Chromatographic characteristics of water-soluble vitamins with irradiation processing and its application

Abstract

This study investigated the chromatographic characteristics of irradiated vitamins and their feasibility for dose measurement by using high performance liquid chromatography (HPLC). Water-soluble vitamins in the B family, including thiamine hydrochloride (TH), riboflavin (RF) and nicotinic acid (NA), were used for comparative study. The results showed that riboflavin was sensitive to irradiation, and the content changes of RF were linearly correlated with irradiation doses. Combining the analyses of ultraviolet and fluorescence spectra, the suitable application range for dose determination was confirmed from 100 to 2000 Gy; moreover, the influencing factors were further discussed. The content of RF could maintain good stability before and after irradiation under low-light conditions. These characteristics make it possible for RF to be used as a material for dose measurement. In addition, this work also provides references for irradiation nutrition research due to the favourable separation and analysis characteristics of HPLC.

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Introduction

With the progress of sterilization technologies, many different physical and chemical methods have been developed, among which, ultraviolet, ozone, high temperature and autoclaving are widely applied. Generally, ultraviolet is effective in eliminating bacteria on the surface of food or in short range space due to its weak penetrating ability. Ozone is a strong oxidizer and corrosive to materials such as metal, fabric and rubber; in addition, it is stimulating and harmful to the respiration system of human operators. For many substances with unstable structures and properties, high pressure and high temperature treatments are destructive factors, and therefore are improper to be adopted. Previous studies showed that irradiation can be applied to sterilization due to the strong penetrating power and ionization effect. This technique is as simple and effective as other methods. Comparatively, irradiation consumes less energy and has been proven to be safe for health.¹ Moreover, no residue or pollutants are generated in the process, and it would not influence the quality of the treated materials appreciably within appropriate dose range, and these advantages make it more acceptable and prevalent. Generally, irradiation doses within 2000 Gy could inhibit the sprouting and maturation process of fruit and vegetables and thus lengthen the storage period.^2,3 In addition to the applications in food preservation, it is also used in drugs and medical apparatus as well.^4–7 With its development during the past 60 years, it has become widely applied and turned out to be worthy of practice.

The most commonly used methods for ionizing radiation dose measurements are silver dichromate spectrophotometry, thermoluminescence, ionization chamber dosimetry and color film dosimetry. Among these methods, the color of dye,^8,9 radiochromic film¹⁰ and photochromic glass¹¹ could change along with the different intensity of irradiation dose, but the stability and accuracy are unsatisfying. Thermoluminescence and ionization chamber dosimetry need to be connected with specialized devices, making them inconvenient to use.

Juanchi¹² studied the irradiation effect of vitamin B-12, and Maged successfully applied it to measure radiation doses in the range of 0.1–2 kGy with spectrophotometer.¹³ According to their results, there was good linearity between doses and variations of vitamin concentrations in solution. In this work, a novel dosimeter is developed by selecting vitamins in the B family and modifying the analysis method, which proves to be more convenient for food irradiation dose measurement.

Experimental procedures

Devices and materials

Thiamine hydrochloride (99%), riboflavin (98%) and nicotinic acid (99%) were purchased from Aladdin Company (Shanghai, China). Equipment used in this work were a high performance liquid chromatograph with a SPD-M 20A photodiode array detector and RF-10A fluorescence detector from the Shimadzu company (Tokyo, Japan), and an INOVA nuclear magnetic resonance from Varian company (California, USA). The gamma radiation (γ) source employed is ⁶⁰Co with intensity of 5.5 × 10¹⁵ Bq, which is provided by the Application of Radiation Technology Research Center in Hunan Province, China.

Irradiation process

Vitamin solutions (thiamine hydrochloride, riboflavin and nicotinic acid) with various concentrations were prepared for sampling. For the selection of concentrations, our previous study showed that there was a remarkable change for TH solution of 0.05 mg ml⁻¹ with a 2000 Gy dose of irradiation, and thus it is sensitive to this dose range and can be used. Another concentration selected in this work was 0.02 mg ml⁻¹. Water solubility of RF is poor, and it is difficult to dissolve when the concentration is above 0.05 mg ml⁻¹. Thus, low concentrations of 0.025 mg ml⁻¹ and 0.05 mg ml⁻¹ RF solution were selected.

Niacin is diffluent in water and has good stability. Different concentrations of NA solution, including 0.02 mg ml⁻¹, 0.1 mg ml⁻¹, 0.5 mg ml⁻¹, and 1.0 mg ml⁻¹, were employed in this work.

Vitamins of TH, RF and NA were accurately weighed and dissolved in distilled water in a volumetric flask. Amber bottles were used to prepare RF solution as it needed to be protected from light. Then, these solutions were divided into the test tubes (with lids) separately. After that, these tubes were sealed up, preserved at room temperature and subjected to irradiation.

The well-packaged tubes were exposed to a prescribed intensity of irradiation over a certain time in order to form a dose. In this experiment, the irradiation dose rate of γ ray was 1 kGy h⁻¹, and the dose range of samples accepted was from 0.1 to 2 kGy.

Results and discussion

Selection of vitamin

Vitamins are divided into water and lipid-soluble varieties according to their chemical characteristics. If fat-soluble vitamins are chosen to be dosimeters, this will interfere with the accuracy of the test due to the volatilization of the organic solvent and the tendency of changing by itself upon irradiation. Thus, water-soluble vitamins are more suitable as the radiation mechanism of water has been extensively studied in the previously studied reports.¹⁴ It is well accepted that water could produce ˙OH and H₂O₂ through the following reactions with the aid of irradiation.

H₂O → ˙H + ˙OH

˙OH + ˙OH → H₂O₂

˙H + ˙OH → H₂O

The oxidation effect of these species could destroy the structure and content of the substance in the water solution. Water-soluble vitamins are generally divided into vitamin B and C families. Our previous research showed that the content decrease of vitamin solution was directly related with the irradiation dose. Therefore, solutions with low concentrations are required for low dose measurements. Vitamin C is readily oxidized by γ rays owing to its oxidizable chemical structure and good solubility in water. However, vitamin C solution is unstable particularly at low concentrations due to the hydrolysis reaction. For this reason, some vitamins in the B family (with a brief introduction in Table 1), such as thiamine hydrochloride (TH), riboflavin (RF) and nicotinic acid (NA), were selected for comparative study in this work.

Table 1 Chemical characteristics of TH, RF and NA

No.	Biochemicals	Molecular formula	Structure	Molecular weight (g mol^−1)	Characteristics
1	TH	C12H17ClN4OS˙HCl		337	Good stability with time, light, and temperature for thiamine combined with HCl
2	RF	C17H20N4O6		376	Yellow powder with fluorescence, sensitive to light and stable at relatively high temperature
3	NA	C6H5NO2		123	Stable towards light and high temperature. No degradation produced in boiling water solution or at melting point

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Content determination and dose response curve

The main apparatus used for vitamin analysis are high performance liquid chromatography (HPLC) and fluorescent spectrometry. With high accuracy and good repeatability, HPLC is widely used in purity determination of substances. Therefore, in this experiment, the HPLC method was employed to determine the content of the three vitamins.

The mobile phase for the determination of TH and RF¹⁵ was 0.05 mol L⁻¹ sodium acetate (pH 4.5)–methanol (65 : 35), and the detection wavelength was 270 nm. For NA, the mobile phase was 0.02 mol L⁻¹ monopotassium phosphate (pH 6.8)–acetonitrile (90 : 10) with a detection wavelength of 261 nm.¹⁶ In this experiment, the injection volume was 20 μL, the flow rate was 1 ml min⁻¹ and the analytical column was Agilent TC-C₁₈ (5 μm, 4.6 × 150 mm).

Under different irradiation doses treatments, the content variation of vitamin solutions is shown in Fig. 1.

Fig. 1 Scatter diagram for irradiation dose and vitamin content. (A) ρ_TH, 0.02 mg ml⁻¹ and 0.05 mg ml⁻¹; (B) ρ_RF, 0.025 mg ml⁻¹ and 0.05 mg ml⁻¹; (C) ρ_NA, 0.02 mg ml⁻¹ and 0.1 mg ml⁻¹.

Among the content changes of these three vitamin solutions before and after irradiation, RF was the most sensitive to γ rays, the decrease in content was evident, and the linear trend between content decrease and radiation dose was good. The RF solution with a concentration of 0.025 mg ml⁻¹ possessed good linearity in the dose range of 100–1500 Gy, and 0.05 mg ml⁻¹ RF for the dose range of 100–2000 Gy, which can be applied in dose determinations both with a relative coefficient of 0.99.

Between the two concentrations of 0.02 mg ml⁻¹ and 0.05 mg ml⁻¹ TH, the content changes of 0.02 mg ml⁻¹ was relatively evident, but as the dose rose up to 1000 Gy, the structure of TH would be significantly destroyed (with degradation occurring on the main peak), which made it unsuitable for analysis. NA solutions with concentrations of 0.02 mg ml⁻¹ and 0.1 mg ml⁻¹ had fluctuant changes upon an irradiation dose range from 100 to 2000 Gy, and the total content decrease was 12%. The content changes of 0.5 mg ml⁻¹ and 1.0 mg ml⁻¹ NA solutions were inconspicuous within the dose range of 2000 Gy. According to the experiments, when the dose rose to 4000 Gy, there were slight changes with concentration variation of 2% and 0.9%. With high irradiation treatment from 100 to 20 000 Gy, the content of the 0.5 mg ml⁻¹ group decreased slowly with a total change of 9.8% and the corresponding change for 1 mg ml⁻¹ NA was 4.4%. These results showed that NA was stable upon irradiation but unsuitable for dose indication.

Comparing the content changes of the three vitamin solutions with different concentrations enduring γ radiation, the sensitivity towards irradiation decreases in the following order: riboflavin > thiamine hydrochloride > nicotinic acid. Through comparison, the content changes of the RF solution exhibited better consistency with the variances of radiation doses; thus, it could be used to indicate the dose changes in the range of 100–2000 Gy.

Radiation doses and spectrogram characteristics of RF

The appearance of the RF solution was bright yellow, and the color gradually faded as the irradiation intensity rose. The conjugated structure of the RF molecule makes it a good absorber of ultraviolet light, and this characteristic can be used in quantitative analysis. The chromatogram of HPLC (Fig. 2) reveals that the content of RF (with a retention time at 5.23 min) continues to decline as the irradiation strengthens, and the decrease shows good relevance within the range of 2000 Gy. As the residual of RF became considerably less, the peak of RF in the HPLC chromatogram disappeared when the dose approached 4000 Gy. Moreover, degradations were effectively separated, shown from 1.5 min to 4 min, and the total content increased with intensified irradiation.

Fig. 2 HPLC chromatogram of RF with different irradiation doses, and curves decline from 1 to 7 denotes 0, 500, 800, 1000, 1500, 2000, 4000 Gy doses, respectively; ρ_RF = 0.05 mg ml⁻¹.

In addition, at an excitation wavelength of 440 nm and emission wavelength of 525 nm, RF is fluorescent, which makes it easy for detection. Thus, samples that had endured irradiation were separated by the HPLC system and tested by the fluorescence detector. The chromatogram variations are shown in Fig. 3.

Fig. 3 Fluorescence variation of RF after irradiation, curve 1 to 6 corresponds to the solutions subjected to 0, 500, 800, 1000, 1500, 2000 Gy doses, respectively (samples need to be diluted 20 times before tests).

The fluorescence changing patterns of RF (with a retention time at 5.20 min) were similar to that of the UV chromatogram, and degradation products were detected at retention times from 1.7 to 3.7 min. As can be seen from the chromatogram, the residual of RF gradually diminished, whereas the degradation product increased, indicating that the structure of the RF molecule was destroyed by γ rays.

Riboflavin has a large conjugated plane on isoalloxazine. With the presence of free radicals and high energy given by γ rays, the integrity of this constitute is easily affected; thus, the qualitative change could be observed through the sensitive spectral characteristic of RF. The structural alteration of irradiated thiamine is a separation of thiazole and pyrimidine segments, but the heterocyclic ring would not be easily affected.¹⁷ For nicotinic acid, pyridine and formic acid are difficult to be oxidized further. As a result, RF is more sensitive towards gamma rays.

Effect of oxygen in irradiation

Oxygen is considered to be a potential factor that influences irradiation in an aqueous solution. Testing was carried out to examine if oxygen had an evident effect on the RF irradiation system. First, 0.5 mg ml⁻¹ RF solution was prepared and delivered into test tubes, and afterwards, high purity of nitrogen (99.99%) was ventilated for 5 min in each tube. The concentration of oxygen was tested by an oxygen meter. The original oxygen content was 7.1 mg L⁻¹ and declined to 1.1 mg L⁻¹ after nitrogen treatment. Samples were sealed with parafilm outside the lids in order to avoid the interference of air outside. The controlling group was the RF solution without nitrogen ventilation. Under the same condition, samples were exposed to irradiation and analyzed with HPLC. Results showed that there were no distinct differences either in the spectrum characteristics of HPLC or content changes between the two groups, indicating that oxygen had no significant effect on this system.

Effects of temperature in dose measurement

The relations of the HPLC characteristic and radiation dose at different temperatures were studied. The content of RF without irradiation remains constant under different conditions due to the stability of the RF solution. Tests revealed that there were no evident differences at 5 °C and 20 °C. As temperatures rose to 30 °C, the residual content of RF was considerably lower. This indicates that the radiation measurement was affected at this temperature. Therefore, if the measurement is carried out at high temperatures above 30 °C, a cooling device is required.

As a result, due to the different conditions of dose measurement (sampling and analyzing), the working curve needs to be calibrated to ensure the accuracy of the results.

Mechanism studies with ¹H-NMR spectrum

After irradiation, the content, as well as the spectrum characteristics of RF, was changed, and degradation products could be detected. In order to study the reaction mechanism of RF upon irradiation, nuclear magnetic resonance (NMR) was utilized.

Samples of the RF solution enduring irradiation and the controlling group were first disposed by a rotary evaporator to remove the solvent (H₂O); afterwards, they were dissolved by DMSO and tested under the same condition. The ¹H-NMR spectrum of RF without irradiation is shown in Fig. 4(A). The chemical shift at δ = 11.35 ppm (1H, s, H-a) was assigned to the proton attached to the nitrogen atom on the heterocyclic ring. The aromatic protons appeared at δ = 7.92 ppm (1H, s, H-b) and δ = 7.89 ppm (1H, s, H-b′). Hydrogens of hydroxyl groups were shown at δ = 4.99–4.24 ppm. The chemical shift at δ = 3.63 ppm and δ = 3.43 ppm corresponded to methylene (H-f, H-g) of the ribitol. δ = 2.44 ppm and δ = 2.40 ppm were attributed to methyl on isoalloxazine (H-h; H-h′), which is thought to be derived from the solvent (DMSO, δ = 2.50 ppm).

Fig. 4 ¹H-NMR spectrum of (A) unirradiated and (B) irradiated RF.

The spectrum of RF subjected to irradiation is shown in Fig. 4(B). Comparing these two spectra, the distribution of hydrogen remains unchanged after irradiation, but the intensity at lower magnetic fields evidently decreased, indicating that the integrity of RF was destroyed. The shielding effect increased, whereas the conjugative effect of ¹H weakened. Combined with the results of the continually decreased UV absorbance and fluorescence intensity, the conjugate planes that formed lumichrome plates were disrupted by γ radiation. The main signals of hydrogen shifted to δ = 3.36 ppm and 2.44 ppm, which were primarily protons of methylene and methyl groups, indicating that the structure tends to be saturated gradually. For the irradiated RF, compared with the signals at δ = 11.35 ppm, δ = 7.92 ppm and δ = 7.89 ppm in downfield, the intensity of signals at the chemical shift from δ = 4.2 ppm to 5.1 ppm are relatively increased. The ratio of integral value for the two parts changed from 3/4 (unirradiated) to 3/16 (irradiated), inferring that hydroxyl substituents increased. These changes illustrated that bond breakage and addition reaction occurred with the ˙H and ˙OH produced by γ rays.

Stability of RF solution

RF is sensitive towards light, which would cause the decrease of the content; therefore, samples should be kept in weak light. When applied in irradiation measurement, in order to ensure the accuracy, a controlling group of RF solution without irradiation could be set to eliminate spontaneous attenuation. Tests showed that simple light protection (e.g. paper wrap) could eliminate the photic influence as shown in Table 2.

Table 2 Stability of 0.05 mg ml⁻¹ RF solution, stored in weak light

Time (Day)	Content (mg ml^−1)	Decrease percent (%)
0	0.0500	0.00
10	0.0497	0.60
15	0.0495	1.05
20	0.0489	2.09

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After irradiation, the concentration of RF in the solution, as well as the fluorescence intensity of RF, decreased. In order to testify if it continues to degrade after irradiation, samples of RF solution were analyzed and content changes were recorded. As shown in Fig. 5, the change was inconspicuous after irradiation. This further demonstrates that the degradation reaction was particularly caused by the γ rays and possesses stability, which would not interfere with irradiation measurement. Chromatographic curves showed that RF solutions of 0.025 mg ml⁻¹ and 0.05 mg ml⁻¹ had good stability. No remarkable content changes were observed within 20 days, and 0.05 mg ml⁻¹ RF solution gave better results. Therefore, in few cases, when samples could not be analyzed instantly by HPLC, they could be stored for a certain amount of time.

Fig. 5 Stability of RF solution (0–20 Days) after irradiation, (A) 0.025 mg ml⁻¹ RF solution, (B) 0.05 mg ml⁻¹ RF solution.

Radiation measurement

The RF dosimeter was applied in the irradiation field and compared with a silver dichromate dosimeter, by which dose distribution was calibrated. Results are shown in Table 3, which indicate that dose values obtained by this method are in good consistency with the standard method.

Table 3 Radiation dose measurement

No.	Calibrated dose (Gy)	RF Dosimeter
		Result^a	RSD (%)
a Note: Mean value of six measurements.
1	100	105.27	1.7
2	500	491.69	0.9
3	1000	1001.15	0.5
4	2000	1975.73	1.1

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Conclusion

Comparing the different irradiation characteristics of TH, RF and NA solutions towards γ rays, RF possesses the best linearity between concentrations variations and dose changes. With good sensitivity, selectivity and stability, 0.05 mg ml⁻¹ RF solution is suitable for 100–2000 Gy irradiation dose measurement. Furthermore, the preparation for measuring is relatively simple and non-toxic, which makes it convenient to use. The accuracy of the measurement can be guaranteed by HPLC, and no other valuable equipment is needed. These merits make RF an ideal material for irradiation dose research.

Acknowledgements

This research was financially supported by the Hunan Nature Science Foundation (11JJ8008). The authors also would like to thank Dr Jingchong Yan at the Chinese Academy of Sciences and Tassie Smith at University of South China for writing assistance.

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