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The release of new olive cultivars with an increased squalene content in their virgin olive oil is considered an important target in olive breeding programs. In this work, the variability of the squalene content in a core collection of 36 olive cultivars was first studied, revealing two olive cultivars, 'Dokkar' and 'Klon-14', with extremely low and high squalene contents in their oils, respectively. Next, four cDNA sequences encoding squalene synthases (SQS) were cloned from olive. Sequence analysis and functional expression in bacteria confirmed that they encode squalene synthases. Transcriptional analysis in distinct olive tissues and cultivars indicated that expression levels of these four SQS genes are spatially and temporally regulated in a cultivar-dependent manner and pointed to OeSQS2 as the gene mainly involved in squalene biosynthesis in olive mesocarp and, therefore, in the olive oil. In addition, the biosynthesis of squalene appears to be transcriptionally regulated in water-stressed olive mesocarp.

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The aim of this work was to understand the actual content of mineral oil hydrocarbons (MOH) in olive pomace oil in order to contribute to the monitoring requested by EFSA for the food groups making a relevant impact on human background exposure. Such information will complement both the information inferred from the limits established by the EU and the interpretation of the coming toxicological risk assessment. At the same time, the origin of such a group of compounds is discussed. From the raw material to the commercial product, olive pomace oils were sampled and analyzed at different points and/or conditions. Through the ultimate online HPLC-GC-FID system, we gathered information on the MOH concentrations and molecular mass profiles (C-fractions), and through GCxGC-TOF/MS, we identified the key structures that prove the innocuousness of the mineral oil aromatic hydrocarbon (MOAH) fraction. Our approaches provided chromatographic signals on the C10-C50 range, rendering 33-205 mg/kg mineral oil saturated hydrocarbon (MOSH) and 2-55 mg/kg MOAH in the commercial product. The results confirmed that the C25-C35 cut is the main fraction to which humans are exposed via olive pomace oil, showing concentrations highly dependent on the extraction process. Moreover, the identification of the main MOAH groups showed that in olive pomace oil, mainly 1- and 2-ring species were present, being virtually free of the carcinogenic 3-7 ring aromatics.

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In this work a SPE/GC-FID method, incorporating the use of a 1-g silica cartridge, for the determination of FAEE in olive oils is presented. The procedure has been fully validated, initially 'in-house' and subsequently by an international validation study involving sixteen laboratories from Europe, the United States of America, and China. Key performance parameters of the method are: (1) Linearity in the 10-134 mg/kg range (R2 > 0.999), (2) LOD and LOQ < 0.5 mg/kg, (3) RSDr < 10%, (4) RSDR < 20% (for 4 out of 5 test materials). In addition, the method has been demonstrated to provide equivalent results to the Official Method (Commission Regulation 2568/91) while providing advantages in terms of reductions in time and solvents and ease of automation. In fact, the proposed protocol requires 30 mL solvents and takes 1.5 h per determination instead of the 350 mL and 6 h needed in the UE Official Method.

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The sinami palm (Oenocarpus mapora H. Karst) is a plant from the South American Amazonia that has great potential for industrial applications in the development of functional foods, nutraceuticals and cosmeceuticals. In this manuscript, the physicochemical properties, total polyphenol content and antioxidant activity of sinami oil that was obtained using four extraction systems, namely expeller press extraction (EPE), cold press extraction (CPE), ultrasound-assisted extraction (UAE) and supercritical fluid extraction (SFE), were studied and compared. The oxidative stability (OSI) was statistically non-significant in EPE and SFE. The chromatic properties (CIELab) were influenced by the extraction methods and SFE presented high values of L* and a lower content of plant pigments. Ultrasound-assisted extraction showed a higher content of polyphenols and higher antioxidant activity. Different analyses for the evaluation of the physicochemical properties, the content of total polyphenols and antioxidant activity were used to classify sinami oil according to chemometrics using principal component analysis (PCA). For example, the sinami oil that was obtained using each extraction method was in a different part of the plot. In summary, sinami oil is an excellent resource for plant pigments. Additionally, the information that was obtained on the quality parameters in this study provided a good foundation for further studies on the characterization of major and minor compounds.

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Mild refined olive oil obtained by neutralization and/or by soft deodorization at a low temperature and its blending with extra virgin olive oil (EVOO) is not allowed and is difficult to detect. Chlorophyll derivatives, pheophytins and pyropheophytin, and their relative proportions were proposed as parameters to detect such processes. The objective of this study is to determine changes in EVOO, in terms of pheophytins and pyropheophytin, occurring after several well-controlled mild refining processes. The changes on those chlorophyll pigments due to the processes depend on the temperature, stripping gas, acidity and oil nature. The data obtained show that, at temperatures below 100 °C, the rate at which pyropheophytin a is formed (Ra) is lower than the rate at which pheophytins a+a' disappear (Ra+a'). As a consequence, the Ra+a' and Ra ratios are considered to be directly linked to pheophytins a+a' decrease instead of to pyropheophytin a formation. Stripping gas very slightly affects the transformation of the chlorophyll pigments; actually both acidity and N2 enhance the increment in the Ra+a' and Ra ratios. In relation to the oil nature, the higher the initial pheophytin a+a' content, the higher the increase in the Ra+a' and Ra relations.

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The detection of soft deodorized olive oils in extra virgin olive oil (EVOO) has become a challenging task ever since it was demonstrated that: 1. The process does not form the typical refining markers, e.g. stigmastadienes, and 2. The determination of the fatty acid alkyl esters renders useful only when the deodorized matrix comes from oils with fermentative defects. Recently researchers have developed strategies to detect such kind of blends, being one of them based on the fact that both diacylglycerol (DAG) and free fatty acids are not interdependent after mild refining activities. Presently, we propose two factors to confirm the absence of soft deodorized oils in EVOO: R1 (10 × free acidity/DAGexp) ≥ 0.23 and R2 (DAGexp-DAGtheor) < 0, in genuine EVOO. We demonstrate that such approach is useful to detect the presence of soft deodorized olive oil when this is at least at 30% in the mixture.

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The commercialization of declared blends of olive oil and seed oil is something long approved by the European Union. There, the olive oil percentage must be at least 50% if the producer aims to advertise its presence on the front label, i.e., somewhere other than in the ingredients list. However, the Regulation did not propose any method to verify such proportion. For this purpose, we recommend the use of decisional trees, being the parameters under study those in which the greatest differences between olive and seed oils are shown: triacylglycerols, acyclic saturated hydrocarbons, free sterols, and tocopherols. In this way, to guarantee the presence of olive oil at 50%: i) palmitodiolein must be above 11-15%; ii) the ß/γ-tocopherol ratio must be below 2.4; iii) the alkane sum C21-C25 should be higher than 3.5-6%; and iv) the total sterol content cannot surpass 2400 mg/kg.

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Sacha inchi (Plukenetia huayllabambana L. and Plukenetia volubilis L.) edible oils were microencapsulated and the lipid fraction of the microparticles was characterized. Hi-cap®, Capsule®, Arabic gum, and the binary combination of Arabic gum + maltodextrin and the ternary combination of Arabic gum + maltodextrin + whey protein isolate, were used as coating materials for the encapsulation process using spray-drying. The surface and the total oils obtained from the microparticles were evaluated in terms of fatty acid composition, minor glyceride polar compounds, polymers, oxidized triglycerides, diglycerides, monoglycerides, and free fatty acids, along with their unsaponifiable components, sterols, and tocopherols. Differences between the original oils and the microencapsulated ones were determined. The most remarkable results included the presence of polymers when there were none in the original oils, the slight loss in ω3-fatty acids, up to 6%, the loss in tocopherols, in some of the cases around 30%, the maintaining of the phytosterol in their initial levels and the presence of cholesterol in the oils encapsulated with whey protein isolate.

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Olive fruits contain an n-alkane series of saturated hydrocarbons mainly in the pulp. Lower amounts of a complex mixture of paraffins, unresolved by gas chromatography (UCM--unresolved complex mixture), have been found in cuticle, stone (woody shell and seed), olive leaves, and talc used as an aid to olive oil extraction. The amounts of both kinds of hydrocarbons are related to the olive cultivar and are transferred to oils in a proportion depending on the oil-obtaining process (centrifugation or solvent extraction). In olive oil obtained by centrifugation, only n-alkanes were detected. However, in olive oil extracted by second centrifugation, small amounts of UCM paraffins were detected together with the n-alkanes. Olive pomace oils showed a very variable content of both types of hydrocarbons according to the different obtaining process, such as double centrifugation, solvent extraction or centrifugation followed by solvent extraction. 'White mineral oil' used in oil extraction machinery is the source of the high concentrations of UCM paraffins found in some olive and olive pomace oils. In the case of second centrifugation olive oil, a maximum limit of 50 mg kg(-1) of UCM is suggested, whereas in the case of crude olive pomace oil, it amounts to 250 mg kg(-1) plus an additional minimum of 1.0 for the n-alkanes/UCM ratio.

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This work deals with the characterization of the main glyceridic and unsaponifiable components of oils obtained from Sacha inchi (Plukenetia huayllabambana L.) seed ecotypes collected during two harvests in the Department of Amazonas in Peru. The seed-oil yield was 30.3-41.2%; standing out are the high percentages of the ω3- and ω6-fatty acids series whose ranges lie within those of the present Regulation for Sacha inchi oils. Triacylglycerols with even equivalent carbon number (ECN; 36-42) were the main components. Minor glyceridic polar compounds such as oxidized triglycerides, diglycerides, monoglycerides, and free fatty acids were determined by high-performance size exclusion chromatography. The low campesterol/stigmasterol ratio (1:6), unusual in the majority of vegetable oils, stands out. Regarding aliphatic hydrocarbons, these oils showed a particular profile for the saturated series of odd and even carbon atom numbers. According to our results Sacha inchi P. huayllabambana oils can be offered as a good alternative to P. volubilis, the species mainly commercialized for this vegetable oil.

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Plant sterols and their derivatives are minor compounds that have been extensively studied in vegetable oils, mainly in olive oil, where they are closely related with its identity. The objective of this work is to determine the content of free and esterified steryl glucosides and their profiles in olive oil in relation to different geographical situation of olive orchards, cultivar, farming modality, and sampling time. The orchards under study were located in the outer ring of the submetropolitan area of Madrid (Spain), where olives from Cornicabra, Manzanilla Cacereña, Manzanilla Castellana, and Picual varieties were grown under traditional and organic modes, and harvested in four different samplings. Conclusions state that cultivar, farming mode, and light exposure do not have outstanding effects, whereas pedoclimate might affect the steryl glucoside presence in a substantial way. Further studies are being carried out presently in order to confirm such statement. Also glucoside derivative profiles are discussed, and reasons for differences with results in previous studies pointed out.

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This work provides a short and easy protocol that allows the analysis of both methanol and ethanol in the static headspace of olive oil. The procedure avoids any kind of sample pre-treatment beyond that of heating the oil to allow a maximum volatile concentration in the headspace of the vials. The method's LOD is 0.55 mg kg(-1) and its LOQ is 0.59 mg kg(-1). Advantages of this method are:•Simultaneous determination of methanol and ethanol (the pre-existing Spanish specification UNE-EN 14110 only analyses methanol).•No need of equipment modifications (standard split injectors work perfectly). Use of a highly polar capillary GC column, leading in most cases to chromatograms in which only three dominant peaks are present - methanol, ethanol, and propanol (that is extremely positive for easy interpretation of results).•Use of an internal standard (1-propanol) to determine the concentration of the analytes, reducing the presence of error sources.

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This work covers two important gaps in the field of micronutrient databases: herein we describe a short and easy protocol that allows the analysis of both free and esterified steryl gulcosides in olive oil. By utilising accurate quantitative methods we achieve a better understanding of olive oil composition and health promoting properties. The procedure consists of isolating the fraction of interest through solid phase extraction, and using gas chromatography-flame ionisation detection for both identification and quantification of the derivatised species. Additionally, mass-spectrometry detection has been utilised for confirming the identity of the individual esterified steryl glucosides in some cases. The method's limit of detection has been set at 0.37mg/kg for each free steryl glucoside and 0.20mg/kg for each esterified steryl glucoside, whereas the recoveries are around 96% and 77%, respectively. Finally, we provide a complete analysis of the commercial standard for esterified steryl glucosides, since such information was not yet available.

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The acidity constants of twofold protonated, antivirally active, acyclic nucleoside phosphonates (ANPs), H(2)(PE)(±), where PE(2-)=9-[2-(phosphonomethoxy)ethyl]adenine (PMEA(2-)), 2-amino-9-[2-(phosphonomethoxy)ethyl]purine (PME2AP(2-)), 2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (PMEDAP(2-)), or 2-amino-6-(dimethylamino)-9-[2-(phosphonomethoxy)ethyl]purine (PME(2A6DMAP)(2-)), as well as the stability constants of the corresponding ternary Cu(Arm)(H;PE)(+) and Cu(Arm)(PE) complexes, where Arm=2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen), are compared. The constants for the systems containing PE(2-)=PMEDAP(2-) and PME(2A6DMAP)(2-) have been determined now by potentiometric pH titrations in aqueous solution at I=0.1M (NaNO(3)) and 25°; the corresponding results for the other ANPs were taken from our earlier work. The basicity of the terminal phosphonate group is very similar for all the ANP(2-) species, whereas the addition of a second amino substituent at the pyrimidine ring of the purine moiety significantly increases the basicity of the N(1) site. Detailed stability-constant comparisons reveal that, in the monoprotonated ternary Cu(Arm)(H;PE)(+) complexes, the proton is at the phosphonate group, that the ether O-atom of the -CH(2)-O-CH(2)-P(O)(2)(-)(OH) residue participates, next to the P(O)(2)(-)(OH) group, to some extent in Cu(Arm)(2+) coordination, and that π-π stacking between the aromatic rings of Cu(Arm)(2+) and the purine moiety is rather important, especially for the H·PMEDAP(-) and H·PME(2A6DMAP)(-) ligands. There are indications that ternary Cu(Arm)(2+)-bridged stacks as well as unbridged (binary) stacks are formed. The ternary Cu(Arm)(PE) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO(3)) species, where R-PO(3)(2-) represents a phosph(on)ate ligand with a group R that is unable to participate in any kind of intramolecular interaction within the complexes. The observed stability enhancements are mainly attributed to intramolecular-stack formation in the Cu(Arm)(PE) complexes and also, to a smaller extent, to the formation of five-membered chelates involving the ether O-atom present in the -CH(2)-O-CH(2)-PO(3)(2-) residue of the PE(2-) species. The quantitative analysis of the intramolecular equilibria involving three structurally different Cu(Arm)(PE) isomers shows that, e.g., ca. 1.5% of the Cu(phen)(PMEDAP) system exist with Cu(phen)(2+) solely coordinated to the phosphonate group, 4.5% as a five-membered chelate involving the ether O-atom of the -CH(2)-O-CH(2)-PO(3)(2-) residue, and 94% with an intramolecular π-π stack between the purine moiety of PMEDAP(2-) and the aromatic rings of phen. Comparison of the various formation degrees of the species formed reveals that, in the Cu(phen)(PE) complexes, intramolecular-stack formation is more pronounced than in the Cu(bpy)(PE) species. Within a given Cu(Arm)(2+) series the stacking intensity increases in the order PME2AP(2-)

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The acidity constants of the two-fold protonated acyclic 9-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(9,8aPMEA)(+)(-), and its 8-isomer, 8-[2-(phosphonomethoxy)ethyl]-8-azaadenine, H2(8,8aPMEA)(+)(-), both abbreviated as H2(PA)(+)(-), as well as the stability constants of their M(H;PA)+ and M(PA) complexes with the metal ions M2+=Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+ or Cd2+, have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO3) and 25 degrees C. Application of previously determined straight-line plots of log K(M)M(R-PO3) versus pK(H)H(R-PO3)for simple phosph(on)ate ligands, R-PO3(2-), where R represents a residue without an affinity for metal ions, proves that for all M(PA) complexes a larger stability is observed than is expected for a sole phosphonate coordination of the metal ion. This increased stability is attributed to the formation of five-membered chelates involving the ether oxygen present in the aliphatic residue (-CH2-O-CH2-PO3(2-)) of the ligands. The formation degrees of these chelates were calculated; they vary between about 13% for Ca(8,8aPMEA) and 71% for Cu(8,8aPMEA). The adenine residue has no influence on complex stability except in the Cu(9,8aPMEA) and Zn(9,8aPMEA) systems, where an additional stability increase attributable to the adenine residue is observed and equilibria between four different isomers exist. This means (1) an open isomer with a sole phosphonate coordination, M(PA)op, where PA(2-)=9,8aPMEA2-, (2) an isomer with a five-membered chelate involving the ether oxygen, M(PA)cl/O, (3) an isomer which contains five- and seven-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)cl/O/N3, and finally (4) a macrochelated isomer involving N7, M(PA)cl/N7. For Cu(9,8aPMEA) the formation degrees are 15, 30, 48 and 7% for Cu(PA)op, Cu(PA)cl/O, Cu(PA)cl/O/N3 and Cu(PA)cl/N7, respectively; this proves that the macrochelate involving N7 is a minority species. The situation for the Cu(PMEA) system, where PMEA2- represents the parent compound, i.e. the dianion of 9-[2-(phosphonomethoxy)ethyl]adenine, is quite similar. The relationship between the antiviral activity of acyclic nucleoside phosphonates and the structures of the various complexes is discussed and an explanation is offered why 9,8aPMEA is biologically active but 8,8aPMEA is not.

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The acidity constants of the 2-fold protonated (1H-benzimidazol-2-yl-methyl)phosphonate, H2(Bimp)(+/-), are given, and the stability constants of the M(H;Bimp)+ and M(Bimp) complexes with the metal ions M2+ = Mg2+, Ca2+, Ba2+, Mn2+, Co2+, Cu2+, Zn2+, or Cd2+ have been determined by potentiometric pH titrations in aqueous solution at I = 0.1 M (NaNO3) and 25 degrees C. Application of previously determined straight-line plots of log KM(M(Bi-R)) versus pKH(H(Bi-R)) for benzimidazole-type ligands, Bi-R, where R represents a residue which does not affect metal ion binding, proves that the primary binding site in the M(H;Bimp)+ complexes is (mostly) N3 and that the proton is located at the phosphonate group; outersphere interactions seem to be important, and the degree of chelate formation is above 60% for all metal ion complexes studied, except for Zn(H;Bimp)+. A similar evaluation based on log KM(M(R-PO3)) versus pKH(H(R-PO3)) straight-line plots for simple phosph(on)ate ligands, R-, where R represents a residue which cannot participate in the coordination process, reveals that the primary binding site in the M(Bimp) complexes is (mostly) the phosphonate group with all metal ions studied. In this case, the formation degree of the chelates varies more widely in dependence on the kind of metal ion involved, i.e., from 17 +/- 11% to nearly 100% for Ba(Bimp) and Cu(Bimp), respectively. For all the M(H;Bimp)+ and M(Bimp) systems, the intramolecular equilibria between the isomeric complexes are evaluated in a quantitative manner. The fact that for Bimp2- the metal ion affinity of the two binding sites, N3 and PO3(2-), can be calculated independently, i.e., the corresponding micro stability constants become known, allows us to present for the first time a method for the quantification of the chelate effect solely based on comparisons of stability constants which carry the same dimensions. This effect is often ill defined in textbooks because equilibrium constants of different dimensions are compared, which is avoided in the present case. For the M(Bimp) complexes, it is shown that the chelate effect is close to zero for Ba(Bimp) whereas for Cu(Bimp) it amounts to about four log units. This method is also applicable to other chelating systems. Finally, considering that benzimidazole as well as phosphonate derivatives are employed as therapeutic agents, the potential biological properties of Bimp, especially regarding nucleic acid polymerases, are briefly discussed.

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The acidity constants of the twofold protonated acyclic nucleotide analogue 9-[2-(phosphonomethoxy)- ethyl]-8-azaadenine, H(2)(9,8aPMEA)(+/-), as well as the stability constants of the M(H;9,8aPMEA)(+) and M(9,8aPMEA) complexes with the metal ions M(2+) =Ni(2+), Cu(2+) or Zn(2+), have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO(3)) and 25. The result for the release of the first proton from H(2)(9,8aPMEA)(+) (pK(a)= 2.73), which originates from the (N1)H(+) site, was confirmed by UV-spectrophotometric measurements. Application of previously determined straight-line plots of log KMM(R-PO(3)) versus PKH(3)(R-HPO(3))' for simple phosph(on)ate ligands, R- PO-, where R represents a residue without an affinity for metal ions, proves that the primary binding site of 9,8aPMEA(2-) is the phosphonate group for all three metal ions studied. By stability constant comparisons with related ligands it is shown, in agreement with conclusions reached earlier for the Cu(PMEA) system [PMEA(2-)=dianion of 9-[2- (phosphonomethoxy)ethyl]adenine], that in total four different isomers are in equilibrium with each other, i.e. (i) an open isomer with a sole phosphonate coordination, M(PA)(op), where PA(2-)=PMEA(2-)or 9,8aPMEA(2-), (ii) an isomer with a 5-membered chelate involving the ether oxygen, M(PA)cl/o, (iii) an isomer which contains 5- and 7-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)(cl/O/N3), and finally (iv) a macrochelated isomer involving N7, M(PA)(cl/]N7). The Cu(2+) systems of PMEA(2-) and 9,8aPMEA(2-) behave quite alike; the formation degrees for Cu(PA)(op), CuM(PA)(cl/O), Cu(PA)(cl/O/N3) and Cu(PA)(cl/N3) are approximately 16, 32, 45 and 7%, respectively, which shows that Cu(PA)(cl/N7) is a minority species. In the Ni(2+) and Zn(2+) systems the open isomer is the dominating one followed by M(PA)(cl/O), but there are indications that the other two isomers also occur to some extent.

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The synthesis of (1H-benzimidazol-2-yl-methyl)phosphonic acid, H2(Bimp)+/-, is described: 2-chloromethylbenzimidazole was reacted with ethylchloroformate to give 1-carboethoxy-2-chloromethylbenzimidazole which was treated with trimethyl phosphite and after hydrolysis with aqueous HBr H2(Bimp)+/- was obtained. In H2(Bimp)+/- one proton is at the N-3 site and the other at the phosphonate group; both acidity constants were determined in aqueous solution by potentiometric pH titrations (25 degrees C; I = 0.1 M, NaNO3) and this furnished the pKa values of 5.37 +/- 0.02 and 7.41 +/- 0.02, respectively. The acidity constant for the release of the primary proton from the P(O)(OH)2 group of H3(Bimp)+ was estimated: pKa = 1.5 +/- 0.2. Moreover, Bimp2- can be further deprotonated at its neutral (N-1/N-3)H site to give the benzimidazolate residue, but this reaction occurs only in strongly alkaline solution (KOH); application of the H_ scale developed by G. Yagil (J. Phys. Chem., 1967, 71, 1034) together with UV spectrophotometric measurements gave pKa = 14.65 +/- 0.12. Comparisons with acidity constants taken from the literature show that this latter pKa value is far too large and this allows the conclusion that an intramolecular hydrogen bond is formed between the (N-1/N-3)H site and the phosphonate group of Bimp2-; the formation degree of this hydrogen-bonded isomer is estimated to be 98 +/- 2%. The general relevance of this and the other results are shortly discussed and the species distribution for the Bimp system in dependence on pH is provided.