Ion chromatography coupled with optical emission spectrometry (IC-ICP-OES) methodology for the analysis of inositol phosphates in food and feed

The goal of this study is to develop a robust analytical method using ion chromatography coupled with inductively coupled plasma optical emission spectrometry (IC-ICP-OES) for the simultaneous detection and quantification of inositol phosphates (InsPx) in food and feed. The method separates 28 isomers from InsP6 to InsP2 within 33 minutes using a nitric acid-water gradient and eliminates baseline drift and the need for post-column derivatization, simplifying quantification.

With detection limits as low as 63 μg/L P and high reproducibility across different sample types, the method is suitable for complex matrices and diverse preparation techniques. It enables reliable analysis of enzymatic degradation pathways and provides a practical tool for assessing InsPx content in both food and animal feed.

The original article

Ion chromatography coupled with optical emission spectrometry (IC-ICP-OES) methodology for the analysis of inositol phosphates in food and feed

Corinna Henninger, Tobias Stadelmann, Daniel Heid, Katrin Ochsenreither, Thomas Eisele

Food Chemistry, Volume 463, Part 4, 2025, 141437

https://doi.org/10.1016/j.foodchem.2024.141437

licensed under CC-BY 4.0

Selected sections from the article follow. Formats and hyperlinks were adapted from the original.

Phytic acid (InsP₆, phytate (salt of phytic acid)) is the primary phosphate storage compound in plants, commonly found in grains, vegetables, and nuts. Structurally, phytic acid consists of an inositol ring esterified with six phosphate groups, rendering the contained phosphate unavailable for nutritional absorption. Over a wide pH range, phytic acid exists as a negatively charged molecule, often forming insoluble complexes with various micro-minerals such as iron and zinc (Heighton et al., 2008), macro-minerals like calcium, and also with proteins (Lee & Mitchell, 2019). The considerable chelation potential of phytic acid raises concerns about its impact on the bioavailability of essential minerals, often categorizing it as an antinutritional factor (Gharibzahedi & Jafari, 2017). Diets high in phytate have been linked to deficiencies in zinc and iron, crucial minerals for human health (Kumar et al., 2010; Schlemmer et al., 2009).

The analysis of these highly charged inositol phosphate 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 (Graf, 1983). Additionally, food and feed matrices containing high levels of salts, ions, proteins, fats, and starch (Graf, 1983) interfere with sample preparation and analytical separation. HPLC (Sandberg & Ahderinne, 1986) and HPTLC (Henninger, Hoferer, et al., 2023; Henninger, Spangenberg, et al., 2023) can distinguish between isomeric pools of inositol phosphates based on the number of remaining phosphate groups. However, the separation of individual isomers is only achievable through ion chromatography, initially established by Phillippy and Johnson (1985). Ion chromatography is frequently coupled with post-column derivatization using Wade reagent, which forms iron(III) sulfosalicylic acid complexes with phytic acid for visualization (Q. Chen & Li, 2003; Oates et al., 2014; Skoglund et al., 1998). Post-column derivatization techniques, while effective, involve complex setups and require disposal of used chemicals, adding to the operational burden and environmental impact.

Previously, inductively coupled plasma optical emission spectrometry (ICP-OES) has been employed for the analysis of phytic acid in urine (Grases & Llobera, 1996), as it is a routine, fast and simple method with high sensitivity (low LODs of 0.15 mg/L phytic acid). However, without prior separation, ICP-OES, similar to the precipitation method, tends to overestimate phytate content due to the presence of free phosphate and other inositol phosphates. Furthermore, the coupling of IC with inductively coupled plasma mass spectrometry (ICP-MS) has been demonstrated for phosphorus-containing analytes (Z. Chen et al., 2009; Guo et al., 2005; Rugova et al., 2014). However, detecting phosphorus with ICP-MS is challenging due to interference from ions such as nitrogen. This limitation necessitates the use of individual calibration curves for each analyte or the implementation of specialized techniques to mitigate these interferences. Such techniques may include using He or H₂ as a purging gas, employing collisionally induced dissociation and kinetic energy discrimination, or utilizing chemical reactions (Z. Chen et al., 2009).

In this study, we propose coupling ion chromatography with an ICP-OES system as an elegant and efficient method for selectively measuring inositol phosphates in complex matrices, eliminating the need for post-column reactions. Additionally, we investigate various sample extraction techniques. This approach aims to enhance the accuracy and reliability of phytate and inositol phosphate quantification in various food and feed samples.

2. Methods

2.1.1. High performance ion chromatography

The separation of InsP_x was achieved with a ThermoFisher Scientific (Dreieich, Germany) ion-chromatography system Dionex ICS-5000⁺ DC paired with the autosampler Dionex AS-AP (ThermoFisher Scientific, Dreieich, Germany). The ion chromatography system was equipped with a CarboPac PA100 analytical column (4 × 250 mm), a CarboPac PA100G (4 × 50 mm) and an IONPAC NG1–10 UM GUARD COLUMN (4 × 35 mm) (ThermoFisher Scientific, Dreieich, Germany). The column temperature was maintained at 30 °C and the injection volume of standard and sample solution was 100 μL, injected with the Dionex AS-AP autosampler. Analytes were eluted with 1 mL/min with a 33-min gradient program consisting of a mixture of (A) H₂O and (B) 0.5 M HNO₃. The mobile phase gradient is shown in Table 1. Chromeleon (Version 7.2) was employed for instrument control.

2.1.2. ICP-OES detection

For detection of inositol-phosphates an ICP-OES spectrometer iCAP 7000 (ThermoFisher Scientific, Dreieich, Germany) was employed. To stabilize and enhance the sensitivity of the solid-state charge injection device (CID) the optical system (Echelle spectrograph) was purged with a constant stream of nitrogen for a minimum of 12 h before measuring. The ICP was operated at 1150 W with an argon (Ar) flow of 12 L/min and an axial view of the plasma was utilized. The sample was transported from the IC column to the nebulizer in a capillary (ThermoFisher Scientific, Dreieich, Germany) with a diameter of 0.125 mm. A quartz cyclonic spray chamber for aqueous solutions was used for sample nebulization before introduction into the plasma with a nebulization gas flow of 0.45 L/min. The ICP-OES system was controlled with iTEVA iCAP Software (version 2.8.0.97). For the coupling of IC-ICP-OES, the autosampler of the ICP system was disabled and the sample supply was set to continuous operation mode. The recording of the analysis was started automatically via a trigger signal of the IC system during sample injection. The phosphorous signal was detected using the spectral lines at 177.495 nm and 213.618 nm.

3. Results and discussion

The pivotal role of phytic acid in human and animal nutrition stems from its function as the primary storage form of P in plants, thereby serving as the main or sole source of this essential nutrient. In human nutrition, comprehending the role of phytic acid degradation products is crucial for understanding its metabolic impact. Likewise, in animal nutrition, the effective supply of P through the degradation of phytic acid is vital, ensuring adequate levels of free phosphate without the risk of accumulation of lower inositol-phosphates.

Phytic acid consists of an inositol ring to which six phosphate groups are connected via ester bonds. The molecule has a chiral center and is symmetrical on both sides of the axis. In theory, 63 different myo-inositol phosphate molecules exist in total (Q. Chen & Li, 2003; Wundenberg & Mayr, 2012). Enantiomers however, cannot be separated on ion-exchange stationary phase. Subtracting the enantiomers, 39 myo-inositol-phosphate molecules remain, that may in theory be separated with ion-chromatography (IC) including 1 InsP₆, 4 InsP₅, 9 InsP₄, 12 InsP₃, 9 InsP₂ and 4 InsP₁ isomers due to their different charges, which results from the spatial arrangement.

3.1. Analytical system

Ion chromatography is currently the only method available for the separation of inositol phosphate isomers. An overview of this methodology is presented in Table 2. This study employs a CarboPac PA100 column with a nitric acid gradient for the separation process. Following the optimization process, our in-house reference standard mix was separated into 28 distinct inositol-phosphates within a total runtime of 33 min, including InsP₆, 4 InsP₅, 9 InsP₄, 8 InsP₃, and 6 InsP₂ isomers, shown in Fig. 1. InsP₁ isomers co-eluted with the phosphate (P_i) peak. The elution order of inositol phosphates corresponded to the increasing number of phosphate groups on the inositol ring, aligning with methodologies utilizing acidic elution systems (Skoglund et al., 1998; Carlsson et al., 2001; Q. Chen & Li, 2003; Oates et al., 2014). Isomeric InsP_x pools appear in clearly distinguishable clusters, which are defined in Table 3.

Food Chemistry, Volume 463, Part 4, 2025, 141437: Fig. 1. Chromatogram of in-house reference sample separated with a CarboPac PA100 column and HNO3/water gradient coupled with detection by ICP-OES. Peaks: (1) Pi with InsP1, (2–7) InsP2, (8–15) InsP3, (16–24) InsP4, (25–28) InsP5 and (29) InsP6.

3.1.2. Detection system

Inositol phosphates and P_i show no characteristic absorption spectra in the ultraviolet or visible region. Consequently, post-column derivatization utilized, typically using a solution of iron(III) nitrate [Fe(NO₃)₃] and perchloric acid is to visualize InsP_x after their separation on an IC system (Blaabjerg et al., 2010; Q. Chen, 2004; Oates et al., 2014; Skoglund et al., 1998).

In this study, IC was directly coupled with the iCAP 7000 ICP-OES, enabling direct detection without the need for post-column derivatization. Although IC-ICP-MS has been previously utilized for detecting InsP₆ and free phosphate in soil and plants (Rugova et al., 2014), IC coupled with ICP-OES is primarily employed for the separation of metal ions such as the separation of Cr³⁺/Cr⁶⁺ (Narukawa et al., 2007) or Fe²⁺/Fe³⁺ (Fernsebner et al., 2014). To the best of our knowledge, the use of IC-ICP-OES for the visualization of inositol phosphates has not been reported in the literature before.

For the detection of inositol phosphates and free phosphate, the phosphorus (P) signal is recorded at wavelengths of 177.495 nm and 213.618 nm. The operating parameters of the ICP-OES were calibrated and optimized by the manufacturer to ensure the phosphorus signal was at its maximum sensitivity. The resulting parameters are described in method section. The ICP-OES used in these experiments was equipped with two optimized slits that focus ultraviolet or visible light, enhancing analytical sensitivity. However, this feature limits the ability to conduct time-resolved and simultaneous analysis of UV and visible emissions (Manard et al., 2019). A flow rate of 1 mL/min was established on IC system to ensure an adequate supply to the ICP-OES. Fernsebner et al. (2014) used a slightly lower flow rate of 0.8 mL/min to feed the ICP-OES connected to the IC, similar to Manard et al. (2019), who diluted the sample post-separation on the IC to achieve the same flow rate. Since ICP-OES detection is not influenced by the eluent system used, the baseline remains constant and does not drift, a common issue in other published methodologies (Q. Chen, 2004; Skoglund et al., 1998). Blaabjerg et al. (2010) developed a system using a PA1 column in combination with methane sulfonic acid and UV detection after post-column derivatization to minimize baseline drift.

3.5.2. Phytate hydrolysis in animal feed

Unprocessed grains and legumes primarily contain phytic acid along with minor amounts of InsP₅. However, during food processing and preparation techniques, such as germination, soaking and fermentation, the phytic acid content can be significantly reduced due to naturally contained phytases or microbial phytase activity leading to various degradation products (Schlemmer et al., 2009). The addition of exogenous phytases to the diet of monogastric animals is more effective for phosphate release from phytate than relying on intrinsic phytases (Riviere et al., 2021). Therefore, an accurate and reliable technique to measure the performance of phytases in feed matrices is essential for optimizing nutrient availability and improving the nutritional quality of animal feed.

4. Conclusion

This study presents the first application of ion chromatography coupled with inductively coupled plasma optical emission spectrometry (IC-ICP-OES) for the quantitation of inositol phosphates (InsP_x), achieving the separation of 28 InsP_x isomers and inorganic phosphate (P_i) with InsP₁ within 33 min. The ICP-OES system enables specific measurement of InsP_x, allowing for direct quantification without relying on estimations based on the InsP₆ signal. This method demonstrates faster and more sensitive detection compared to existing techniques and simplifies the analytical process by eliminating the need for individual standards, correction factors, or the removal of interfering ions, offering a more streamlined and potentially more accurate approach to InsP_x quantification.

The proposed methodology holds significant potential for various applications, such as enhancing the understanding of phytases and the phytic acid degradation process, as well as the production of specific InsP_x isomers. Additionally, it effectively manages complex sample matrices such as feed and food. The utilization of IC coupled with ICP-OES represents an elegant and powerful analytical system with substantial potential.