Anion-exchange high-performance liquid chromatography with post-column detection for the analysis of phytic acid and other inositol phosphates

The use of gradient anion-exchange HPLC, with a simple post-column detection system, is described for the separation of myo-inositol phosphates, including "phytic acid" (myo-inositol hexaphosphate). Hexa-, penta-, tetra-, tri- and diphosphate members of this homologous series are clearly resolved within 30 min. This method should facilitate analysis and quantitation of "phytic acid" and other inositol phosphates in plant, food, and soil samples.     

Preparation of samples for high-performance liquid chromatography of inositol phosphates.

A simple method is described for the removal of extraneous material from tissue extracts prior to anion-exchange high-performance liquid chromatography of inositol phosphates. Samples are prepared by extraction with trichloroacetic acid or perchloric acid followed by removal of the excess acid. The extracts are then passed through small Dowex-50 cation-exchange columns and eluted with water. Dowex-50 pretreatment removes most of the ultraviolet absorbing material and cations from the samples but does not alter the content of inositol phosphates. This treatment results in improved reliability of chromatography, especially with respect to weakly retained molecules such as adenosine 5'-phosphate and the isomers of inositol monophosphate. In addition, sample pretreatment improves the useful lifetime of the analytical anion-exchange columns.     

Quantitative analysis of inositol lipids and inositol phosphates in synaptosomes and microvessels by column chromatography: comparison of the mass analysis and the radiolabelling methods.

Chromatographic methods that measure both the mass and the radiolabelling of various inositol lipids and inositol phosphates in tissues have been developed. The mass of phosphatidylinositol (PtdIns), phosphatidylinositol-4-monophosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] was quantitated by measuring the inorganic phosphate, whereas inositol monophosphate (IP), inositol bisphosphate (IP2), inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) were quantitated by using an enzymic method. The radiolabelling of various inositol lipids and inositol phosphates was determined by incubating the tissue samples with [3H]myo-inositol, separating individual inositol lipids and inositol phosphates, and measuring the radioactivity in each compound. Although the mass analysis method was sensitive enough to measure low levels of inositol lipids or inositol phosphates, the method was laborious and time-consuming. Compared with the enzymic method, the radiolabelling method was simple and fast, but it gave variable results. This study demonstrated differences in inositol lipid and inositol phosphate levels by radiolabelling and mass measurements, and agonist-stimulated phosphatidylinositol turnover of synaptosomes versus the blood-brain barrier as represented by microvessels. Although the mass of PtdIns, PtdIns(4)P and PtdIns(4,5)P2 was comparable in synaptosomes and microvessels, the incorporation of [3H]myo-inositol into phosphorylated PtdIns in microvessels was less than that in synaptosomes.     

Rapid and selective isolation of radiolabelled inositol phosphates from cancer cells using solid-phase extraction.

A method is described for rapid and selective determination of radiolabelled inositol phosphates in cancer cells using solid-phase extraction with Bond Elut strong anion-exchange minicolumns. The inositol phosphates IP1, IP2 and IP3 are selectively eluted with 0.05, 0.3 and 0.8 M ammonium formate-0.1 M formic acid, respectively. Cancer cells are extracted with 10% perchloric acid which is then neutralised prior to loading samples on to the minicolumns. Recovery is 54.1, 66.6 and 61.3% for IP1, IP2 and IP3 with between-day coefficients of variation of 7.6, 6.8 and 1.9%, respectively. When the method was applied to cancer cells high-performance liquid chromatographic analyses confirmed both the identity of the IP1, IP2 and IP3 fractions and showed that there was no detectable cross contamination of these inositol phosphates with each other.     

Structural distinction among inositol phosphate isomers using high-energy and low-energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization

Electrospray (ESI) collisional-activated dissociation (CAD) tandem mass spectrometric methods for the structural characterization of inositol phosphates (InsPs) using both quadrupole and sector mass spectrometers are described. Under low-energy CAD, the [M + H](+) ions of the positional isomers of inositol phosphates, including inositol mono-, bis- and trisphosphates, yield distinguishable product-ion spectra, which are readily applicable for isomer differentiation. In contrast, the product-ion spectra arising from high-energy CAD (2 keV collision energy, floating at 50%) tandem sector mass spectrometry are less applicable for isomer identification. The differences in the product-ion spectrum profiles among the aforementioned InsP isomers become more substantial and differentiation of positional isomers can be achieved when the collison energy is reduced to 1 keV (floating at 75%). These results demonstrate that the applied collision energies play a pivotal role in the fragmentations upon CAD. The product-ion spectra are similar among the positional isomers of inositol tetrakisphosphates and of inositol pentakisphosphates. Thus, isomeric distinction for these two inositol polyphosphate classes could not be established by the tandem mass spectrometric methods that have achieved such distinctions for the less highly phosphorylated inositol phosphate classes. Under both high- and low-energy CAD, the protonated molecular species of all InsPs undergo similar fragmentation pathways, which are dominated by the consecutive losses of H(2)O, HPO(3) and H(3)PO(4).     

Determination of myo-inositol (free and bound as phosphatidylinositol) in infant formula and adult nutritionals by liquid chromatography/pulsed amperometry with column switching: first action 2011.18

Myo-inositol is a 6-carbon cyclic polyalcohol also known as meso-inositol, meat sugar, inosite, and i-inositol. It occurs in nature in both free (myo-inositol) and bound (inositol phosphates and phosphatidylinositol) forms. For the determination of free myo-inositol, samples are mixed with dilute hydrochloric acid to extract myo-inositol and precipitate proteins, diluted with water, and filtered. For the determination of myo-inositol bound as phosphatidylinositol, samples are extracted with chloroform, isolated from other fats with silica SPE cartridges, and hydrolyzed with concentrated acid to free myo-inositol. Prepared samples are first injected onto a Dionex CarboPac PA1 column, which separates myo-inositol from other late-eluting carbohydrates. After column switching, myo-inositol is further separated on a CarboPac MA1 column using a 0.12% sodium hydroxide mobile phase; strongly retained carbohydrates are eluted from the PA1 column with a 3% sodium hydroxide mobile phase. Eluant from the CarboPac MA1 analytical column passes through an electrochemical detector cell where myo-inositol is detected by pulsed amperometry using a gold electrode. The method showed appropriate performance characteristics versus selected established standard method performance requirement parameters for the determination of myo-inositol: linear response; repeatability (RSDr) of 2%; and intermediate precision (RSDir) of 2.5%. Instrument LOD and LOQ were 0.0004 and 0.0013 mg/100 mL, respectively, and correspond to a free myo-inositol quantitation limit of 0.026 mg/100 g and a phosphatidylinositol quantitation limit of 0.016 mg/100 g. Correlation with the reference microbiological assay was good. The proposed method has been accepted by the Expert Review Panel as an AOAC First Action Method, suitable for the routine determination of myo-inositol in infant formula and adult nutritionals.     

Analytical Methods for Determination of Phytic Acid and Other Inositol Phosphates: A Review

From the early precipitation-based techniques, introduced more than a century ago, to the latest development of enzymatic bio- and nano-sensor applications, the analysis of phytic acid and/or other inositol phosphates has never been a straightforward analytical task. Due to the biomedical importance, such as antinutritional, antioxidant and anticancer effects, several types of methodologies were investigated over the years to develop a reliable determination of these intriguing analytes in many types of biological samples; from various foodstuffs to living cell organisms. The main aim of the present work was to critically overview the development of the most relevant analytical principles, separation and detection methods that have been applied in order to overcome the difficulties with specific chemical properties of inositol phosphates, their interferences, absence of characteristic signal (e.g., absorbance), and strong binding interactions with (multivalent) metals and other biological molecules present in the sample matrix. A systematical and chronological review of the applied methodology and the detection system is given, ranging from the very beginnings of the classical gravimetric and titrimetric analysis, through the potentiometric titrations, chromatographic and electrophoretic separation techniques, to the use of spectroscopic methods and of the recently reported fluorescence and voltammetric bio- and nano-sensors.     

Thermal degradation of phytate produces all four possible inositol pentakisphosphates as determined by ion chromatography and 1H and 31P NMR spectroscopy

Abstract

Decomposition of phytate has recently been shown to occur under mild conditions in the solid state, giving rise to a complex mixture of lower inositol phosphates. In this study, the reaction products of this thermal, abiotic degradation of phytate were separated using ion chromatography and the most highly phosphorylated products subsequently identified using 1D and 2D NMR spectroscopy. Two late eluting fractions were each shown to be a mixture of two specific inositol pentakisphosphate isomers. The peak with shorter retention time contained Ins(1,2,3,4,6)P₅ and DL-Ins(1,2,3,4,5)P₅, while the later eluting fraction contained Ins(1,3,4,5,6)P₅ and DL-Ins(1,2,4,5,6)P₅. The formation of all four possible inositol pentakisphosphate isomers by thermal degradation of phytate contrasts with the selective production of unique inositol pentakisphosphate isomers during enzymatic phytate degradation and therefore suggests a method for differentiating abiotic and biotic processes in environmental samples, including soils and decomposing plant biomass.     

Ion chromatographic determination of sugar phosphates in physiological samples

Ion chromatography is shown to be capable of simultaneous determination of biologically important anions. Application of this technique is illustrated for the separation and quantification of the major anions present in rat brain and liver tissues. Sugar phosphates and carboxylic acids are separated on high-performance anion-exchange columns and are detected using chemically suppressed conductivity. Detection limits range from 20 to 100 pmol for the anions tested, including inositol phosphates, lactate, pyruvate, glucuronic acid-1-phosphate, fructose-6-phosphate and glucose-6-phosphate. The coefficient of variation for the determination of most anions was in the range 5-10%. Many of these anions are either difficult to separate with other methods, or require expensive radiochemical techniques for detection. This method should be applicable to other biological studies, from the flow of carbons in photosynthesis to the study of synaptic transmission.     

Anion-exchange high-performance liquid chromatography with conductivity detection for the analysis of phytic acid in food

A sensitive method for the accurate determination of phytic acid in food samples is described. The proposed procedure involves the anion-exchange liquid chromatography with conductivity detection. Initially, two methods of determination of phytic acid were compared: absorptiometry and high-performance ion chromatography (HPIC) with chemically suppressed conductivity detector. Unlike most conventional methods involving precipitation by FeCl3, the simpler and more reliable HPIC assay avoids the numerous assumptions inherent in the iron precipitation and the accuracy is independent of the phytate content. The protocol was also applied to a survey of phytic acid concentration in some cereal, oil and legume seeds.     

Determination of myo-inositol phosphates in food samples by flow injection-capillary zone electrophoresis

A capillary electrophoresis (CE) method with indirect photometric detection was developed to identify and quantify myo-inositol phosphates in food samples. A flow-injection (FI) system including a micro-column containing anionic exchange resin was used for the solid-phase extraction of the myo-inositol phosphates with a view to their preconcentration. The FI system was automatically coupled to CE equipment via a mechanical interface. The overall analysis time was shortened by incorporating an FI system for myo-inositol hexakisphosphate monitoring. The limit of detection for myo-inositol phosphates as determined by FI-CE ranged from 11 to 26 micromol/L and the coefficient of variation from 3.9 to 5.0%. On the other hand, the limit of detection and coefficient of variation for myo-inositol hexakisphosphate as monitored by the FI system were 75 micromol/L and 2.9%, respectively. The proposed method was successfully applied to a variety of food samples with recoveries ranging from 96.0 to 107.7% and the precision from 3.9 to 7.9%. Based on the results, the content of myo-inositol hexakisphosphate in nuts was two or three times higher than that in legumes.     

The separation of rhodium and iridium by anion-exchange

The separation of rhodium and iridium in amounts of 100–1000 μg was achieved with a strongly basic anion-exchange resin. Rhodium was eluted with 0.8 M hydrochloric acid containing cerium(IV) sulfate. Iridium was recovered by eluting with concentrated nitric acid at 74°C, which minimized the formation of hydrolysis products. Recoveries of 98% or more were obtained for both metals.     

Facile syntheses of all possible diastereomers of conduritol and various derivatives of inositol stereoisomers in high enantiopurity from myo-inositol

Phosphoinositide-based signaling processes are crucially important in intracellular signal transduction events. Inositol phosphate analogues have been useful in probing the structure-activity relationships between inositol phosphates and biomacromolecules, and in studying biological functions of newly found inositol phosphates. Thus, a systematic and ready access to inositol stereoisomers is highly desirable. And practical and convenient syntheses of conduritols and related compounds are also important because of their biological activities and their synthetic utilities in the preparation of other bioactive molecules. We herein report the first syntheses of all possible diastereomers of conduritol and various derivatives of eight inositol stereoisomers in high enantiopurity from myo-inositol, which involve efficient enzymatic resolution of the intermediates conduritol B and C derivatives, followed by oxidation-reduction or the Mitsunobu reaction, and cis-dihydroxylation in stereo- and regioselective manners.     

Evaluation of titanium and titanium oxides as chemo-affinity sorbents for the selective enrichment of organic phosphates.

Titanium (Ti) and TiO, Ti2O3, Ti3O5 and TiO(1.98) as well as TiO2 have been evaluated as chemo-affinity sorbents for the selective enrichment of organic phosphates. A column-switching high-performance liquid chromatography (HPLC) system, constructed with a precolumn (4.6 mm i.d. x 10 mm) packed with the titanium sorbents, an anion-exchange analytical column and a UV detector (215 nm) was used. When an aqueous 0.015% trifluoroacetic acid (TFA) was used as a sample-loading solution, O-phospho-L-tyrosine (P-Tyr), phenyl phosphate and phenylphosphonic acid were adsorbed onto all of the titanium sorbents with recoveries of 60.9 - 102.9%. Some acidic compounds other than phosphates, such as benzenedicarboxylic acid (BDA) isomers, were also adsorbed onto all of the titanium sorbents. To improve the selectivity to organic phosphates, various sample-loading solutions were examined using a Ti precolumn, two phosphorylated peptides [Ile-Ser(p)-Val-Arg (PP1) and Gln-Ile-Ser(p)-Val-Arg (PP2)], P-Tyr, BDA isomers and diglutamic acid (Glu-Glu) as test compounds. Among the sample-loading solutions tested, such as TFA, HClO4, organic acids, boric acid and NaCl, the use of 100 mM NaCl in 10 mM boric acid was found to be effective. The recoveries of PP1, PP2 and P-Tyr were 73.0, 88.3 and 71.5%, respectively, whereas those of Glu-Glu and BDAs were suppressed to only below 10%.     

HPLC with inductively coupled plasma optical emission spectrometric detection for the analysis of inositol phosphates

The use of inductively coupled plasma optimal emission spectroscopy as a detector for the high-performance liquid chromatographic analysis of inositol phosphates is studied. It is found that separation of different inositol phosphates with a mobile phase consisting of tetraethylammonium (0.14%, w/v), methanol (5%, v/v), and formic acid (0.18%, w/v) may be obtained on a PRP-1 column with an analysis time of 18 min. In addition, high specificity and sensitivity of the detection system used permits detection of the inositol phosphates from bi- to hexaphosphate free from interference of other chromatographic peaks, which could be from the sample or mobile phase. Additionally, it is possible to use less sample because of the high sensitivity of the detection system.     

Design and synthesis of biotinylated inositol phosphates relevant to the biotin-avidin techniques

Six bifunctional molecules containing biotin and various inositol phosphates were synthesized. These compounds were designed on the basis of X-ray structures of the complexes of D-myo-inositol 1,4,5-triphosphates (IP(3)) and phospholipase C delta pleckstrin homology domain (PLCdelta PH) considering the application to the biotin-avidin techniques. The building blocks of the inositol moiety were synthesized starting with optically resolved myo-inositol derivatives and assembled to the biotin linker through a phosphate linkage.     

Determination of nitrite, nitrate, and glucose-6-phosphate in muscle tissues and cured meat by IC/MS

The endogenous nitrate concentration in fresh meat and the residual nitrate and nitrite contents after curing are related to food quality and safety. Most ion chromatography (IC) methods suffer from interferences, especially in fresh meat samples, in which the endogenous nitrate content is low, and in cured meat products, in which other nitrogenous compounds can interfere with the separation of inorganic anions. One of the major classes of interfering compounds in fresh meat are sugar phosphates, which originate from glycolysis during the conversion of muscle glycogen to lactic acid. Nitrate can be separated from interfering compounds with a high-capacity anion-exchange column that was manufactured for use with hydroxide eluents (i.e., hydroxide-selective). This column has a different selectivity than traditional IC columns that use carbonate eluents and facilitates the determination of nitrate in both fresh and cured meats. Nitrate was detected by both suppressed conductivity measurement and mass spectrometry (MS). The identifications of nitrate and glucose-6-phosphate were confirmed by MS detection. The described IC/MS method is robust, sensitive to nitrate concentrations as low as 0.10 mg/kg, and can determine sugar phosphates that are useful for monitoring meat freshness. We successfully used this method to determine nitrate in nearly 100 muscle tissues and cured meat samples.     

Ion chromatographic determination of inositol in infant formulae and clinical products for enteral feeding

An ion chromatographic method is described for the determination of inositol in infant formula and products for enteral feeding. A two-step procedure for hydrolysis and extraction of total inositol has been developed, involving alkaline hydrolysis with 3 M potassium hydroxide and enzymatic dephosphorylation. Substances having a long chromatographic retention time were removed with an ion-exchange resin. Inositol was separated on a high-resolution ion-exchange column and detected by pulsed amperometric detection. Phytic acid interferes only slightly in the analysis. This method can be used for determination of total inositol in infant formulae, and enteral feeding products. The analytical method gave an average recovery of 94% from infant formula samples spiked with inositol and a recovery of 86+/-3% from products spiked with lecithin.     

The “Other” Inositols and Their Phosphates: Synthesis,Biology, and Medicine (with Recent Advances in myo‐Inositol Chemistry)

Cell signaling via inositol phosphates, in particular via the second messenger myo‐inositol 1,4,5‐trisphosphate, and phosphoinositides comprises a huge field of biology. Of the nine 1,2,3,4,5,6‐cyclohexanehexol isomers, myo‐inositol is pre‐eminent, with “other” inositols (cis‐, epi‐, allo‐, muco‐, neo‐, l ‐chiro‐, d ‐chiro‐, and scyllo‐) and derivatives rarer or thought not to exist in nature. However, neo‐ and d ‐chiro‐inositol hexakisphosphates were recently revealed in both terrestrial and aquatic ecosystems, thus highlighting the paucity of knowledge of the origins and potential biological functions of such stereoisomers, a prevalent group of environmental organic phosphates, and their parent inositols. Some “other” inositols are medically relevant, for example, scyllo‐inositol (neurodegenerative diseases) and d ‐chiro‐inositol (diabetes). It is timely to consider exploration of the roles and applications of the “other” isomers and their derivatives, likely by exploiting techniques now well developed for the myo series.     

Studies of the separation and characterisation of mixtures of starch and cellulose derivatives by use of chromatography and mass spectrometry

In this work a method was developed for characterisation of commercially available polymers consisting of mixtures of substituted cellulose and starch. Selective hydrolysis with specific enzymes was used to achieve separation of the two polymers in the mixture. Enzymes hydrolysing (1→4)-α-D and (1→6)-α-D-glycosidic bonds were used for the starch part and enzymes hydrolysing (1→4)-β-D-glycosidic bonds for the cellulose part. The hydrolysed fraction was separated from the unhydrolysed fraction and characterised by use of size-exclusion chromatography (SEC), to confirm that enzyme hydrolysis of the different polymers had occurred. High-performance anion-exchange chromatography (HPAEC) was performed to determine the amount of unmodified glucose units (UGU) in the fractions. Electrospray ionisation mass spectrometry (ESIMS) was used for determination of the substituents. All products were converted to monomers by acid hydrolysis to simplify mass spectral identification of the substituents. The monomers were further subjected to acetylation with acetic acid anhydride to facilitate identification of the substituents. By combining the results from the different analytical techniques a picture of the samples was obtained.