Introduction

Phytate, myo-inositol hexaphosphoric acid (InsP6), a fully phosphorylated form of inositol [1], is a naturally occurring component and the principal storage source of phosphorus in many plants, such as cereals, soybeans, legumes, oil seeds, pollens, nuts, fruits and vegetables [2–4]. In InsP6 molecule, there are 12 replaceable protons, among which six are strongly (pK a < 3.5) and six are weakly (pK a = 4.6–10) dissociated [5]. InsP6, including its deprotonated species, possesses the high affinity for chelating metal ions such as Fe2+, Fe3+, Zn2+, Mn2+, Cu2+, Mg2+ and Ca2+, as well as proteins and starch [6, 7]. InsP6 binds with the essential elements in human body, forming complexes with low solubility at physiological pH [8, 9], and thus potentially influences their bioavailability [10]. Hence, InsP6 has long been considered solely as an antinutrient and related studies attracted a great deal of interest in human nutrition [11]. However, a variety of epidemiological and studies on living organisms demonstrated that InsP6 and its lower phosphorylated forms have beneficial functions. For example, it could reduce the risk of cancers [12–14], heart disease, diabetes, and renal calculi [15, 16]. In addition, InsP6 can also bind potentially toxic mineral elements such as Cd2+ and Pb2+ in human body, and thus influences their toxicity and facilitates their excretion [17].

Subsequently, determination of phytic acid level in dietary products becomes a very important task. Some methods can be used to determine phytic acid concentration in cereal products, biological and urine samples. The analysis of InsP6 in various matrices was usually performed by means of chromatography [18–20] and capillary isotachophoresis [21]. Among them, ion pair chromatography [22] and high-performance ion chromatography [23] are the commonly used methods, in which most are capable of simultaneous separation and determination of InsP6 and its lower phosphorylated species. Other methods are based on the direct/indirect determination of phosphorus [24] or inositol [25] in InsP6 or its quantitative hydrolytic products by spectrophotometry [26], NMR spectroscopy [27] and inductively coupled plasma atomic emission spectrometry [28]. Moreover, a fluorimetric measurement procedure for InsP6 in human urine and food samples were developed [29, 30]. However, it has no report that determination of InsP6 by synchronous fluorescence spectroscopy method according to our knowledge.

1,10-Phenanthroline (phen), an N-donor ligand with aromatic rings, is known to form the protonated species in the range of pH = 2–7, i.e., H(phen)+ and H(phen)2+. For H(phen)2+, two 1,10-phenanthroline molecules are stacked through their aromatic rings, and it is known that the ligand favorably forms intercalated complexes [31]. Because of their inherent pocket structure, [M(phen)3]n+ complexes have the ability to form outer sphere complexes with anions, neutral molecules, or combination of them [32]. It was found that the synchronous fluorescence of phen will be decrease when Fe3+ was added, and the characteristic wavelength with red-movement. Moreover, the synchronous fluorescence increased by addition of InsP6 subsequently, and the characteristic wavelength was also changed. In this procedure, the synchronous fluorescence is constant wavelength synchronous luminescence. By this mean, a novel procedure for the determination of InsP6 in food was developed in this paper. The synchronous fluorescence method has high precision and accuracy, simple, rapid, and it can eliminate the Rayleigh interference effectively. In the proposed procedure, the sensitivity is excellent, but the selectivity can be better. Although this procedure has some disadvantage, we hope it can offer some new thought for the quantification of phytic acid.

Experimental

Apparatus

The fluorescence spectra were recorded on a JASCO FP-6500 spectrofluorometer equipped with a thermostated compartment using 1.0 cm quartz cuvette. The pH measurements were carried out on a PHS-3C Exact Digital pH meter equipped with Phonix Ag–AgCl reference electrode (Cole-Parmer Instrument Co.), which was calibrated with standard pH buffers. The control experiments were carried out on a SHIMADZU UV-2450 UV–visible spectrophotometer. A CN column (25 cm × 4.6 mm i.d.) with 5 μm cyanopropylsilica packing material (Spherisorb) was used in the separation.

Reagents

Sodium InsP6 was purchased from Sigma. 1,10-Phenanthroline (phen) was obtained commercially from Sigma (www.sigmaaldrich.com). A 1,10-Phenanthroline (phen; 0.01 mol L−1) ethanol–water solution was prepared. An aqueous standard solution of sodium InsP6 (0.1000 mol L−1) was prepared and stored in refrigerator prior to use. FeCl3 solution (0.01 mol L−1) was prepared to quench the synchronous fluorescence of phen. Acetate buffer (pH 6.0) as used to maintain the solution pH value throughout the experimental procedure. The cations examined in the disturbance experiments were all chloride salts and the anions were all sodium compounds. FeCl3·6H2O and 5-sulfosalicylic acid was obtained commercially and used for determination of InsP6 in food by UV-spectrophotometry method. All reagents and solvents were of analytical grade and used without further purification unless otherwise noted. All aqueous solutions were prepared using newly double-distilled and deionized water. The AG1-X8 anion-exchange resin (chloride form, 200–400 mesh) was purchased from Sinopharm Chemical Reagent Co., Ltd (www.sinoreagent.com).

Fluorimetric detection

A solution of 3.00 mL 1,10-Phenanthroline (phen) (0.001 mol L−1) containing acetate buffer (pH 6.0, 15 mmol L−1) was transferred to a quartz cell, and then an appropriate aliquot of Fe3+ solution (0.01 mol L−1) was added. The resultant mixture was allowed to equilibrate for 3 min at ambient temperature and scanned on the fluorophotometer in the range of 320–420 nm with the Δλ = 40.0 nm. For constant wavelength synchronous luminescence was adopted, Δλ is wavelength space between the excitation wavelength and the emission wavelength. The spectral bandwidths of excitation and emission slits were both 5.0 nm. The synchronous fluorescence of the solution was detected. The variation of synchronous fluorescence of phen against the Fe3+ concentration could be observed.

Some 10 mL volumetric flasks were prepared. 1 mL phen solution (0.01 mol L−1) and 1 mL acetate buffer (pH 6.0) was transferred to every volumetric flask, and 0.3 mL FeCl3 solution was added to every flask. Then 5 μL to 400 μL sodium InsP6 standard solution (0.1000 mol L−1) was added to the flasks respectively. After 10 min, the synchronous fluorescence of the solution was recorded and the fluorescent intensity was measured.

Sample treatment

The treatment of food samples was carried out following the literature described procedure with minor modifications as follows [29]. A 0.5–0.25 g amount of the sample was ground and extracted with 50 ml of 0.5 mol L−1 HCl for 3 h at room temperature, according to recommended procedures [33]. The suspension was centrifuged and an aliquot of the solution obtained after centrifugation, and then was purified by means of anionic-exchange treatment. The resin was washed with 50 mL of 0.05 mol L−1 HCl at 0.4 mL min−1. The retained phytate was eluted with 5 mL of 2 mol L−1 HCl at 0.25 mL min−1. The collected solution was neutralized with 2 mol L−1 NaOH and purified via the same process to separate phosphate completely. Then the 2 mol L−1 HCl solution containing InsP6 was lyophilized and reconstituted with 5 mL of HCl–NaCl solution (pH 6.6, 10−3 mol L−1 NaCl). An aliquot of this solution was taken to analyze the content of InsP6.

Results and discussion

Fluorimetric detection

A solution of 1,10-Phenanthroline (phen; 0.001 mol L−1) contain 15 mmol L−1 acetate buffer (pH 6.0) show strong synchronous fluorescence with characteristic wavelength at 362.0 nm, when the Δλ is 40.0 nm. Upon addition of Fe3+ (0.01 mol L−1) to the solution, the fluorescence intensity decreased gradually with a little red shift in the characteristic wavelength to 364.0 nm. As the Fig. 1 show, when the concentration of the added Fe3+ achieved a certain degree, the fluorescence intensity reached the minimum. Moreover, the peak at 362.0 nm has disappeared, and only a weak peak at 382.0 nm, as shown in the plot of the Fig. 1. It can be calculated the molar ratio of phen to Fe3+ is 1:3. So the following experiments all adopted this ratio.

Fig. 1
figure 1

The synchronous fluorescence of phen was quenched by Fe3+. The inset is curves of the last five additions of Fe3+. Phen: 0.001 mol L−1, Fe3+: 0.01 mol L−1, acetate buffer (pH 6.0)

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Then, the synchronous fluorescence of the different concentration InsP6 and phen (0.001 mol L−1and Fe3+ (0.003 mol L−1) mixture solution was tested. We can see from the Fig. 2, at 364.0 nm, a peak was arisen. Furthermore, the fluorescence intensity continually increased with the augment of InsP6 concentration. However, the fluorescence intensity could not recover to the original level, and the characteristic wavelength is 364.0 nm not 362.0 nm. For 1,10-Phenanthroline has the inherent pocket structure, phen metal complexes have the ability to form outer sphere complexes with anions, neutral molecules, or combination of them [34]. Consequently, it could be deduced that InsP6, phen and Fe3+ formatted a ternary complex. It made the synchronous fluorescence intensity of the solution increasing.

Fig. 2
figure 2

The variation of the fluorescence intensity of different InsP6 concentration. The dashed line a is the sample no InsP6 added, b~s: 0.33 mg ~32.34 mg InsP6. phen: 0.001 mol L−1, Fe3+: 0.003 mol L−1, acetate buffer (pH 6.0)

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It was revealed that the formation of InsP6-metal complexes depends on conditions such as pH and the InsP6-to-metal molar ratio [35, 36]. Many results demonstrated that InsP6 can be deprotonated in aqueous solution, giving rise to different anionic species over a wide pH range [37]. And when the pH is about 6.0, it has a good ability of InsP6 binding Fe3+, and the InsP6 – Fe3+ complex is more onefold [8], so the pH 6.0 was adopted in this study.

Calibration and linearity

As stated in above, the fluorescence of 1,10-Phenanthroline will be quenched with the addition of Fe3+. However, as it shown in Fig. 2, with the concentration of InsP6 increasing, the fluorescence intensity at 364.0 nm was enhanced continuously. And the fluorescence intensity enhancement was proportional with the increasing InsP6 concentration under the stated conditions. Plotting of the fluorescent intensity versus the InsP6 concentration gave rise to a calibration curve that could use to quantitative analysis for the linearity (Fig. 3). The linearity range is 0.33~32.34 mg and with the linear equation of I f = 8.770 + 2.980c (R 2 > 0.9994; I f represents the fluorescent intensity of the solution at 364.0 nm and c represents the concentration of the InsP6 expressed as mg L−1). Above these concentrations the calibration of InsP6 is not linear, and the probably reason is that InsP6, phen and Fe3+ formatted a ternary complex not replacement reaction.

Fig. 3
figure 3

The calibration curve. The fluorescence intensity is at 364.0 nm. phen: 0.001 mol L−1, Fe3+: 0.003 mol L−1, acetate buffer (pH 6.0)

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Variables affecting the fluorescence

The variables that affect the synchronous fluorescence of the experiment such as Δλ and foreign ions, were investigated. In this experiment, constant wavelength synchronous luminescence was adopted, the value of Δλ was considered. Generally, the Stokes shift is the best choice. Consequently, the excitation spectrum and emission spectrum were detected, and the Stokes shift is 40 nm. In addition, the synchronous fluorescence was measured with Δλ equal 10.0 nm, 20.0 nm, 30.0 nm, 40.0 nm, 50.0 nm, 60.0 nm, 70.0 nm, 80.0 nm, 90.0 nm respectively, and it was found that 40.0 nm was the most appropriate result. So the Δλ was set 40.0 nm in the study.

The effect of foreign ions and typical chelating agent EDTA on the fluorescence of phen–Fe3+–InsP6 were investigated. The examined foreign ions include K+, Ba2+, Ca2+, Mg2+, Cu2+, Zn2+, Fe2+, Al3+, \({\text{CO}}_{\text{3}}^{{\text{2}} - } \), \({\text{PO}}^{{3 - }}_{4} \), \({\text{SO}}_4^{{\text{2}} - } \), \({\text{P}}_{\text{2}} {\text{O}}_{\text{7}}^{{\text{4 - }}} \), and EDTA. The final concentration of each cation solution was ten times that of the previously added Fe3+, and each anion solution was ten times excess of the added InsP6. As shown in Table 1, among the cation ions, Ba2+, Cu2+, Zn2+ can weaken the fluorescence of the phen–Fe3+–InsP6 solution significant, whereas the Al3+ and Mg2+ can reversely strengthen the fluorescence. The possible reason is that Ba2+, Cu2+and Zn2+ have a strong ability combining 1,10-Phenanthroline, and the Al3+ and Mg2+ had a intense hydrolysis reaction with great influence to the complex reaction. All other cations had little influence on the fluorescence of the system. On the other hand, among the anion ions, only \({\text{PO}}_4^{{\text{3}} - } \) has remarkable influence on the fluorescence of phen–Fe3+–InsP6, the \({\text{PO}}_4^{{\text{3}} - } \)would strengthen the fluorescence intensity. The possible reason is the \({\text{PO}}_4^{{\text{3}} - } \) also can format a complex with phen–Fe3+ as InsP6, thus enhanced the fluorescence intensity. However, during the preparation of the food samples, the anion-exchange resin was used and the effect of these foreign ions could be ignored.

Table 1 Effect of foreign species on the fluorescence of phen–Fe3+–InsP6
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Reproducibility of the measurements

Reproducibility is basic for establishing a method for determining an analyte and is one of guarantees for a method precision. The reproducibility of measurements was checked by monitoring the synchronous fluorescent of a phen–Fe3+ solution with a certain InsP6 (15 mg L−1) for five replicates every day within a week. RSD was used to present the reproducibility and it was calculated from five (n = 5) replicated measurement. The average RSD of the obtained results at 95% confidence interval was found to be less than 2.24%. The result indicated that the reproducibility of the proposed method was excellent.

Determination of InsP6 in food

In order to determine the range of applicability of this method, six different food including rice, peanut, wheat flour, soybean, feed (pig diet), rapeseed, were treated by the aforementioned procedure. The InsP6 level in each sample was measured according to the proposed method. In order to evaluate the recovery of expected InsP6 concentration, an aliquot of 10.0 mg L−1 of InsP6 was added into each sample solution and then analyzed. Results are given in Table 2 as the mean of five replicates. Recoveries (%) are in the range of 92.2%–98.3% for InsP6 obtained by subtracting its initial level in samples. The UV-spectrophotometry method [26, 38] was used to compare to the proposed method for determination of InsP6 in the food, and the deviation of the two methods is less than 5%. As can be seen in Table 2, good agreement can be observed between the two methods, thus demonstrating the suitability of the proposed procedure. The results indicated that the proposed method is reliable to be applied for the determination of InsP6 in food. Compared the two method, the synchronous fluorescence method is simple and sensitive, the selectivity is its disadvantage, and should be improved.

Table 2 Results obtained for the determination of InsP6 in food by the proposed method and by an UV-spectrophotometry method (mean ± S.E., n = 3)
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Conclusions

A reliable method was developed for determination of phytic acid in food samples based on the synchronous fluorescence measurement. The calibration graph showed good linearity in the InsP6 concentration range of 0.33–32.34 mg L−1 with a linear equation of I f = 8.770 + 2.980c (R 2 > 0.9994). The reproducibility is satisfied. The method was applied for determination of InsP6 in some food samples, and the suitability of the proposed procedure was also demonstrated.