Ni speciation in tea infusions by monolithic chromatography—ICP-MS and Q-TOF-MS

For humans, Ni is not considered to be an essential trace element. Its compounds, at levels present in foodstuffs and drinks, are generally considered to be safe for consumption, but for individuals who already suffer from contact allergy to Ni and may be subject to develop systemic reactions from its dietary ingestion, dietary exposure to Ni must be kept under control. Being the second most popular beverage, tea is a potential source of dietary Ni. Present knowledge on its speciation in tea infusions is poor. Therefore, complete speciation analysis, consisting of separation by liquid chromatography using a weak CIM DEAE-1 monolithic column, “on-line” detection by inductively coupled plasma mass spectrometry (ICP-MS) and “off-line” identification of ligands by hybrid quadrupole time-of-flight mass spectrometry (Q-TOF MS), was implemented for the first time to study Ni speciation in tea infusions. Total concentrations of Ni in dry leaves of white, green, oolong and black tea (Camellia sinensis) and flowers of herbal chamomile (Matricaria chamomilla) and hibiscus (Hibiscus sabdariffa) tea were determined after microwave digestion by ICP-MS. They lay between 1.21 and 14.4 mg?kg^?1. Good agreement between the determined and the certified values of the Ni content in the standard reference material SRM 1573a tomato leaves confirmed the accuracy of the total Ni determination. During the infusion process, up to 85 % of Ni was extracted from tea leaves or flowers. Separation of Ni species was completed in 10 min by applying aqueous linear gradient elution with 0.6 mol?L^?1 NH₄NO₃. Ni was found to be present in the chromatographic fraction in which quinic acid was identified by Q-TOF in all the tea infusions analysed, which had pH values between 5.6 and 6.0. The only exception was the infusion of hibiscus tea with a pH of 2.7, where results of speciation analysis showed that Ni is present in its divalent ionic form.     

Evaluation of an in vitro method to estimate trace elements bioavailability in edible seaweeds

Raw edible seaweed harvested in the Galician coast (Northwestern Spain), including two red seaweed types (Dulse and Nori), three brown seaweed (Kombu, Wakame and Sea Spaghetti), one green seaweed (Sea Lettuce) and one microalgae (Spirulina platensis) were studied to assess trace elements bioavailability using an in vitro method (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked seaweed sample (canned in brine) consisting of a mixture of two brown seaweed (Sea Spaghetti and Furbelows) and a derived product (Agar-Agar) from the red seaweed Gelidiumm sesquipedale, were also included in the study. The total trace element content as well as the non-dialyzable fractions was carried out after a microwave acid digestion of the seaweed samples by inductively coupled plasma-mass spectrometry (ICP-MS). The dialyzable fraction was determined without any pre-treatment by ICP-MS.PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a NIES-09 certified reference material (Sargasso seaweed). The accuracy of the in vitro procedure was established by a mass balance study which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). The highest dialyzability percentages (100 ± 0.2%) were obtained for Dulse in Mn and V.     

Multielement determination and speciation of major-to-trace elements in black tea leaves by ICP-AES and ICP-MS with the aid of size exclusion chromatography.

A multielement determination of major-to-trace elements in black tea leaves and their tea infusions was carried out by ICP-AES (inductively coupled plasma atomic emission spectrometry) and ICP-MS (inductively coupled plasma mass spectrometry). Tea infusions were prepared as usual tea beverage by brewing black tea leaves in boiling water for 5 min. About 40 elements in tea leaves and tea infusions could be determined over the wide concentration range in 8 orders of magnitude. The extraction efficiency of each element was estimated as the ratio of its concentration in tea infusions to that in tea leaves. From the experimental results for the extraction efficiencies, the elements in black tea leaves were classified into three characteristic groups: (i) highly-extractable elements (>55%): Na, K, Co, Ni, Rb, Cs and Tl, (ii) moderately-extractable elements (20-55%): Mg, Al, P, Mn and Zn, and (iii) poorly-extractable elements (<20%): Ca, Fe, Cu, Sr, Y, Zr, Mo, Sn, Ba and lanthanoid elements. Furthermore, speciation of major-to-trace elements in tea infusions was performed by using a combined system of size exclusion chromatography (SEC) and ICP-MS (or ICP-AES). As a result, many diverse elements were found to be present as complexes associated with large organic molecules in tea infusions.     

Aluminium in tea—concentrations, speciation and bioavailability

Tea (Camellia sinensis) is one of a few plants accumulating aluminium (Al), making tea a major source of dietary Al intake. This paper reviews published studies on the concentrations, speciation and bioavailability of Al in tea. With very few exceptions, the total concentration of Al in tea infusions is in the range 1-6 mg l⁻¹. Probably more than 90% of this Al is bound to organic matter, but the nature of the organic species is unclear. Three studies using size exclusion chromatography provide evidence for Al species in the molecular mass (MM) range 4000-8500 Da, probably polyphenolic complexes. Two ultrafiltration studies indicate the presence of Al species with MMs above 10,000. The relative amount of the different organic Al species in tea infusions is unclear, and even the identity of any of these has not been demonstrated with certainty. A possible exception is Al trioxalate, which may be an important species based on evidence from two ²⁷Al-NMR studies. It seems fairly well established that drinking tea leads to measurable, but moderate increases in urinary Al excretion. However, the Al present in tea does not seem to be much more bioavailable than that from other dietary sources. Even so, it cannot be dismissed that tea infusions may contain particularly bioavailable and neurotoxic compounds such as Al maltolate, but this is at present speculative.     

A dynamic continuous-flow dialysis system with on-line electrothermal atomic-absorption spectrometric and pH measurements for in-vitro determination of iron bioavailability by simulated gastrointestinal digestion

A dynamic continuous-flow dialysis (CFD) method with on-line electrothermal atomic absorption spectrometric (ETAAS) and pH measurements for study of simulated gastrointestinal digestion has been developed for prediction of iron bioavailability. The method used to estimate mineral bioavailability was based on gastric digestion in a batch system then dynamic continuous-flow intestinal digestion. The intestinal digestion was performed in a dialysis bag placed inside a chamber containing a flowing stream of dialyzing solution. Mineral concentration and dialysate pH were monitored by ETAAS and use of a pH meter, respectively. The amount of dialyzed minerals in the intestinal digestion stage was used to evaluate the dialyzability. The dialysis profile and pH change can be used to understand or examine differences between the dialyzability of different food samples. To test the proposed system it was used to estimate the iron dialyzability of different kinds of milk. Iron dialyzability for powdered cow milk, cereal milk, and two brands of soymilk was found to be 1.7, 20.4, 24.9, and 37.7%, respectively. The developed CFD–ETAAS–pH system is a simple, rapid, and inexpensive tool for bioavailability studies, especially for minerals at ultratrace levels.     

Study on essential and toxic elements intake from drinking of Chinese tea

Twenty different brands of Chinese tea were analyzed for multiple trace elements, including some essential and toxic elements, by neutron activation analysis (NAA). A comparison among tea brands from China, India, US and other countries was made for the ranges and averages of concentrations for Na, K, Mn, Cu, and Br. It has been observed that the trace element contents in tea leaves are largely dependent upon the soil and the environment where the tea grows. Chinese tea is rich in Mn and Cu comparing with those of other counties surveyed, but is indigent in Na. The transference ratio for each element determined (i.e., the fraction of an element in tea leave transferred into solution when tea is leached by percolation) is also reported. Adult daily intakes of some essential and toxic elements from tea drinking were also estimated.     

Comparison of heating extraction procedures for Al, Ca, Mg, and Mn in tea samples.

A focused-microwave-assisted procedure was adopted for the extraction of Al, Ca, Mg, and Mn in tea leaves. The efficiency of extraction was evaluated using diluted acids and a water-soluble alkaline tertiary-amines solution. The extraction procedure was implemented in 5 min. A conventional hot-plate digestion procedure was employed for a comparison. A colorless digest was obtained for all tea samples and the total contents determined for each analyte were employed for estimating the efficiency of extraction. Tea infusions were also prepared. Subsequent determinations of Al, Ca, Mg, and Mn were carried out using flame atomic absorption spectrophotometry (FAAS) and inductively coupled plasma optical emission spectrometry (ICP-OES). In most cases, quantitative, or at least semi-quantitative, extractions were attained for Ca, Mg, and Mn. Lower recoveries were attained to Al, which seems to be related to compounds that bind this element in the sample matrix. Large variations in the trace-element composition of teas were observed; these results are discussed with reference to both extraction media and type of tea.     

In vitro evaluation of Cu and Fe bioavailability in cashew nuts by off-line coupled SEC-UV and SIMAAS

In this work Cu and Fe bioavailability in cashew nuts was evaluated using in vitro method. Extractions with simulated gastric and intestinal fluids and dialysis procedures were applied for this purpose. The proteins separation and quantification were performed by size exclusion chromatography (SEC) coupled on-line to ultra-violet (UV) and off-line to simultaneous multielement atomic absorption spectrometry (SIMAAS). The SEC-UV and SIMAAS profiles of the protein fractions obtained by alkaline extraction (NaOH) and precipitation with HCl indicated the presence of high and low molecular weight species in the range between >75 kDa and 9.3 kDa. Almost 83% of Cu and 78% of Fe were extracted during cashew nut digestion and 90% of both elements were dialyzed. With these results it is possible to assume that 75% of Cu and 70% of Fe present in cashew nut could be bioavailable. The SEC-UV and SIMAAS chromatographic profiles obtained after in vitro gastrointestinal digestion reveal that Cu and Fe not dialyzed can be associated to a compound of 9.2 kDa.     

Flow field-flow fractionation-inductively coupled optical emission spectrometric investigation of the size-based distribution of iron complexed to phytic and tannic acids in a food suspension: implications for iron availability

Flow field–flow fractionation–inductively coupled plasma optical emission spectrometry (FlFFF–ICP–OES) was applied to achieve the size-based fractionation of iron in a food suspension in order to gain insights into iron availability. The binding of iron with phytic and tannic acids, employed as model inhibitors of iron availability in foods, was investigated at pH 2.0 (representing stomach fluid), pH 5.0 (the transition stage in the upper part of the duodenum), and pH 7.0 (the small intestine). In the presence of phytic acid, iron was found as a free ion or it was associated with molecules smaller than 1 kDa at pH 2.0. Iron associated with molecules larger than 1 kDa when the pH of the mixture was raised to 5.0 and 7.0. In the presence of tannic acid, iron was again mostly associated with molecules smaller than 1 kDa at pH 2.0. However, at pH 5.0, iron and tannic acid associated in large molecules (∼25 kDa), while at pH 7.0, most of the iron was associated with macromolecules larger than 500 kDa. Iron size-based distributions of kale extract and tea infusion containing phytic and tannic acids, respectively, were also examined at the three pH values, with and without enzymatic digestion. Without enzymatic digestion of the kale extract and the tea infusion at pH 2.0, most of the iron was released as free ions or associated with molecules smaller than 1 kDa. At other pH values, most of the iron in the kale extract and the tea infusion was found to bind with ~2 kDa and >500 kDa macromolecules, respectively. Upon enzymatic gastrointestinal digestion, the iron was not observed to bind to macromolecules >1 kDa but <500 kDa, due to the enzymatic breakdown of large molecules to smaller ones (<1 kDa). Figure Flow field–flow fractionation was exploited in order to achieve size-based iron fractionation and thus investigate iron-binding behavior under gastrointestinal conditions     

Simultaneous determination of Cd, Pb, Cu, Sb, Bi, Se, Zn, Mn, Ni, Co and Fe in water samples by differential pulse stripping voltammetry at a hanging mercury drop electrode

A highly sensitive and selective voltammetric procedure is described for the simultaneous determination of eleven elements (Cd, Pb, Cu, Sb, Bi, Se, Zn, Mn, Ni, Co and Fe) in water samples. Firstly, differential pulse anodic stripping voltammetry (DPASV) with a hanging mercury drop electrode (HMDE) is used for the direct simultaneous determination of Cd, Pb, Cu, Sb and Bi in 0.1 M HCI solution (pH = 1) containing 2 M NaCl. Then, differential pulse cathodic stripping voltammetry (DPCSV) is used for the determination of Se in the same solution. Zn is subsequently determined by DPASV after raising the pH of the same solution to pH 4. Next, the pH of the medium is raised to pH 8.5 by adding NH3/NH4Cl buffer solution for the determination of Mn by DPASV. Ni and Co are determined in the same solution by differential pulse adsorptive stripping voltammetry (DPAdSV) after adding DMG (1 x 10(-4) M). Finally, 1 x 10(-5) M 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) is added to the solution for the determination of Fe by DPAdSV. The optimal conditions are described. Relative standard deviations and relative errors are calculated for the eleven elements at three different concentration levels. The lower detection limits for the investigated elements range from 1.11 x 10(-10) to 1.05 x 10(-9)M, depending on the element determined. The proposed analysis scheme was applied for the determination of these eleven elements in some ground water samples.     

Dynamic continuous-flow dialysis method to simulate intestinal digestion for in vitro estimation of mineral bioavailability of food

A system for dynamic continuous-flow dialysis during intestinal digestion for an in vitro simulation of gastrointestinal digestion is presented as an alternative to human and animal in vivo methods for estimation of the bioavailability of minerals. The method is based on the in vitro batch dialysis method described by Miller, which was developed into a continuous-flow system of a simple design to perform dynamic dialysis in the intestinal digestion stage. A flow dialysis system has the advantages of simulation being close to in vivo physiological conditions because pH change during dialysis is gradual and dialyzed components are continuously removed. The proposed new design performed dialysis during a continuous flow of dialyzing solution (NaHCO₃) around a dialysis bag containing peptic digest, which is placed inside a glass dialysis chamber. Gradual change of dialysis pH, similar to that occurring in the gastrointestinal tract, was obtained by optimization of flow rate and concentration of NaHCO₃. The dialysate collected in fractions was analyzed to determine dialyzed minerals and pH change in the course of dialysis. The method was tested by determination of calcium bioavailability of powder milk and calcium carbonate tablets.     

Study of complex formation in the manganese(II)/azide system

The complex formation between Mn(II) cations and N(3)(-) anions was studied in aqueous medium at 25 degrees C and ionic strength 2.0 M (NaClO(4)). Data of average ligand number, n (Bjerrum's function), were obtained from pH measurements on the Mn(II)/N(3)(-)/HN(3) system followed by integration to obtain Leden's function, F(0)(L). Graphical treatment of data and a matrix solution of simultaneous equations have given the following overall formation constants of mononuclear stepwise complexes: beta(1)=4.15+/-0.02 M(-1), beta(2)=6.61+/-0.04 M(-2), beta(3)=3.33+/-0.02 M(-3), beta(4)=0.63+/-0.01 M(-4). A linear plot of log K(n) vs. (n-1) shows no change in the configuration during complex formation. Slow spontaneous oxidation of solutions to Mn(III) occurs when the N(3)(-) concentration is greater than 1.0 M.     

原子吸收光谱法测定茶叶中铅和镉时消解方法的优化

采用多种方法对同一种茶叶进行消解,用原子吸收光谱法测定不同方法消解的茶叶中有害微量元素铅和镉的含量并进行对比,探索了茶叶中微量元素铅和镉低损耗的最佳消解条件.实验结果表明,不同的消解方式对茶叶中微量元素的测定结果影响很大.测定茶叶中铅和镉元素时,采用硝酸-高氯酸加盖浸泡过夜方法较为理想.     

Protective effect of Boldo and tea infusions on the visible light-mediated pro-oxidant effects of vitamin B2, riboflavin

The effect of Boldo and black tea infusions on the pro-oxidant effects of vitamin B2, riboflavin (RF), when exposed to the action of visible light was studied. The amounts of antioxidants present in Boldo and tea infusions were evaluated by a procedure based on the bleaching of preformed 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cations and were expressed as 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid equivalent concentrations. The quenching rate constants of singlet oxygen (1O2; [kq]Boldo = 6.0 x 10(7) M(-1) s(-1) and [kq]Tea = 3.2 x 10(7) M(-1) s(-1)) and triplet RF (3RF; [3RFkq]Boldo = 10 x 10(8) M(-1) s(-1) and [3RFkq]TEA = 3.2 x 10(8) M(-1) s(-1)) with Boldo and tea were determined by flash photolysis. These data allow a quantitative interpretation of the results obtained. Our data suggest that most of the oxygen consumption observed in the photolysis of RF in the presence of tea and Boldo infusions is caused by 1O2 reactions. The oxygen consumption quantum yield is considerably smaller than the fraction of RF triplets trapped by the additives (AH) present in the infusion, indicating that their interaction with 3RF does not lead to chemical reactions or that the AH*+ radical ions initially formed participate in secondary processes that do not consume oxygen. Boldo and tea infusions have a significant protective effect when a system containing RF and tryptophan (Trp) is exposed to visible light, not only by quenching the 1O2 and interfering with the Type-I mechanism but also by repairing the damage to Trp molecules associated with the latter mechanism.     

Extraction of PCDD/PCDF from soil with supercritical CO2: Optimization by a three-level factorial design approach

A highly sensitive and selective voltammetric procedure is described for the simultaneous determination of eleven elements (Cd, Pb, Cu, Sb, Bi, Se, Zn, Mn, Ni, Co and Fe) in water samples. Firstly, differential pulse anodic stripping voltammetry (DPASV) with a hanging mercury drop electrode (HMDE) is used for the direct simultaneous determination of Cd, Pb, Cu, Sb and Bi in 0.1 M HCl solution (pH = 1) containing 2 M NaCl. Then, differential pulse cathodic stripping voltammetry (DPCSV) is used for the determination of Se in the same solution. Zn is subsequently determined by DPASV after raising the pH of the same solution to pH 4. Next, the pH of the medium is raised to pH 8.5 by adding NH₃/NH₄Cl buffer solution for the determination of Mn by DPASV. Ni and Co are determined in the same solution by differential pulse adsorptive stripping voltammetry (DPAdSV) after adding DMG (1 × 10^–4 M). Finally, 1 × 10^–5 M 2-(5-bromo-2-pyridylazo)-5-diethylaminophenol (5-Br-PADAP) is added to the solution for the determination of Fe by DPAdSV. The optimal conditions are described. Relative standard deviations and relative errors are calculated for the eleven elements at three different concentration levels. The lower detection limits for the investigated elements range from 1.11 × 10^–10 to 1.05 × 10^–9 M, depending on the element determined. The proposed analysis scheme was applied for the determination of these eleven elements in some ground water samples.     

First hydrolysis constants of hexaaquacobalt(III) and -manganese(III): longstanding issues resolved

The acid dissociation constants, Kh, of the hexaaquacobalt(III) and -manganese(III) ions are reported under conditions which minimize the problems of instability of these species at modest acidities (pH 1-4). A rapid and convenient preparation of aqueous Co(III) has been used, and the latter has been reacted with aqueous Mn(II) to form aqueous Mn(III). The noncoordinating buffer H2PIPPS (1,4-bis(3-sulfopropyl)piperazine) has been used to react with H+ and provide some control of the pH. Spectrophotometric changes, after stopped-flow mixing for Co(III) and conventional mixing for Mn(III), were used to determine the Kh values. The pKh values are as follows: Co(III), 3.10 +/- 0.07 (6.0 degrees C) and 2.92 +/- 0.04 (20.0 degrees C) in 1.0 M H+, Na+, ClO4-; Mn(III), 0.22 +/- 0.04 (7.6 degrees C) and 0.08 +/- 0.04 (20.0 degrees C) in approximately 4 M H+, Mn2+, ClO4-. Global fits of the data for each metal have been used to estimate the DeltaH degree and DeltaS degree values for Kh. It is suggested that the unusually high acidity of Mn(OH2)6(3+) may be due to a Jahn-Teller distortion effect.     

Application of high-performance liquid chromatography--inductively coupled plasma mass spectrometry to the investigation of cadmium speciation in pig kidney following cooking and in vitro gastro-intestinal digestion

The speciation of cadmium in retail pig kidney has been examined by size-exclusion chromatography (SEC) coupled directly to inductively coupled plasma mass spectrometry (ICP-MS). Approximately 35% of the cadmium from uncooked kidney was soluble after aqueous extraction at pH 8 and SEC - ICP-MS revealed three discrete peaks whose retention times corresponded to estimated relative molecular masses of 1.2 x 10(6), 7.0 x 10(4) and 6 x 10(3)-9 x 10(3). In the cooked kidney, 35% of the Cd was soluble and was all associated with a peak of a relative molecular mass (Mr) of 6 x 10(3)-9 x 10(3). After simulated gastric digestion of cooked pig kidney at pH 2.5, 60% of the cadmium was solubilised and associated with a species of Mr less than 1 x 10(3). When the digest was also subjected to simulated intestinal digestion at pH 6.8, a single peak, which corresponded to 20% of the original cadmium, was eluted. This peak co-eluted with the single peak extracted at pH 8.0 from the cooked kidney. It was also of similar estimated Mr to the single broad peak observed after simulated gastro-intestinal digestion of equine renal metallothionein (Mr = 1.1 x 10(4]. The results suggest that the majority of soluble cadmium in retail pig kidney is associated with a metallothionein-like protein that survives both cooking and simulated in vitro gastro-intestinal digestion.     

Profiling of In Vitro Bioaccessibility and Intestinal Uptake of Flavonoids after Consumption of Commonly Available Green Tea Types

The aim of this study was to profile the bioaccessibility and intestinal absorption of epicatechins and flavonols in different forms of green tea and its formulation: loose leaf tea, powdered tea, 35% catechins containing GTE, and GTE formulated with green tea-derived polysaccharide and flavonols (CATEPLUS™). The bioaccessibillity and intestinal absorption of epicatechins and flavonols was investigated by using an in vitro digestion model system with Caco-2 cells. The bioaccessibility of total epicatechins in loose leaf tea, powdered tea, GTE, and CATEPLUS™ was 1.27%, 2.30%, 22.05%, and 18.72%, respectively, showing that GTE and CATEPLUS™ had significantly higher bioaccessibility than powdered tea and loose leaf tea. None of the flavonols were detected in powdered tea and loose leaf tea, but the bioaccessibility of the total flavonols in GTE and CATEPLUS™ was 85.74% and 66.98%, respectively. The highest intestinal absorption of epicatechins was found in CATEPLUS™ (171.39 ± 5.39 ng/mg protein) followed by GTE (57.38 ± 9.31), powdered tea (3.60 ± 0.67), and loose leaf tea (2.94 ± 1.03). The results from the study suggest that formulating green tea extracts rich in catechins with second components obtained from green tea processing could enhance the bioavailability of epicatechins.     

微波辅助消解-电感耦合等离子体原子发射光谱法(ICP-AES)测定几种血藤中17种元素

建立电感耦合等离子体原子发射光谱法(ICP-AES)测定血藤类中药中K,Ca,Mg,P,Sr,Fe,Mn,Zn,Cu,Co,Cr,Al,Ba,Ni,As,Pb和Cd 17种常量和微量元素含量的分析方法。样品用微波辅助消解,ICP-AES法检测几种血藤中17种元素的含量,方法的检出限为0.000 03~0.009 81μg/mL,加标回收率88.37%~110.00%,相对标准偏差(RSD)均小于5.0%,方法可快速、灵敏、准确测定血藤类中药中多种常量、微量元素的含量。血藤样品中除含有人体必需的常量元素K,Ca,Mg外,还含有Fe,Zn,Mn,Cr,Co等必需的微量元素和其它元素P,Ni,Ba,Al,Sr,Pb,Cd,Ba等。     

The nature of the intermediates in the reactions of Fe(III)- and Mn(III)-microperoxidase-8 with H(2)O(2): a rapid kinetics study

Kinetic studies were performed with microperoxidase-8 (Fe(III)MP-8), the proteolytic breakdown product of horse heart cytochrome c containing an octapeptide linked to an iron protoporphyrin IX. Mn(III) was substituted for Fe(III) in Mn(III)MP-8.The mechanism of formation of the reactive metal-oxo and metal-hydroperoxo intermediates of M(III)MP-8 upon reaction of H(2)O(2) with Fe(III)MP-8 and Mn(III)MP-8 was investigated by rapid-scan stopped-flow spectroscopy and transient EPR. Two steps (k(obs1) and k(obs2)) were observed and analyzed for the reaction of hydrogen peroxide with both catalysts. The plots of k(obs1) as function of [H(2)O(2)] at pH 8.0 and pH 9.1 for Fe(III)MP-8, and at pH 10.2 and pH 10.9 for Mn(III)MP-8, exhibit saturation kinetics, which reveal the accumulation of an intermediate. Double reciprocal plots of 1/k(obs1) as function of 1/[H(2)O(2)] at different pH values reveal a competitive effect of protons in the oxidation of M(III)MP-8. This effect of protons is confirmed by the linear dependence of 1/k(obs1) on [H(+)] showing that k(obs1) increases with the pH. The UV-visible spectra of the intermediates formed at the end of the first step (k(obs1)) exhibit a spectrum characteristic of a high-valent metal-oxo intermediate for both catalysts. Transient EPR of Mn(III)MP-8 incubated with an excess of H(2)O(2), at pH 11.5, shows the detection of a free radical signal at g approximately equal to 2 and of a resonance at g approximately equal to 4 characteristic of a Mn(IV) (S = 3/2) species. On the basis of these results, the following mechanism is proposed: (i) M(III)MP-8-OH(2) is deprotonated to M(III)MP-8-OH in a rapid preequilibrium step, with a pK(a) = 9.2 +/- 0.9 for Fe(III)MP-8 and a pK(a) = 11.2 +/- 0.3 for Mn(III)MP-8; (ii) M(III)MP-8-OH reacts with H(2)O(2) to form Compound 0, M(III)MP8-OOH, with a second-order rate constant k(1) = (1.3 +/- 0.6) x 10(6) M(-1) x s(-1) for Fe(III)MP-8 and k(1) = (1.6 +/- 0.9) x 10(5) M(-1) x s(-1) for Mn(III)MP-8; (iii) this metal-hydroperoxo intermediate is subsequently converted to a high-valent metal-oxo species, M(IV)MP-8=O, with a free radical on the peptide (R(*+)). The first-order rate constants for the cleavage of the hydroperoxo group are k(2) = 165 +/- 8 s(-1) for Fe(III)MP-8 and k(2) = 145 +/- 7 s(-1) for Mn(III)MP-8; and (iv) the proposed M(IV)MP-8=O(R(*+)) intermediate slowly decays (k(obs2)) with a rate constant of k(obs2) = 13.1 +/- 1.1 s(-)(1) for Fe(III)MP-8 and k(obs2) = 5.2 +/- 1.2 s(-1) for Mn(III)MP-8. The results show that Compound 0 is formed prior to what is analyzed as a high-valent metal-oxo peptide radical intermediate.