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Article

Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry

by
Ping Guo
1,2,†,
Sijie Zhu
1,2,† and
Bo Chen
1,2,*
1
Key Laboratory of Phytochemical R&D of Hunan Province, Hunan Normal University, No. 36, Lushan Road, Changsha 410081, China
2
Key Laboratory of Chemical Biology & Traditional Chinese Medicine Research of Ministry of Education, Hunan Normal University, No. 36, Lushan Road, Changsha 410081, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2026, 31(5), 799; https://doi.org/10.3390/molecules31050799
Submission received: 20 January 2026 / Revised: 19 February 2026 / Accepted: 26 February 2026 / Published: 27 February 2026
(This article belongs to the Special Issue Advanced Analytical Methods in Food Chemistry)
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Abstract

Phytic acid (PA), owing to its strong acidity and multidentate metal-chelating properties, readily forms multiple adduct/complex ions in mass spectrometry and is prone to pronounced matrix effects, resulting in complicated spectra and compromised sensitivity and quantitative robustness, which poses a major challenge for rapid and accurate PA quantification. Herein, we developed a rapid quantitative method for PA based on trimethylsilyldiazomethane (TMSD) methyl-ester derivatization coupled with paper spray mass spectrometry (PS–MS). PA was derivatized with TMSD to yield the methylated product (PA-Me), and the derivative solution was purified via “post-derivatization nitrogen blow-down followed by water reconstitution”, thereby markedly reducing background interference. In positive-ion mode, the stable sodium adduct ion [PA-Me+Na]+ (m/z 851.04) was used as the quantifier, enabling fast quantification with selected ion monitoring (SIM). PS–MS was performed with a 15 μL spotting volume and methanol/water (90/10, v/v, containing 0.1% formic acid) as the spray solvent, allowing rapid analysis without chromatographic separation. The method exhibited good linearity over 0.125–30 μg/mL (R2 ≥ 0.9965), with a limit of detection (LOD, S/N = 3) of 0.080 μg/mL and a limit of quantification (LOQ, S/N = 10) of 0.270 μg/mL. The intra-day and inter-day precision values were both < 10% (RSD), and recoveries ranged from 87.2% to 122.4%. This LC-free strategy features low solvent consumption and high analytical throughput, and was validated using rice bran protein and rice bran polysaccharide samples, providing technical support for rapid screening and quality control of PA in complex food/plant matrices.

1. Introduction

Phytic acid (PA), also known as inositol hexakisphosphate (IP6), is a ubiquitous organic phosphate widely distributed in nature. It is commonly present in plant-derived foods (e.g., cereals, legumes, nuts, and oilseeds), where it serves as a major storage form of phosphorus [1,2,3,4,5,6]. PA is primarily synthesized and accumulated during seed maturation and can account for 60–90% of total phosphorus in dormant seeds [1,4]. Traditionally, PA has been regarded as an anti-nutritional factor because it can chelate minerals and interact with proteins and digestive enzymes, thereby reducing the bioavailability of minerals and protein-associated nutrients [2,3,4,5,6,7,8,9,10,11,12,13,14,15,16]. In addition, PA readily forms insoluble complexes with multivalent metal ions, including Fe, Zn, Mg, Ca, Mn, and Cu [17,18,19,20]; given the limited endogenous phytase activity in humans and monogastric animals, PA is often insufficiently hydrolyzed, which compromises nutrient absorption and may increase phosphorus discharge via excretion [3,4,5,6,21,22,23,24]. Therefore, developing a rapid, sensitive, and matrix-tolerant quantitative method for PA is of considerable importance [4,6,25].
Early methods for PA determination mainly relied on precipitation and colorimetric assays. However, during food processing, PA can be partially degraded to lower inositol phosphates (IP5–IP1) [7,8,14]. These classical assays generally fail to discriminate PA from its degradation products, resulting in limited accuracy and specificity [1,25,26,27], and they are therefore unsuitable for PA determination in complex matrices [4,27,28]. Accordingly, more robust analytical approaches have been developed, including high-performance liquid chromatography (HPLC), ion chromatography (IC), and liquid chromatography–mass spectrometry (LC–MS) [4,29,30,31,32,33]. Nevertheless, PA contains six phosphate groups and is highly acidic and strongly hydrophilic, leading to insufficient retention on conventional reversed-phase columns and consequently challenging chromatographic separation [25,28]. As a result, polar chromatographic modes (e.g., hydrophilic interaction liquid chromatography, HILIC) are frequently employed in LC–MS(/MS) workflows for PA and related inositol phosphates to improve retention and separation [34]. Although LC–MS offers high sensitivity and selectivity, PA readily forms multiple adduct/complex ions with protons and metal ions, producing complicated mass spectra and potentially severe ion suppression, which compromises sensitivity and quantitative robustness [21,25,29]. To improve ionization and mitigate matrix interference, trimethylsilyldiazomethane (TMSD) derivatization has been applied to convert PA into methylated derivatives; this chemical modification reduces polarity and acidity, thereby enhancing MS response. However, such workflows typically still depend on chromatographic separation, with relatively long analysis time and substantial solvent consumption, limiting their suitability for high-throughput testing [25,35].
In contrast to conventional LC–MS workflows for PA, which typically require extraction/cleanup steps and chromatographic separation (often with specialized conditions) to manage the high polarity and adduct heterogeneity, our method couples TMSD methylation with paper-spray MS to enable direct, chromatography-free quantification [24]. Compared with LC-based methods, this approach reduces sample-handling complexity and eliminates LC run time and column maintenance, thereby lowering the instrumental barrier and improving throughput. Regarding matrix effects, LC–MS often mitigates suppression/enhancement mainly through separation and internal-standard correction, whereas in the present PS–MS workflow we address matrix-driven variability by derivatization, matrix-matched calibration for the protein workflow, and recovery/precision validation in representative matrices. Overall, the streamlined “derivatize–spray–measure” format is practical for routine quality-control screening where rapid turn-around and low solvent consumption are prioritized [36]. Paper spray mass spectrometry (PS–MS) as a representative ambient ionization technique enables rapid analysis with minimal sample consumption and without chromatographic separation, making it attractive for fast qualitative/quantitative analysis and high-throughput screening in complex matrices [37,38,39,40]. In this study, we developed a rapid PA determination method by coupling TMSD methylation derivatization with PS–MS, and applied it to industrial rice bran protein and rice bran polysaccharide samples. After derivatization with TMSD, PA is converted to its methylated product (PA-Me) (Figure 1), with reduced polarity and acidity, which improves ionization behavior and enhances MS response [29,41]. In positive-ion mode, a stable sodium-adduct ion was employed as the diagnostic signal for PA identification and quantification, providing methodological support for quality and safety evaluation of rice bran protein, rice bran polysaccharide, and related products [36,37,38,39,42].

2. Results and Discussion

2.1. PS–MS Confirmation of the Derivatized Product of PA

PA contains six phosphate groups; its strong acidity and multidentate chelation capability make it prone to forming multiple adduct/complex ions with protons and metal ions in positive-ion mode, resulting in complicated mass spectra and reduced signal stability due to pronounced matrix effects. To improve ionization behavior and enhance the mass spectrometric response [21,25,43], TMSD was employed to methylate PA, thereby decreasing its polarity and acidity and improving analytical sensitivity and repeatability [44,45].
The PS–MS full-scan spectrum of the PA-Me acquired in positive-ion mode (Figure 2) showed two characteristic ions at mass-to-charge ratio (m/z) 829.15 and 851.04, corresponding to [PA-Me+H]+ and [PA-Me+Na]+, respectively. The sodium-adduct ion is more intense and exhibits better stability, making it suitable for subsequent quantification using selected ion monitoring (SIM). To further support the assignment of the quantifier ion (m/z 851.04, [PA-Me+Na]+), MS/MS confirmation was performed on a triple-quadrupole instrument (Shimadzu LCMS-8050) using the nominal m/z 851 precursor, yielding diagnostic product ions at m/z 473.20 and 325.10; the confirmatory MRM transitions (m/z 851.20 → 473.20 and 851.20 → 325.10) are provided in the Supporting Information (Figure S2). To assess the influence of sodium availability on the quantifying ion, we evaluated the effect of NaCl added to the spray solvent on the response of [PA-Me+Na]+ (m/z 851.04) and observed stable signals at low added salt levels but pronounced suppression at ≥ 1 mM (Figure S1). Because PS–MS is a direct ionization technique without chromatographic separation, derivatization by-products and background signals from the paper substrate/solvent may elevate the baseline-particularly in the low m/z region—and induce ion suppression. Therefore, post-derivatization treatment and ion selection should be further optimized to improve the signal-to-noise ratio (S/N) and ensure robust quantification.

2.2. Optimization of Post-Derivatization Treatment and Selection of Qualifier/Quantifier Ions

Termination of the TMSD derivatization with formic acid generated gaseous nitrogen, methyl formate, and siloxane-related byproducts, which can increase background signals and suppress the response of the target ions under direct ionization conditions [35]. Therefore, a nitrogen blow-down step was introduced to remove volatile/semi-volatile residues, thereby reducing baseline noise and alleviating ion suppression. The effect of reconstitution solvent after nitrogen blow-down was further evaluated by comparing methanol versus water reconstitution of the PA-Me (Figure 3). Nitrogen blow-down markedly cleaned up the spectral background and improved the S/N of the target ions.
Under SIM, using 100 μg/mL PA-Me as an example, the S/N of m/z 851.04 was 34.7 after methanol reconstitution, whereas water reconstitution increased the S/N of m/z 851.04 to 686.5 (approximately 20-fold), demonstrating that the “nitrogen blow-down-water reconstitution” workflow is critical for improving the quantitative sensitivity of PS–MS [13,36,37,38,39]. In addition, a signal at m/z 436.95 was observed after water reconstitution and can be assigned to [PA-Me+2Na]2+, which is likely promoted by trace sodium ions in the aqueous system facilitating multi-sodium adduct formation. Considering signal intensity, background interference, and stability, m/z 851.04 and 436.95 were selected as qualifier ions; among them, m/z 851.04 ([PA-Me+Na]+) showed the highest S/N and the most stable response, and was therefore chosen as the quantifier ion for this method.

2.3. Derivatization Conditions and PS–MS Parameter Optimization

2.3.1. Derivatization Time and TMSD Volume

In this study, we employed TMSD as the derivatization reagent for PA because it enables rapid, reproducible methyl-esterification under mild, operationally convenient conditions [24,46]. Compared with alternative methylation/permethylation reagents such as diazomethane or methyl iodide-based alkylation, which may require higher-risk handling and/or more demanding procedures, TMSD is well suited to a routine, high-throughput derivatization–PS–MS workflow.
To improve derivatization efficiency while maintaining analytical throughput, the effect of derivatization time on the peak area of the quantifier ion m/z 851.04 was evaluated (Figure 4a). As the reaction time increased, the peak area first increased and then slightly decreased, reaching a maximum at 60 min. This indicates that 60 min is sufficient to achieve efficient derivatization while avoiding potential side reactions and/or ion suppression associated with excessively long reaction times; therefore, 60 min was selected as the optimal derivatization time. Minor additional ions were observed at 30 min and are reported descriptively as putative incompletely derivatized species or in-source ions. At 60 min, the spectrum was dominated by the characteristic adduct ions (m/z 851.04 and 829.08), supporting 60 min for quantification.
The volume of TMSD was further investigated (80–160 μL, Figure 4b). These volumes correspond to ~1.1 × 103–2.1 × 103 equiv. of TMSD relative to PA under the conditions described in Section 3.5 (with 120 μL corresponding to ~1.6 × 103 equiv.). When the volume was <120 μL, the peak area increased with increasing TMSD. When the volume was ≥120 μL, the response approached a plateau and slightly decreased. Considering derivatization efficiency (plateauing response) and reagent economy, 120 μL was chosen as the optimal TMSD volume.

2.3.2. Spray Voltage and Cone Voltage

Voltage parameters directly affect spray stability and ion transmission efficiency. The spray voltage was optimized over 2.0–4.5 kV (Figure 5a) by monitoring the peak area of the quantifier ion (m/z 851.04). The peak area increased markedly with increasing voltage and reached a maximum at 4.0 kV; a further increase led to a decrease, suggesting that excessively high voltage may destabilize the spray and/or cause ion losses. Therefore, 4.0 kV was selected as the optimal spray voltage.
The cone voltage was optimized over 20–70 V (Figure 5b). The peak area first increased and then decreased, with a maximum at 50 V. An overly high cone voltage may induce excessive collisional activation and fragmentation, thereby attenuating the target-ion signal. Consistently, at 70 V we observed additional cone-voltage-dependent signals in the positive-ion full-scan spectrum (Figure S3), including several higher-m/z ions (e.g., m/z 815.00, 743.01, 721.04, and 702.89) together with enhanced low-m/z ions, which are reported descriptively as putative in-source ions (including adducts) and/or background ions from the paper/solvent. Accordingly, 50 V was chosen as the optimal cone voltage.

2.3.3. Effect of Urea Concentration on the PA Response in a Protein Matrix

In the rice bran protein matrix, PA can readily associate with proteins, which compromises extraction efficiency and derivatization yield [43]. Urea can relax protein structure by weakening hydrogen bonding and ionic interactions, thereby facilitating the release of protein-bound PA [47]. As shown in Figure 6, the peak area of the quantifier ion at m/z 851.04 increased continuously with increasing urea concentration; the maximum response was obtained at 8 mol/L, close to the solubility limit. Therefore, 8 mol/L urea was selected as the optimal treatment condition for rice bran protein samples. This matrix-matching strategy improves the analytical sensitivity and robustness of PA determination in complex protein matrices [47,48].

2.4. Method Validation

2.4.1. Linearity, Limit of Detection (LOD), and Limit of Quantification (LOQ)

Under the optimized conditions, calibration curves were established (Figure 7) and evaluated using water and 8 mol/L urea aqueous solution as the solvent systems (Table 1). Each calibration level was analyzed in triplicate (n = 3), and the mean peak area was used for regression. Both solvent systems exhibited good linearity over 0.125–30 μg/mL (initial PA concentration before derivatization), with R2 ≥ 0.9965. The regression equations obtained for the water and urea systems were y = 17,689.22x − 889.51 and y = 31,066.97x + 3651.60, respectively. The higher slope observed for the urea system indicates an enhanced ionization/response under these conditions. The LOD and LOQ were 0.080 and 0.270 μg/mL, respectively, as defined by S/N = 3 and S/N = 10 in SIM mode. Although the calibration was evaluated over 0.125–30 μg/mL, routine quantification is recommended at concentrations ≥ LOQ (0.270 μg/mL, S/N = 10).

2.4.2. Precision and Recovery

Precision was evaluated at three concentration levels (0.5, 5, and 30 μg/mL) (Table 2). For the water system, the intra-day relative standard deviation (RSD) ranged from 2.2 to 3.5%, and the inter-day RSD ranged from 5.7 to 8.9%. For the urea system, the intra-day RSD ranged from 1.4 to 2.7%, and the inter-day RSD ranged from 4.3 to 6.6%. Overall, the precision met the requirements for quantitative analysis.
Spike-recovery experiments were performed at 0.5, 1.0, and 1.5 μg/mL (Table 3). Recoveries of 91.6–110.8% were obtained for the water system, whereas those for the urea system were 87.2–122.4%. The slightly elevated recoveries observed at certain levels in the urea system suggest that some degree of matrix enhancement may occur under strongly matrix-matched and signal-enhancing conditions. Given the direct-ionization nature of PS–MS, such modest enhancement is not unexpected. Nevertheless, the proposed method provided acceptable accuracy and repeatability in both solvent systems. For routine quantitative analysis and quality control, matrix-matched calibration (standards prepared in 8 mol/L urea) is recommended for urea-rich protein matrices, and standard addition or periodic QC spike checks may be used when matrix composition varies between batches.

2.5. Application to Industrial Samples: PA Content in Rice Bran Protein and Rice Bran Polysaccharide

The proposed method was applied to determine the PA content in industrially produced rice bran protein and rice bran polysaccharide samples (Table 4). The PA contents ranged from 0.84 to 31.78 g/kg in rice bran protein samples and 0.36 to 0.48 g/kg in rice bran polysaccharide samples. Notably, rice bran protein 1 exhibited a substantially higher PA level than the commercial products, which may be associated with co-precipitation of protein–PA complexes during the alkali extraction–acid precipitation process. The relatively elevated PA levels observed in the polysaccharide samples further suggest that co-enrichment of PA can occur during the preparation of rice bran protein/polysaccharide products. These findings highlight the necessity of monitoring PA during product development and quality control, and of evaluating process strategies for dephytinization or for reducing bound PA to meet quality and safety requirements.
In this context, the proposed TMSD-PS–MS workflow enables rapid PA quantification without a chromatographic separation step, which is advantageous for routine screening and QC-oriented analysis. By combining derivatization-enhanced ionization with direct PS–MS readout, the method reduces analytical complexity and supports high-throughput operation on a routine single-quadrupole platform. These features make the approach practical for frequent monitoring of PA during process optimization and quality control of food- and plant-derived products.

3. Materials and Methods

3.1. Reagents and Materials

Methanol (HPLC grade; Beijing InnoChem Science & Technology Co., Ltd., Beijing, China), formic acid (HPLC grade, >99%; Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), urea (analytical grade; Shanghai Macklin Biochemical Co., Ltd., Shanghai, China), phytic acid standard (≥98%; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and TMSD (2 M, n-hexane solution; Shanghai Adamas-Beta Chemical Reagents Co., Ltd., Shanghai, China) were used. Distilled water (double distillation) was used throughout. Chromatography paper (Xinyang Paper Industry Co., Ltd., Hangzhou, China) was employed as the paper substrate for PS–MS.

3.2. Instruments and Equipment

A high-speed centrifuge (Sorvall ST 16R, Thermo Fisher Scientific, Waltham, MA, USA), an ultrasonic cleaner (F-030SD, Shenzhen Fuyang Technology Group Co., Ltd., Shenzhen, China), an analytical balance (AP135W/ATY224, Shimadzu, Kyoto, Japan), an automatic double-distillation water apparatus (SZ-93, Shanghai Yarong Biochemical Instrument Co., Ltd., Shanghai, China), and a nitrogen blow-down concentrator (MTN-2800D, Tianjin Autoscience Instruments Co., Ltd., Tianjin, China) were used.
Mass spectrometric analysis was performed on a Waters ZQ 2000 single-quadrupole mass spectrometer (Waters Corporation, Milford, MA, UK) equipped with MassLynx™ 4.0 software. For PS–MS, a high-voltage power supply was connected to the paper substrate via a copper clip to apply high voltage directly to the paper. A three-dimensional translation stage was used to adjust the spatial position between the paper tip and the MS cone inlet. Paper substrates were cut using a cutting plotter (CUTOK DC 330, Hefei CNC Equipment Co., Ltd., Hefei, China).

3.3. Preparation and Pretreatment of Paper Substrates

Chromatography paper was ultrasonically cleaned in methanol for 30–60 min, removed and air-dried, and then dried in an oven at 100–110 °C to constant weight. After drying, the paper was cut into isosceles triangles (height 10 mm; base width 5 mm) for PS–MS analysis.

3.4. Sample Source and Preparation

3.4.1. Rice Bran Protein Samples

Rice bran protein 1 was prepared in-house using an alkaline extraction–acid precipitation procedure, whereas rice bran protein 2 and rice bran protein 3 were commercially available products.

3.4.2. Rice Bran Polysaccharide Samples

Rice bran polysaccharide was prepared in-house. Briefly, crude polysaccharides were obtained by alkaline extraction. Deproteinization was performed by adsorption with FePO4·4H2O (160 mg/mL, room temperature, 1 min), followed by centrifugation and collection of the supernatant. The polysaccharides were then precipitated with ethanol, washed, and dried under nitrogen to afford rice bran polysaccharide.

3.5. Solution Preparation and TMSD Derivatization

An 8 mol/L urea solution was prepared. The PA standard stock solution (10 mg/mL) was prepared in water or in urea solution. The working solution was prepared by diluting with methanol to 100 μg/mL.
An aliquot of 1.0 mL of 100 μg/mL PA in methanol was mixed with 120 μL TMSD (2 M in n-hexane; ~1.6 × 103 equiv. relative to PA) and allowed to react at room temperature for 60 min. The reaction was quenched by adding 50 μL formic acid. Subsequently, 200 μL of the derivatized solution was evaporated to dryness under a nitrogen stream and reconstituted in 200 μL water for further analysis.

3.6. Pretreatment of Real Samples for PS–MS

For real samples, rice bran protein and rice bran polysaccharide were solubilized by ultrasonication at 25 °C for 20 min (until complete dissolution), and the resulting solutions were used directly without further clarification. For rice bran protein samples, 5.0 mg protein was dissolved in 1.0 mL 8 mol/L urea. For rice bran polysaccharide samples, 5.0 mg polysaccharide was dissolved in 1.0 mL water. Then, 10 μL of each resulting solution was diluted to 1.0 mL with methanol, followed by derivatization according to Section 3.5, nitrogen blow-down, and reconstitution in 200 μL water for further analysis.

3.7. PS–MS Analytical Conditions

PS–MS was performed in positive-ion mode. The distance between the paper tip and the MS cone was approximately 4–6 mm. A triangular paper substrate (10 mm height × 5 mm base) was mounted using a copper clip connected to the high voltage supply and positioned in front of the MS sampling cone using an in-house 3D translation stage (with an angle adjuster). A 15 μL aliquot of sample was spotted onto the paper substrate and allowed to stand for 30–60 s. Spraying was initiated by adding the spray solvent methanol/water (90/10, v/v, containing 0.1% formic acid), and the spray voltage was set to 4.0 kV. High voltage was applied directly to the paper via the copper clip to initiate paper spray ionization (PSI). Data acquisition combined full-scan (m/z 200–1000) and SIM. The cone voltage was 50 V. The source temperature was 120 °C, and the desolvation gas temperature was 300 °C. The gas flow rate was 400 L/h, and the cone gas flow was 50 L/h. Quantification was performed using the peak area of m/z 851.04. A photograph of the external PSI setup and a schematic diagram are provided in the Supporting Information (Figures S4 and S5).

3.8. Method Validation Experimental Design

Under the optimized conditions, the method was validated in terms of linear range, LOD, LOQ, precision, and spiked recovery. Unless otherwise stated, all experiments were performed using three independent sample preparations, and the results are reported as mean ± SD (n = 3). Quantification was performed in positive-ion mode using the sodium adduct ion of the PA-Me, m/z 851.04 ([PA-Me+Na]+). The peak area obtained in selected SIM mode was used as the quantitative signal.

3.8.1. Linear Range, LOD, and LOQ

The PA stock standard solution was diluted with methanol to 100 μg/mL and derivatized with TMSD according to Section 3.5, followed by nitrogen blow-down and reconstitution in water. The derivatized solution was then serially diluted to prepare a set of standards at 0.125, 0.25, 0.5, 1, 2, 5, 10, 20, 30 μg/mL. Two calibration curves were constructed by preparing the standards in water and in 8 mol/L urea solution, respectively; the urea system was used for matrix-matched quantification in the protein-matrix (rice bran protein) workflow. All standards were analyzed under the PS–MS conditions described in Section 3.7, and the SIM peak area of m/z 851.04 was recorded. Linear regression was performed using the standard concentration (x) and peak area (y). Each concentration level was measured in triplicate (n = 3), and the mean peak area was used for regression. Although an isotopically labeled internal standard would be preferred to correct potential inter-day variations in paper-spray ionization, such a standard was not available in this study; therefore, robustness was supported by matrix-matched calibration for the protein workflow (standards prepared in 8 mol/L urea), along with replicate measurements (n = 3, mean ± SD) and recovery/precision validation under the defined conditions. The LOD and LOQ were defined as the lowest concentrations corresponding to S/N = 3 and S/N = 10 in SIM mode, respectively (S/N was calculated from the quantifier-ion signal of low-concentration standards). Accordingly, under comparable noise conditions, the LOQ is expected to be approximately three times the LOD.

3.8.2. Precision

Precision was evaluated using PA standards at 0.5, 5, 30 μg/mL. Intra-day precision was determined by triplicate analyses within the same day; inter-day precision was determined by one analysis per day over three consecutive days. Results were expressed as RSD.

3.8.3. Spiked Recovery

Spiked recovery was assessed in real sample matrices at three fortification levels (0.5, 1.0, 1.5 μg/mL), with three replicates at each level. Spiking was performed prior to derivatization; samples were processed in parallel following the sample pretreatment procedure in Section 3.6 and the derivatization procedure in Section 3.5, and then analyzed by PS–MS. Recovery was calculated as: Recovery (%) = (measured content in spiked sample − native content in unspiked sample)/theoretical spiked amount × 100%, and RSD (%, n = 3) was reported.

4. Conclusions

Addressing the analytical bottlenecks of PA—namely complex adduct/chelate ion formation arising from its strong acidity and multidentate coordination, pronounced matrix effects, and the time- and solvent-intensive nature of conventional chromatographic workflows—this study proposes and validates a rapid quantitative strategy that couples TMSD methyl-esterification derivatization with PS–MS. By reducing PA polarity and acidity via derivatization, enabling chromatography-free direct ionization through PS–MS, and incorporating a simplified post-derivatization treatment step (nitrogen blow-down followed by water reconstitution), the method delivers stable and selective quantitative signals even on a routine single-quadrupole platform. In addition, this method integrates a rapid chemical derivatization step with direct MS analysis, offering a more efficient and cost-effective alternative to traditional LC–MS workflows. Overall, the proposed approach features a streamlined workflow, fast analysis, and low solvent consumption, demonstrating clear potential for high-throughput screening and quality control in complex food/plant matrices. Although demonstrated here using rice bran protein and polysaccharide matrices, the proposed strategy is in principle applicable to other food and plant-derived matrices where PA is commonly present. As a direct-ionization technique, PS–MS remains sensitive to matrix composition; therefore, matrix-matched calibration (or standard addition when needed) and routine QC spike checks are recommended, especially for high-salt/high-lipid or highly co-extractive samples that may exhibit stronger ion suppression/enhancement. Collectively, this work effectively integrates the selectivity advantages of chemical derivatization with the speed of PS–MS direct analysis, providing a practical analytical solution for rapid PA monitoring in complex matrices.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31050799/s1. Figure S1. Effect of NaCl added to the spray solvent on the PS–MS response of [PA-Me+Na]+ (m/z 851.04). The y-axis shows the normalized peak area of m/z 851.04, with the “No added NaCl” condition (0 mM NaCl added to the spray solvent) set to 1.00. The “No added NaCl” condition corresponds to 0 mM added NaCl; background trace Na+ may still be present from solvents, reagents, or the sample matrix. Data are presented as mean ± SD (n = 3). The response remains essentially unchanged in the low-salt range (0–100 μM), whereas higher NaCl levels (≥1 mM) cause pronounced ion suppression, with substantial signal loss at 10 mM. Lines are guides to the eye. Figure S2. MS/MS confirmation of the nominal m/z 851 ion (assigned as [PA-Me+Na]+). Product-ion spectra of the precursor m/z 851.20 were acquired in positive-ion mode on a triple-quadrupole mass spectrometer (Shimadzu LCMS-8050) at different collision energies (e.g., 35 and 65 eV), showing diagnostic product ions at m/z 473.20 and 325.10. These ions were used to define confirmatory MRM transitions (m/z 851.20→473.20 and 851.20→325.10). Note: the precursor is monitored as m/z 851.04 on the Waters ZQ 2000 for quantification; the small difference in reported m/z values arises from instrument-dependent mass calibration. Figure S3. Positive-ion full-scan spectrum of the derivatized PA (PA-Me) acquired at cone voltage = 70 V. The characteristic ions m/z 851.04 ([PA-Me+Na]+) and m/z 829.08 ([PA-Me+H]+) are observed together with additional cone-voltage-dependent signals in the higher m/z region (e.g., m/z 815.00, 743.01, 721.04, and 702.89), reported here as putative in-source ions consistent with in-source activation. Several low-m/z ions (e.g., m/z 326.96, 348.92, 568.00, and 590.01) are also enhanced at 70 V and are reported descriptively as putative cone-voltage-dependent in-source ions (including adducts) and/or background ions from the paper/solvent. No definitive structural assignment is claimed for these ions in the absence of dedicated MS/MS. Figure S4. Photographs of the in-house paper spray ionization (PSI) setup coupled to a Waters ZQ 2000 mass spectrometer. (A) Close-up view of the triangular paper substrate positioned in front of the MS sampling cone (tip-to-cone distance ≈ 4–6 mm); high voltage is applied directly to the paper via a copper clip. (B) In-house 3D translation stage with an angle adjuster used to reproducibly position the paper substrate. Figure S5. Schematic of the paper spray ionization (PSI) configuration used in this study. A triangular paper substrate serves as the spray emitter; the sample and spray solvent are applied to the paper, and a high voltage (4.0 kV) is applied to generate an ion plume from the paper tip. The resulting ions are sampled directly into the MS sampling cone.

Author Contributions

B.C.; methodology, P.G. and S.Z.; investigation, P.G. and S.Z.; formal analysis, P.G. and S.Z.; data curation, P.G. and S.Z.; writing—original draft preparation, P.G. and S.Z.; writing—review and editing, B.C.; supervision, B.C.; project administration, B.C. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge support from the National Natural Science Foundation of China (Grant No. 22276049) and the National Sustainable Development Demonstration Zone Project (Chenzhou, Grant No. 2022sfq49).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Molecules 31 00799 g001
Figure 1. Reaction scheme for TMSD methyl esterification of PA. Reaction conditions: PA (100 μg/mL) dissolved in methanol (MeOH), reacted with 2 M TMSD in n-hexane (120 μL) at room temperature for 60 min, and quenched with 50 μL formic acid. Under these conditions, 120 μL of 2 M TMSD corresponds to ~1.6 × 103 equiv. relative to PA (based on 1.0 mL of 100 μg/mL PA). Chemical structures: PA = inositol hexakisphosphate (IP6), PA-Me = methyl-ester derivative of PA generated under the selected TMSD derivatization conditions; reaction solvent is methanol.
Figure 1. Reaction scheme for TMSD methyl esterification of PA. Reaction conditions: PA (100 μg/mL) dissolved in methanol (MeOH), reacted with 2 M TMSD in n-hexane (120 μL) at room temperature for 60 min, and quenched with 50 μL formic acid. Under these conditions, 120 μL of 2 M TMSD corresponds to ~1.6 × 103 equiv. relative to PA (based on 1.0 mL of 100 μg/mL PA). Chemical structures: PA = inositol hexakisphosphate (IP6), PA-Me = methyl-ester derivative of PA generated under the selected TMSD derivatization conditions; reaction solvent is methanol.
Molecules 31 00799 g001
Molecules 31 00799 g002
Figure 2. PS–MS full-scan mass spectrum of the PA-Me acquired in positive-ion mode. Ordinate: Signal intensity (unit: counts, shown as arbitrary units); abscissa: m/z. Characteristic peaks: m/z 829.15 corresponds to the proton adduct ion [PA-Me+H]+, and m/z 851.04 corresponds to the sodium adduct ion [PA-Me+Na]+ (quantifier ion for subsequent quantification).
Figure 2. PS–MS full-scan mass spectrum of the PA-Me acquired in positive-ion mode. Ordinate: Signal intensity (unit: counts, shown as arbitrary units); abscissa: m/z. Characteristic peaks: m/z 829.15 corresponds to the proton adduct ion [PA-Me+H]+, and m/z 851.04 corresponds to the sodium adduct ion [PA-Me+Na]+ (quantifier ion for subsequent quantification).
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Molecules 31 00799 g003
Figure 3. Effect of post-derivatization treatment on PS–MS spectra of PA-Me in positive-ion mode: (a) methanol reconstitution after nitrogen blow-down; (b) water reconstitution after nitrogen blow-down. Ordinate: Signal intensity (arbitrary units); abscissa: m/z. Key indicator: S/N of the quantifier ion m/z 851.04, with water reconstitution showing 20-fold higher S/N than methanol reconstitution. Nitrogen blow-down was used to remove volatile byproducts.
Figure 3. Effect of post-derivatization treatment on PS–MS spectra of PA-Me in positive-ion mode: (a) methanol reconstitution after nitrogen blow-down; (b) water reconstitution after nitrogen blow-down. Ordinate: Signal intensity (arbitrary units); abscissa: m/z. Key indicator: S/N of the quantifier ion m/z 851.04, with water reconstitution showing 20-fold higher S/N than methanol reconstitution. Nitrogen blow-down was used to remove volatile byproducts.
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Molecules 31 00799 g004
Figure 4. Optimization of derivatization conditions based on the peak area of the quantifier ion (m/z 851.04): (a) derivatization time; (b) TMSD volume. Derivatization time: Ordinate = Peak area of m/z 851.04 (×105); abscissa = Reaction time (min), optimized to 60 min (maximum peak area). TMSD volume: Ordinate = Peak area of m/z 851.04 (×105); abscissa = Volume of 2 M TMSD (μL) (corresponding to 1.1 × 103–2.1 × 103, equiv. relative to PA; Section 3.5), optimized to 120 μL (TMSD/PA ~ 1.6 × 103, equiv.). All experiments were performed in triplicate (n = 3), and error bars represent standard deviation (SD).
Figure 4. Optimization of derivatization conditions based on the peak area of the quantifier ion (m/z 851.04): (a) derivatization time; (b) TMSD volume. Derivatization time: Ordinate = Peak area of m/z 851.04 (×105); abscissa = Reaction time (min), optimized to 60 min (maximum peak area). TMSD volume: Ordinate = Peak area of m/z 851.04 (×105); abscissa = Volume of 2 M TMSD (μL) (corresponding to 1.1 × 103–2.1 × 103, equiv. relative to PA; Section 3.5), optimized to 120 μL (TMSD/PA ~ 1.6 × 103, equiv.). All experiments were performed in triplicate (n = 3), and error bars represent standard deviation (SD).
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Molecules 31 00799 g005
Figure 5. Optimization of PS–MS instrumental parameters in positive-ion mode: (a) spray voltage; (b) cone voltage. Spray voltage: Ordinate = Peak area of quantifier ion m/z 851.04 (×105); abscissa = Spray voltage (kV), optimized to 4.0 kV (stable spray and maximum ion transmission). Cone voltage: Ordinate = Peak area of m/z 851.04 (×105); abscissa = Cone voltage (V), optimized to 50 V (minimal ion fragmentation). All experiments were conducted with 100 μg/mL PA-Me, in triplicate (n = 3).
Figure 5. Optimization of PS–MS instrumental parameters in positive-ion mode: (a) spray voltage; (b) cone voltage. Spray voltage: Ordinate = Peak area of quantifier ion m/z 851.04 (×105); abscissa = Spray voltage (kV), optimized to 4.0 kV (stable spray and maximum ion transmission). Cone voltage: Ordinate = Peak area of m/z 851.04 (×105); abscissa = Cone voltage (V), optimized to 50 V (minimal ion fragmentation). All experiments were conducted with 100 μg/mL PA-Me, in triplicate (n = 3).
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Figure 6. Influence of urea concentration on the PS–MS response of PA-Me (quantifier ion: m/z 851.04). Ordinate = Peak area of quantifier ion m/z 851.04 (×105); abscissa = Concentration of urea (mol/L). Urea functions to disrupt protein–PA binding via weakening hydrogen bonds and ionic interactions; optimized concentration = 8 mol/L (maximum response, close to urea solubility limit). Experiments were performed with rice bran protein samples spiked with PA, in triplicate (n = 3).
Figure 6. Influence of urea concentration on the PS–MS response of PA-Me (quantifier ion: m/z 851.04). Ordinate = Peak area of quantifier ion m/z 851.04 (×105); abscissa = Concentration of urea (mol/L). Urea functions to disrupt protein–PA binding via weakening hydrogen bonds and ionic interactions; optimized concentration = 8 mol/L (maximum response, close to urea solubility limit). Experiments were performed with rice bran protein samples spiked with PA, in triplicate (n = 3).
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Molecules 31 00799 g007
Figure 7. Calibration curves for PA determination under the optimized conditions using different solvent systems: (a) water; (b) 8 mol/L urea aqueous solution. Water system (for non-protein matrices, e.g., rice bran polysaccharide); 8 mol/L urea system (for protein matrices, e.g., rice bran protein). PA was derivatized to its methylated product (PA-Me) prior to PS–MS analysis; thus, the ordinate corresponds to the peak area of the PA-Me quantifier ion at m/z 851.04, whereas the abscissa indicates the initial PA concentration before derivatization (μg/mL). Each calibration level was prepared and analyzed in triplicate using three independent sample preparations (n = 3), and the data are presented as mean ± SD.
Figure 7. Calibration curves for PA determination under the optimized conditions using different solvent systems: (a) water; (b) 8 mol/L urea aqueous solution. Water system (for non-protein matrices, e.g., rice bran polysaccharide); 8 mol/L urea system (for protein matrices, e.g., rice bran protein). PA was derivatized to its methylated product (PA-Me) prior to PS–MS analysis; thus, the ordinate corresponds to the peak area of the PA-Me quantifier ion at m/z 851.04, whereas the abscissa indicates the initial PA concentration before derivatization (μg/mL). Each calibration level was prepared and analyzed in triplicate using three independent sample preparations (n = 3), and the data are presented as mean ± SD.
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Table 1. Analytical performance of the proposed PS–MS quantification method for PA in different solvent systems. Solvent systems: Water (for polysaccharide samples) and 8 mol/L urea (for protein samples). Unless otherwise stated, all measurements were performed using three independent sample preparations (n = 3), and results are reported as mean ± SD where applicable. Quantification is recommended for concentrations ≥ LOQ (S/N = 10).
Table 1. Analytical performance of the proposed PS–MS quantification method for PA in different solvent systems. Solvent systems: Water (for polysaccharide samples) and 8 mol/L urea (for protein samples). Unless otherwise stated, all measurements were performed using three independent sample preparations (n = 3), and results are reported as mean ± SD where applicable. Quantification is recommended for concentrations ≥ LOQ (S/N = 10).
SolventLinear Range
(μg/mL)		Calibration EquationR2LOD
(μg/mL)		LOQ
(μg/mL)
Water0.125–30y = 889.51 + 17,689.22x0.99650.0800.270
8 mol/L urea solution0.125–30y = 3651.60 + 31,066.97x0.99660.0800.270
Table 2. Intra-day and inter-day precision for PA determination in different solvent systems (RSD%, n = 3). Precision was evaluated at three PA concentration levels: 0.5 μg/mL, 5 μg/mL, and 30 μg/mL. Intra-day precision: Triplicate analyses performed on the same day; inter-day precision: One analysis per day for three consecutive days. RSD = relative standard deviation (%).
Table 2. Intra-day and inter-day precision for PA determination in different solvent systems (RSD%, n = 3). Precision was evaluated at three PA concentration levels: 0.5 μg/mL, 5 μg/mL, and 30 μg/mL. Intra-day precision: Triplicate analyses performed on the same day; inter-day precision: One analysis per day for three consecutive days. RSD = relative standard deviation (%).
SolventIntra-Day Precision (RSD%, n = 3)Inter-Day Precision (RSD%, n = 3)
0.5 μg/mL5 μg/mL30 μg/mL0.5 μg/mL5 μg/mL30 μg/mL
Water3.52.22.88.96.45.7
8 mol/L urea solution2.71.41.66.64.35.9
Table 3. Recovery and precision of the spiked PA assay in different solvent systems (RSD%, n = 3). Spiking levels: 0.5 μg/mL, 1.0 μg/mL, and 1.5 μg/mL (added prior to derivatization). Water system: for polysaccharide matrices; 8 mol/L urea system: for protein matrices.
Table 3. Recovery and precision of the spiked PA assay in different solvent systems (RSD%, n = 3). Spiking levels: 0.5 μg/mL, 1.0 μg/mL, and 1.5 μg/mL (added prior to derivatization). Water system: for polysaccharide matrices; 8 mol/L urea system: for protein matrices.
SolventRecovery (%, n = 3)RSD (%, n = 3)
0.5 μg/mL1.0 μg/mL1.5 μg/mL0.5 μg/mL1.0 μg/mL1.5 μg/mL
Water93.2110.891.66.54.32.6
8 mol/L urea solution122.487.295.85.83.24.4
Table 4. Determination of PA content in real rice bran protein and rice bran polysaccharide samples. Sample sources: Rice bran protein 1 (in-house prepared via alkali extraction–acid precipitation), rice bran protein 2–3 (commercially available); rice bran polysaccharide 1–2 (in-house prepared via alkaline extraction and FePO4·4H2O deproteinization). PA content: Expressed as mean ± SD (g/kg, dry weight basis); n = 3 (triplicate analyses per sample).
Table 4. Determination of PA content in real rice bran protein and rice bran polysaccharide samples. Sample sources: Rice bran protein 1 (in-house prepared via alkali extraction–acid precipitation), rice bran protein 2–3 (commercially available); rice bran polysaccharide 1–2 (in-house prepared via alkaline extraction and FePO4·4H2O deproteinization). PA content: Expressed as mean ± SD (g/kg, dry weight basis); n = 3 (triplicate analyses per sample).
SamplePA Content (g/kg)
Rice bran protein 131.8 ± 2.3
Rice bran protein 21.08 ± 0.28
Rice bran protein 30.84 ± 0.32
Rice bran polysaccharide 10.36 ± 0.02
Rice bran polysaccharide 20.48 ± 0.03
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Guo, P.; Zhu, S.; Chen, B. Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry. Molecules 2026, 31, 799. https://doi.org/10.3390/molecules31050799

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Guo P, Zhu S, Chen B. Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry. Molecules. 2026; 31(5):799. https://doi.org/10.3390/molecules31050799

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Guo, Ping, Sijie Zhu, and Bo Chen. 2026. "Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry" Molecules 31, no. 5: 799. https://doi.org/10.3390/molecules31050799

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Guo, P., Zhu, S., & Chen, B. (2026). Rapid Analysis of Phytic Acid by Paper Spray Mass Spectrometry. Molecules, 31(5), 799. https://doi.org/10.3390/molecules31050799

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