPO inhibitors 5, 8 and 9 were not toxic to human neutrophils at 50 µM, as well as displayed weak 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH•) and hypochlorous acid (HOCl) scavenger abilities. Docking simulations indicated binding mode of MPO inhibitors, evidencing hydrogen bonds between the amino group at 4'position (ring A) of chalcones with Gln91, Asp94, and Hys95 MPO residues. In this regard, the efficacy and low toxicity promoted aminochalcones and arylic analogues to the rank of hit compounds in the search for new non-steroidal anti-inflammatory compounds.

Crotalaria spectabilis as a source of pyrrolizidine alkaloids and phenolic compounds: HPLC-MS/MS dereplication and monocrotaline quantification of seed and leaf extracts.

INTRODUCTION: Crotalaria spectabilis is an important species used as a pre-plant cover for soybean crops to control the proliferation of endoparasitic nematodes. Species from the Crotalaria genus are known for presenting pyrrolizidine alkaloids (PAs) in their composition, however, C. spectabilis is still considered chemically under-explored. OBJECTIVE: The goal of this manuscript is the development and validation of a method for PAs and flavonoids identification and quantification of C. spectabilis seeds and leaves, a toxic plant used for nematode proliferation control in soil, especially in soybean crops. MATERIALS AND METHODS: Seeds and leaves extracts were analysed by high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) for the identification of the compounds. RESULTS: PAs and phenolic compounds could be identified in both samples based on the MS/MS fragmentation pattern. Molecular formulas of the annotated compounds were confirmed by ultra-high-performace liquid chromatography-quadrupole time-of-flight (UHPLC-QToF), and monocrotaline could also be confirmed by standard comparison. The quantification of monocrotaline was performed by HPLC-MS/MS, resulting in 123 times higher monocrotaline content in seeds than in the leaves, which could explain its efficiency in combating nematode proliferation in soil. CONCLUSION: This was the first report of phenolic compounds in C. spectabilis. The current study highlights the importance of C. spectabilis for nematode control due to the presence of toxic PAs, and the employment of analytical techniques for identification and quantification of compounds present in the extracts.

Dereplication by HPLC-DAD-ESI-MS/MS and Screening for Biological Activities of Byrsonima Species (Malpighiaceae).

INTRODUCTION: Byrsonima species have been used in the treatment of gastrointestinal and gynecological inflammations, skin infections and snakebites. Based on their biological activities, it is important to study other organisms from this genus and to identify their metabolites. OBJECTIVES: To determine the metabolic fingerprinting of methanol and ethyl acetate extracts of four Byrsonima species (B. intermedia, B. coccolobifolia, B. verbascifolia and B. sericea) by HPLC-DAD-ESI-MS/MS and evaluate their in vitro antioxidant, anti-glycation, anti-inflammatory and cytotoxic activities. MATERIALS AND METHODS: Antioxidant activity was determined by DPPH˙, ABTS˙+ and ROO˙ scavenging assays. Anti-glycation activity was evaluated by the ability to inhibit the formation of advanced glycation endproducts (AGEs). Anti-inflammatory activity was evaluated using a murine macrophage cell line (RAW 264-7) in the presence of lipopolysaccharide (LPS). Tumour necrosis factor alpha (TNF-α) and nitrite (NO2- ) production were measured by ELISA and the Griess reaction, respectively. The compounds present in the extracts were tentatively identified by HPLC-DAD-ESI-MS/MS. RESULTS: The evaluation of the biological activities showed the potential of the extracts. The activities were assigned to the presence of glycoside flavonoids mainly derived from quercetin, quinic acid derivatives, gallic acid derivatives, galloylquinic acids and proanthocyanidins. Two isomers of sinapic acid-O-hexoside were described for the first time in a Byrsonima species. CONCLUSION: This research contributes to the study of the genus, it is the first report of the chemical composition of B. sericea and demonstrates the importance of the dereplication process, allowing the identification of known compounds without time-consuming procedures. Copyright © 2017 John Wiley & Sons, Ltd.

Dansylglycine, a fluorescent probe for specific determination of halogenating activity of myeloperoxidase and eosinophil peroxidase.

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCl and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl-) and bromide (Br-), which makes the assay able to distinguish the halogenating from the peroxidase activity. A linear correlation was obtained between the hydrogen peroxide (H2O2) concentration and the fluorescent decay. Similarly, the enzyme activity was measured by keeping constant H2O2. The method was applied for studding MPO/EPO specific inhibitors as 5-fluortryptamine (reversible inhibitor) and 4-hydroxybenzhydrazide (irreversible inhibitor). Differently of the taurine chloramine/3,3',5,5'-tetramethylbenzidine assay, which is among the most used technique, the dansylglycine assay was able to differentiate these inhibitors based on their kinetic behavior. In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCl and HOBr.

Redox-active biflavonoids from Garcinia brasiliensis as inhibitors of neutrophil oxidative burst and human erythrocyte membrane damage.

ETHNOPHARMACOLOGICAL RELEVANCE: Garcinia brasiliensis, a plant native to the Brazilian Amazon Rainforest, is used in traditional medicine to treat inflammation of the urinary tract, peptic ulcers, arthritis and other conditions. AIM OF THE STUDY: The purposes of this study were to analyze the chemical constituents of G. brasiliensis branches and leaves and to evaluate the potential of isolated compounds to act as inhibitors of both the oxidative burst of stimulated neutrophils and oxidative damage in human erythrocyte membranes to verify the antioxidant and anti-inflammatory effects of this plant. MATERIALS AND METHODS: Neutrophils were isolated from the blood of healthy donors by Ficoll-Paque density gradient centrifugation. Superoxide anion and total reactive oxygen species (ROS) produced by stimulated neutrophils were measured by WST-1 reduction and luminol-enhanced chemiluminescence assays, respectively. Radical-induced lipoperoxidation and hemolysis were performed using erythrocytes from the blood of healthy donors. Compounds were isolated from G. brasiliensis branches and leaves by HPLC microfractionation, and structure elucidation of the isolated compounds was performed based on NMR and HR-MS analyses. RESULTS: The biflavonoids procyanidin, fukugetin, amentoflavone and podocarpusflavone isolated from G. brasiliensis showed potent inhibitory effects on the oxidative burst of human neutrophils, inhibiting ROS production by 50% at 1 µmol L(-1). These biflavonoids also proved to be potent inhibitors of hemolysis (with 88 ± 7% inhibition at 50 µmol L(-1) for procyanidin) and lipid peroxidation in human erythrocytes, with a malondialdehyde level (a biomarker of oxidative stress) of 8.5 ± 0.3 nmol/mg Hb at 50 µmol L(-1) for procyanidin. CONCLUSIONS: These findings indicate that the biflavonoids extracted from G. brasiliensis branches and leaves modulate oxidative stress via inhibition of NADPH oxidase and ROS production by stimulated human neutrophils. Furthermore, the biflavonoids exhibited potent inhibition of oxidant hemolysis and lipid peroxidation induced by AAPH in human erythrocytes. Therefore, these studies suggest the use of G. brasiliensis extract as an antioxidant and anti-inflammatory agent.

Hydrophobicity and antioxidant activity acting together for the beneficial health properties of nordihydroguaiaretic acid.

Nordihydroguaiaretic acid (NDGA) and rosmarinic acid (RA), phenolic compounds found in various plants and functional foods, have known antioxidant and anti-inflammatory properties. In the present study, we comparatively investigated the importance of hydrophobicity and oxidisability of NDGA and RA, regarding their antioxidant and pharmacological activities. Using a panel of cell-free antioxidant protocols, including electrochemical measurements, we demonstrated that the anti-radical capacities of RA and NDGA were similar. However, the relative capacity of NDGA as an inhibitor of NADPH oxidase (ex vivo assays) was significantly higher compared to RA. The inhibitory effect on NADPH oxidase was not related to simple scavengers of superoxide anions, as confirmed by oxygen consumption by the activated neutrophils. The higher hydrophobicity of NDGA was also a determinant for the higher efficacy of NDGA regarding the inhibition of the release of hypochlorous acid by PMA-activated neutrophil and cytokine (TNF-α and IL-10) production by Staphylococcus aureus-stimulated peripheral blood mononuclear cells. In conclusion, although there have been extensive studies about the pharmacological properties of NDGA, our study showed, for the first time, the importance not only of its antioxidant activity, but also its hydrophobicity as a crucial factor for pharmacological action.

Antifungal compounds produced by Colletotrichum gloeosporioides, an endophytic fungus from Michelia champaca.

In this study, eight endophytic fungi were isolated from the leaves, stems and roots of Michelia champaca. The isolates were screened and evaluated for their antifungal, anticancer and acetylcholinesterase (AChE) inhibitory activities. All of the extracts exhibited potent activity against two evaluated phytopathogenic fungi. Chemical investigation of EtOAc extracts of the endophytic fungus Colletotrichum gloeosporioides resulted in the isolation of one new compound, 2-phenylethyl 1H-indol-3-yl-acetate (1), and seven known compounds: uracil (2), cyclo-(S*-Pro-S*-Tyr) (3), cyclo-(S*-Pro-S*-Val) (4), 2(2-aminophenyl)acetic acid (5), 2(4-hydroxyphenyl)acetic acid (6), 4-hydroxy- benzamide (7) and 2(2-hydroxyphenyl)acetic acid (8). All of the compound structures were elucidated using 1D and 2D NMR and MS analyses. The antifungal and AChE inhibitory activities of compounds 1-8 were evaluated in vitro. Compound 1 exhibited promising activity against Cladosporium cladosporioides and C. sphaerospermum that was comparable to that of the positive control nystatin.

Caffeic Acid phenethyl ester: consequences of its hydrophobicity in the oxidative functions and cytokine release by leukocytes.

Numerous anti-inflammatory properties have been attributed to caffeic acid phenethyl ester (CAPE), an active component of propolis. NADPH oxidases are multienzymatic complexes involved in many inflammatory diseases. Here, we studied the importance of the CAPE hydrophobicity on cell-free antioxidant capacity, inhibition of the NADPH oxidase and hypochlorous acid production, and release of TNF-α and IL-10 by activated leukocytes. The comparison was made with the related, but less hydrophobic, caffeic and chlorogenic acids. Cell-free studies such as superoxide anion scavenging assay, triene degradation, and anodic peak potential (E pa) measurements showed that the alterations in the hydrophobicity did not provoke significant changes in the oxidation potential and antiradical potency of the tested compounds. However, only CAPE was able to inhibit the production of superoxide anion by activated leukocytes. The inhibition of the NADPH oxidase resulted in the blockage of production of hypochlorous acid. Similarly, CAPE was the more effective inhibitor of the release of TNF-α and IL-10 by Staphylococcus aureus stimulated cells. In conclusion, the presence of the catechol moiety and the higher hydrophobicity were essential for the biological effects. Considering the involvement of NADPH oxidases in the genesis and progression of inflammatory diseases, CAPE should be considered as a promising anti-inflammatory drug.

Bioactive secondary metabolites from Phomopsis sp., an endophytic fungus from Senna spectabilis.

Chemical investigation of an acetonitrile fraction from the endophytic fungus Phomopsis sp. led to the isolation of the new natural product 2-hydroxy-alternariol (7) together with the known compounds cytochalasins J (1) and H (2), 5'-epialtenuene (3) and the mycotoxins alternariol monomethyl ether (AME, 4), alternariol (AOH, 5) and cytosporone C (6). The structure of the new compound was elucidated by using 1-D and 2-D NMR (nuclear magnetic resonance) and high resolution mass spectrometry. The cytochalasins J (1) and H (2) and AOH (5) exhibited potent inhibition of the total ROS (reactive oxygen species) produced by stimulated human neutrophils and acted as potent potential anti-inflammatory agents. Moreover, cytochalasin H (2) demonstrated antifungal and acetylcholinesterase enzyme (AChE) inhibition in vitro.

Chemical composition of the bark of Tetrapterys mucronata and identification of acetylcholinesterase inhibitory constituents.

The secondary metabolite content of Tetrapterys mucronata, a poorly studied plant that is used occasionally in Brazil for the preparation of a psychotropic plant decoction called "Ayahuasca", was determined to establish its chemical composition and to search for acetylcholinesterase (AChE) inhibitors. The ethanolic extract of the bark of T. mucronata exhibited in vitro AChE inhibition in a TLC bioautography assay. To localize the active compounds, biological profiling for AChE inhibition was performed using at-line HPLC-microfractionation in 96-well plates and subsequent AChE inhibition bioautography. The analytical HPLC-PDA conditions were transferred geometrically to a preparative medium-pressure liquid chromatography column using chromatographic calculations for the efficient isolation of the active compounds at the milligram scale. Twenty-two compounds were isolated, of which six are new natural products. The structures of the new compounds (9, 10, 16-18, and 20) were elucidated by spectroscopic data interpretation. Compounds 1, 5, 6, 9, and 10 inhibited AChE with IC50 values below 15 µM.

Apocynin: chemical and biophysical properties of a NADPH oxidase inhibitor.

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (e275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M-1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

Evaluation of the antioxidant activity of passion fruit (Passiflora edulis and Passiflora alata) extracts on stimulated neutrophils and myeloperoxidase activity assays.

The antioxidant activity of methanol extracts from Passiflora edulis and Passiflora alata pulp, and P. edulis rinds, healthy or infected with the passion fruit woodiness virus (PWV), was investigated using the oxidant activities of the neutrophil and the neutrophil granule enzyme myeloperoxidase (MPO), both playing key roles in inflammation. The reactive oxygen species produced by stimulated neutrophils were evaluated by lucigenin-enhanced chemiluminescence (CL) and the activity of purified MPO was measured by SIEFED (Specific Immunological Extraction Followed by Enzymatic Detection), a technique for studying the direct interaction of a compound with the enzyme. The rind extracts of P. edulis possessed higher and dose-dependent inhibitory effects on CL response and on the peroxidase activity of MPO than total pulp extracts from both passion fruit species. The quantification of isoorientin in the extracts showed a correlation with their antioxidant activity, suggesting the potential of P. edulis rinds as functional food or as a possible source of natural flavonoids.

Maracujá: um alimento funcional? / Passion fruit: a functional food?

Este artigo é uma revisão bibliográfica sobre as espécies brasileiras de Passiflora (Passiflora edulis fo. flavicarpa O. Deg., P. alata Curtis e P. edulis fo. edulis). A maioria dos artigos da literatura focaliza somente as folhas de Passiflora, enquanto que esta revisão contém informações sobre a polpa, cascas e sementes dos frutos do maracujá, com destaque para a composição química, estudos nutricionais e farmacológicos. O enfoque nos frutos do maracujá fundamenta-se no amplo consumo do suco de maracujá (fresco ou industrializado) no Brasil e também nas investigações em andamento para avaliar o seu potencial uso como alimento funcional.

This paper consists of a bibliographic review of the most relevant edible Brazilian Passiflora species (Passiflora edulis fo. flavicarpa O. Deg., P. alata Curtis and P. edulis fo. edulis). Most of the reports in the literature focus solely on Passiflora leaves, whereas this review contains information about passion fruit pulp, rind and seeds, highlightening chemical composition, nutritional and pharmacological studies. The emphasis on the "maracujá" fruit is due to the extensive consumption of passion fruit juice (fresh or processed) in Brazil and on ongoing investigations into its potential as a functional food.

Determinação espectrométrica dos flavonóides das folhas de Maytenus (Celastraceae) e de Passiflora (Passifloraceae) e comparação com método CLAE-UV / Spectrometric determination of flavonoids from Maytenus (Celastraceae) and Passiflora (Passifloraceae) leaves and comparison with an HPLC-UV method

Este trabalho apresenta uma modificação dos procedimentos descritos nas Farmacopéias Francesa e Européia para a análise de flavonoides de Passiflora incarnata L., Passifloraceae, por espectrometria UV-Visível e propõe a sua aplicação na determinação dos flavonoides totais das folhas da espinheira-santa (Maytenus aquifolium Mart. e Maytenus ilicifolia (Schrad.) Planch., Celastraceae) e do maracujá (Passiflora edulis Sims. e Passiflora alata Curtis, Passifloraceae). Os resultados obtidos por espectrometria no UV-Visível foram comparados aos obtidos por cromatografia líquida de alta eficiência (CLAE-UV), encontrando-se resultados estatisticamente similares entre os métodos espectrométrico modificado da Farmacopéia Francesa e CLAE-UV.

This paper reports on a modification of the spectrometric procedures originally described in the French and European Pharmacopoeia for the analysis of Passiflora incarnata L. (Passifloraceae) flavonoids, proposing its application in the determination of total flavonoids from "espinheira-santa" (Maytenus aquifolium Mart. and Maytenus ilicifolia (Schrad.) Planch., Celastraceae) and "maracujá" leaves (Passiflora edulis Sims and Passiflora alata Curtis, Passifloraceae). A comparison was made of the results obtained by the spectrometric procedure with those obtained by high performance liquid chromatography (HPLC-UV), which demonstrated complete compatibility between the modified French Pharmacopoeia (spectrometric) and HPLC-UV methods.