Ion chromatography hyphenated with inductively coupled plasma optical emission spectrometry (IC-ICP-OES) for the determination of inositol phosphates in food and feed

Background:
Phytic acid, also known as inositol hexaphosphate, is the principal storage form of phosphorus in many plant tissues, particularly in bran, seeds, legumes, cereals, and grains. Structurally, phytic acid consists of an inositol ring esterified with six phosphate groups, making the phosphate unavailable for absorption by non-ruminant animals.  In contrast, ruminants can digest phytate due to the phytase enzyme produced by rumen microorganisms, which hydrolyses the inositol-phosphate bond. Over a wide pH range, phytic acid exists as a negatively charged molecule, often forming insoluble complexes with various minerals such as iron, zinc, calcium, and proteins. This significant chelation potential raises concerns about its impact on the bioavailability of essential minerals, categorizing it as an antinutritional factor.

Given the significance of inositol phosphates, reliable analytical methodologies are essential for their accurate determination. However, analysing these highly charged molecules is challenging due to their lack of characteristic absorption maxima in the ultraviolet or visible spectrum, necessitating post-column derivatization or other non-optical detection methods. Additionally, the presence of high levels of salts, ions, proteins, fats, and starch in food and feed matrices complicates sample preparation and analytical separation.


The new study:
In reviewing existing analytical methods for separating inositol phosphate molecules, the authors concluded that individual isomer separation is only achievable through ion chromatography. For detection, they proposed ICP-OES, which eliminates the need for post-column derivatization and offers compound-independent calibration.

The method achieved the separation of 28 inositol phosphate isomers using an ion-chromatographic system with a CarboPac PA 100 analytical column (4 x 250 mm) and a nitric acid-water gradient within 33 minutes. This setup included a CarboPac PA100 guard column and an additional guard column to protect against food and feed matrices.

The ICP-OES spectrometer was purged with nitrogen to allow for sensitive detection of the phosphorus UV lines at 177.495 nm and 213.618 nm with axial viewing. The linear working range was 0.062-32 mg/L P, with limits of detection and quantification determined to be 63 µg/L P and 208 µg/L P, respectively.

The researchers also evaluated various acidic and alkaline extraction methods for food and feed samples, finding that the highest phytate extraction yield was achieved using 0.5 M HCl for 1 hour. Sample clean-up, necessary to remove matrix components such as carbohydrates, was performed using C18 SPE columns.

Using this developed methodology, the authors analysed nine samples, including grains, rice, beans, lentils, and pseudo-grains, for their phytate and inorganic phosphate content. The results demonstrated high reproducibility (under 4% for feed samples) and an 80% recovery rate for phytate. The authors concluded that their methodology is suitable for exploring enzymatic degradation pathways and analysing inositol phosphate in complex food and animal feed matrices.