Optimization for quantification of sorghum tannins by Ferric ammonium citrate assay

Xujuan Wang,Xiaoxian Han,Limin Li,Xueling Zheng*

College of Food Science and Technology,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT Due to the complexity of structure,it is difficult to determine the content of sorghum tannins.The current method for the determination of sorghum tannins is the Ferric ammonium citrate assay described in ISO 9648.However,the standard tannic acid(TA)used in the ISO 9648 method is significantly different in both structure and dynamics from sorghum tannins,resulting in inaccurate quantitative result.Furthermore,the extraction solvent,75%dimethylformamide(DMF),used in each step of this method had large toxicity to human.Hence,the objective of this study is to optimize the ISO 9648 method by selecting proper standard and extraction solvents so as to enhance the accuracy and safety.In this study,Sephadex LH-20 chromatography combined with high pressure liquid chromatography was used to purify and identify the extracted sorghum tannin (ST).Then, commercial TA, epicatechin(EA),grape seed procyanidin(PA)were selected as the standard and ST was used as reference for Ferric ammonium citrate assay to compare the effects of the three standards.Furthermore,the extraction rates of sorghum tannins in the presence of several low toxic solvents,such as absolute ethanol,absolute methanol,70%ethanol solution,70%methanol solution and 70%acetone solution,were compared to determine the alternative solvents of 75%DMF solution.The results showed that PA was superior to TA and EA in structure,with satisfactory color yield close to ST,and higher accuracy than TA.In terms of the extraction solvent,70%acetone solution was selected to replace 75%DMF solution because of its higher extraction yield(only next to 75%DMF)and low toxicity.Verification experiment results showed that both the recovery rate and the repeatability of the optimized method met the requirements of AOCO.Moreover,the optimized method,with higher accuracy and safety than the ISO 9648 method,can be applied widely in laboratory.

Keywords: Sorghum Tannin Ferric ammonium citrate assay Optimization

1.Introduction

Sorghum tannins were mixture of oligomers and polymers connected by the flavan-3-ol units with C4-C8 bonds,belonging to B-type procyanidin[1-4].The identificationof sorghum tannins is extremely difficult due to their structural complexity[5].Many analytical methods have been used to quantify sorghum tannins.Normally,these methods are based on colorimetric methods,including Acid-butanol assay[6],Vanillin-HCl/H2SO4assay[7-9],Prussian blue assay[10,11]and Ferric ammonium citrate assay(ISO 9648).Besides,precipitation reactions and high-performance liquid chromatography(HPLC)have also been used.

Due to the complicated stereochemistry structure of sorghum tannins,there is no appropriate commercially available standards to identify sorghum tannins [5,12,13].Cyanidin,delphinidin and quebracho tannin[5,14]have been used as the standards for the Acid-butanol assay,but the color yield of the three reactions differs by more than 30 times [6].Catechin (flavan-3-ol) or epicatechin has been employed as a standard for the Vanillin-HCI/H2SO4assay[7,9,15]or HPLC[1,4,16].Moreover,the catechin,as a monomeric substance,does not represent a standard of polymers,and it differs in the rate of reaction with the associated procyanidin polymers[9].The use of these standards only allows for the evaluation of oligomeric forms rather than their polymeric forms[16].In addition,tannic acid is often selected as the standard in total phenol analysis,such as the Folin-Denis, the Prussian blue assay [17] or the Ferric ammonium citrate assay.But it is a hydrolyzable tannin which has a dramatically distinct structure from sorghum tannins and has never been found in sorghum[18].

The standards for quantitative analysis of sorghum tannins had been mainly limited to tannic acid or catechin,and it has become a trend to use natural polymer as a standard.Strumeyer et al.[19]proposed a method for separating tannins from sorghum, which is based upon the discovery that tannins were adsorbed onto Sephadex LH-20 in 95% aqueous ethanol, and the non-tannin components were washed with ethanol.Then,the tannins were collected and eluted through 50%aqueous acetone.Moreover,Lea[20]tried to separate procyanidins using normal phase HPLC based on degree of polymerization (DP).Later,the decameric standard was obtained by extraction from cocoa and chocolate,and the purity of each oligomeric fraction was evaluated using HPLC and mass spectrometry[16,21] to quantify the tannin content in sorghum [1].However, the standards prepared by the above methods were not widely used in laboratory.Interestingly,the commercially available procyanidins standard extracted from grape seeds is structurally similar to sorghum tannins[3,22],and its purity is ≥95%,so it can be tried as a standard for the determination of sorghum tannins.

The Ferric ammonium citrate assay has been extensively used in many areas,but the selection of standard still needs further exploration.Meanwhile,the extraction solvent used throughout the whole experiment in this method is 75%DMF solution,which is highly toxic and mainly damaged the human digestive system and potential functions.Therefore,it is necessary to choose a low-toxicity extraction solvent to substitute for the toxic solvent of 75%DMF solution.

The purpose of this study is to establish a high-acccuracy and high-safety method for quantitative measurement of sorghum tannins based on the Ferric ammonium citrate assay described in ISO 9648 through re-selecting an appropriate standard and low-toxicity extraction solvent.The optimized method has relatively high accuracy and safety,and can be used widely in laboratory.

2.Materials and methods

2.1.Materials

Three varieties of sorghum were obtained from Liaoning(brown sorghum, LN-BS), Guizhou (white sorghum, GZWS) and Shandong (black sorghum, SD-HS) provinces,China.

2.2.Chemicals and reagents

TA and EA were purchased from Commeo (Tianjin,China) and Solarbio (Beijing, China), respectively.Four kinds of PA samples isolated from grape seeds were provided by Bomei(PA-BM,Anhui,China),Desite(PA-LM,Sichuan, China), Meilunbio (PA-ML, Liaoning, China) and Yuanye(PA-YY,Shanghai,China),respectively.The purity of all compounds measured by HPLC and UV scanning in the commercial products is ≥95%.

Ferric citrate was supplied by Commeo(Tianjin,China).The methanol,acetonitrile,and acetic acid are HPLC grade.Other reagents are analytical grade.Sephadex LH-20 gel was purchased from Solarbio(Beijing,China).

2.3.Extraction and separation of sorghum tannin

Sorghum tannin(ST)was extracted from LN-BS by the method described by Strumeyer et al.[19].Next, the above extract was re-dissolved in 95% aqueous ethanol and added to a Sephadex LH-20 column equilibrated with 95%aqueous ethanol.After removing monomeric phenols and low molecular weight procyanidins with absolute ethanol,the ST was recovered with 50%aqueous acetone[19].Finally, the acetone was removed by rotary evaporation,and the remaining material was freeze dried and stored at-20°C for later use.

2.4.HPLC analysis of standards and tannins

The purity of the extracted ST and commercially purchased EA,PA-BM,PA-ML,PA-YY,PA-LM were analyzed by Agilent 1260 HPLC system (Agilent Technologies,Santa Clara, CA, USA), a Develosil (4.6 × 250 mm 100 Diol,5 μm particle size)was used.

HPLC analysis was performed according to the report of Robbins et al.[23]with slight modification.The wavelength of UV detection was 280 nm.The chromatographic mobile phase was a binary gradient (solvents A and B), which consisted of an acidic acetonitrile(A,V(CH3CN):V(HOAc)=98:2)and acidic aqueous methanol(B,V(CH3OH):V(H2O):V(HOAc)=95:3:2).Column temperature was 35°C and flow rate was 1.0 mL/min.The gradient elution procedure was shown in Table 1.

The ST recovered with 50%aqueous acetone sample was extracted and dissolved into a methanol solution and filtered through a 0.45 μm PTFE syringe filters prior to injection.The estimation of the polymeric portion was made by subtracting the sum of resolved peak area(monomeric portions)from the entire elution peak areas and calculating thepolymer content(ST).Specifically,the integration method of Gu et al.[4]was adopted.Based on the plane baseline,the peak curve,two vertical lines by the flat baseline were integrated.According to the peak time of catechins,the substances after the peak of catechins were all tannins,and the peak areas were sequentially expressed as S1, S2, S3…Sn,and the total peak area was expressed as S.

Table 1 Gradient elution procedure.

2.5.Standard curves establishment

Preparation of the standard solution was comprised of the following steps: TA, EA, PA-BM, PA-ML, PA-YY, PALM and ST were respectively dissolved in 2 g/L of given stock solutions.Wherein each stock solution was diluted to establish a standard curve.Firstly,1 mL of 0.1,0.2,0.3,0.4,0.5 mg/mL standard solution was transferred to a test tube,and 5 mL of distilled water,1 mL of 3.5 g/L ferric ammonium citrate and 1 mL of 8 g/L NH3were added into the test tube.After reacting for 10 min at 25°C in dark condition, the absorbance of the solution was measured at 525 nm by a UV-762 ultraviolet spectrophotometer(Shanghai, China).Meanwhile, sample blanks were included in which the ferric reagent was replaced by distilled water.Lastly,taken the absorbance value of each solution in the standard series as the ordinate, and drawn a standard curve with the corresponding standard concentration(mg/mL)as the abscissa.The standard curve does not pass through the origin and need not to be corrected through the origin.

2.6.Optimization of extraction solvent

The sorghum grain was ground at high speed in the presence of liquid nitrogen and passed through a 0.5 mm sieve.Briefly,milled grain(0.5 g)was extracted with 20 mL absolute ethanol,absolute methanol,70%ethanol solution,70%methanol solution,70%acetone solution and 75%DMF solution at room temperature for 30 min and centrifuged at 3,000 r/min for 10 min, respectively.Then 1 mL of the above extraction solution was mixed with 5 mL of distilled water,1 mL of 3.5 g/L ferric ammonium citrate,and 1 mL of 8 g/L NH3.After reacting for 10 min at 25°C in dark condition, the absorbance of the solution was measured at 525 nm with UV-762 ultraviolet spectrophotometer.Meanwhile,sample blanks were included in which the ferric reagent was replaced by distilled water.

The tannin content will use the standard equivalent(%,dry weight)value.

where c is the standard concentration(mg/mL)of the test solution; m is the mass (g) of the test portion; H is the water content of the test sample(%).As a result,the arithmetic mean of the three determinations provides a requirement for repeatability.

2.7.Verification of optimized methods

2.7.1.Limit of detection

The limit of detection(LOD)was determined from the linear regression analysis using the following equation[24]:

Here SD and AS are the standard deviation of the intercept and the average slope of the linear regressions,respectively.

2.7.2.Recovery rate determination

The recovery rate was determined by adding five levels of standards (0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL), which covered the working range of the balnk matrix, to the corresponding sorghum samples.Concretely,recovery rate was expressed as the percentage difference between the detected concentration and the added concentration.

where c1is extract concentration(mg/mL)of the test solution;c2is control concentration(mg/mL)of the test solution; cnis the concentration of the added standard(mg/mL) of the test solution.As a result, the arithmetic mean of the three determinations provides a requirement for repeatability.

2.7.3.Repetition rate determination

The repetition rate of the method was examined using the interday and intraday repeatability[25].In intraday repeatability experiment,eleven parallel experiments were carried out on the three sorghum samples.Then,interday repeatability was determined by measuring distinctive batches of three sorghum samples on different dates.The results were expressed as the relative standard deviations(RSD%)of the measurements.Finally,the tannin content in ten sorghum samples was measured using ISO 9648 and the optimized methods for validation.

2.8.Statistical analysis

Analytical determinations for the samples were performed in triplicate and standard deviations were reported.All the data of variance(ANOVA)were performed to compare treatment means and differences were considered at the significant level of 95%(P ＜0.05)by Duncan's multiple range tests.Software of SPSS Statistics 17.0 and Origin 8.0 was used to analyze data for this study.

3.Results and discussion

3.1.HPLC analysis of commercially purchased standards and ST

The normal phase HPLC chromatograms of EA,PA-BM,PA-ML, PA-YY, PA-LM and ST were shown in Fig.1.In a diol stationary phase chromatography column in normal phase mode, the binary gradient of acidified acetonitrile and methanol-water was a normal phase separation of procyanidins based upon their DP under conventional chromatographic conditions.Non-tannin phenolic substances in the solution were first eluted and then the elution order increased with the DP increasing[23,26].The retention time of EA(Fig.1e)was 5.35 min.According to the peak time of EA,the substances after the peak of EA were all tannins.As shown in Fig.1,the ST(Fig.1f)was mainly composed of high molecular weight(DP ＞10)polymers which resolved to a single peak at 65.46 min, the result was consistent with the research of Gu et al.[4].Each peak was detected by UV scanning at 280 nm and,compared with literature[21,27],the purity of ST was calculated as 98.62%by the purity calculation formula.The peak diagram of EA was significantly different from that of ST,indicating that EA had the greatest difference in structure with ST.However, it could be observed from the HPLC diagram that PA was similar in structure to ST and had diverse degrees of polymerization.

3.2.Screening of standards

According to the basic steps of the ISO 9648,the absorbance of TA, EA, PA-BM, PA-ML, PA-YY, PA-LM (purity≥95%)and ST were determined using 75%DMF solution as solvent,and the standard curves were shown in Fig.2.All curves showed a good linear relationship,and the adjusted R2for each of the regression equations was greater than 0.99.In colorimetric assays,differences in standards had a large effect on color yield.The results emerged that at the same concentration,the TA which had the greatest difference in structure from ST possessed the highest absorbance and the maximum color yield after the reaction in these three standards,followed by EA,PA.As the concentration increased,ST absorbance showed a slight increasing trend.In other words,compared to the slope values of ST,the slopes of the TA,EA and PA-ML standard curves were significantly distinct.Sorghum tannins were composed of epicatechin and catechin subunits[21,28-30].Importantly,the catechins as monomeric substances did not represent the standard of the polymer,and its reaction kinetics were also different from that of the procyanidin polymers[9,29].And more notably,the structure of sorghum tannins were very similar to that of the procyanidins extracted from grape seeds, but molecular weight characteristics of procyanidins extracted sorghum and grape seeds were diverse[22].Concretely,sorghum tannins were mostly comprised of high DP (＞10) units, in contrast,the degrees of polymerization of grape seed procyanidins were relatively small.Moreover,tannins with different DP values have distinct molar absorption coefficients,because additional or side reactions appear to take place at sites other than the terminal units[31].This phenomenon indicated that the chromogenic reactions of different standards were distinctive at the same concentration,which was similar to the research of standards by Giner et al.[29,32,33].It could be determined from Fig.1 that PA was closer to ST than EA as a standard in sorghum tannins assays.

Fig.1.Normal-phase HPLC chromatograms for PA-BM(a),PA-ML(b),PA-YY(c),PA-LM(d),EA(e)and ST(f).

To further determine the variation of molar absorption coefficients of specific tannin,the absorbance of three sorghum samples(GZ-WS,LN-BS and SD-HS)were measured using the Ferric ammonium citrate assay and the corresponding different standard equivalent values (%, dry weight) were calculated.As shown in Table 2, there was about 3.5-fold difference in response between the most reactive compound (TA) and the least reactive compound(ST) in Ferric ammonium citrate assay.In addition, the use of TA,EA and PA-ML as standard in this assay resulted in an underestimation of sorghum tannins.It was known that suitable tannin standards for determination of the tannin content could be obtained by purifying the standard from the sorghum.However, that approach was very hard to achieve, because sorghum of different varieties and from different sources had different purity of tannin polymers, and the purification process was complex, making it difficult to obtain “standard” sorghum tannins.So, there was no commercially attainable standard to represent the complex structure of tannin in sorghum.Since a single purified standard was not easily obtained, PA was recommended as the standard for the determination of sorghum tannins by Ferric ammonium citrate assay.

3.3.Optimization of extraction solvent

It is vital to select a low-toxicity solvent for the determination of tannin content in sorghum because the currently used solvent (DMF) is toxic and slightly irritating to the eyes during the experiment.In this study,we selected several commonly used solvents with low toxicity and high safety,such as absolute ethanol,absolute methanol,70%ethanol solution,70%methanol solution and 70%acetone solution,tested their extraction effects of tannins-ferric ammonium citrate reaction and compared with that of 75%DMF solution.The solvent with higher extraction rate was chosen as the alternative extraction solvent in the Ferric ammonium citrate assay.

Fig.2.Regression curves for different standards in Ferric ammonium citrate assay.

The standard curves of PA-ML determined in different solvents were shown in Fig.3.The results of analysis of variance showed no significant difference between the extraction solvents under the same substance(P ＜0.05).These results showed that,in the Ferric ammonium citrate assay,the type of extraction solvent did not affect the absorbance of the reaction.As shown in Table 3,the GZ-WS sorghum sample had a relatively low tannin content,and the methanol extraction effect reached the same as 75%DMF solution.However, for the other two sorghum samples, the tannin content (%, dry weight) of sorghum samples extracted with 70%acetone solution was the second highest,only next to 75%DMF solution,which was significantly superior to methanol water system and ethanol water system.It was observed that 70%acetone solution was preferable as an alternative extraction solvent of this method.

Therefore,we selected PA as the optimized standard to replace the standard tannic acid in ISO 9648,and 70%acetone solution as the optimal extraction solvent.Other conditions were described as above.

3.4.Verification of optimized methods

3.4.1.Limit of detection

The limit of detection (LOD) in the optimized method was calculated to be 0.0144 g/100 g,and there was no significant difference with the detection limit before improvement.

3.4.2.Recovery rate

The recovery rate of the optimized method was compared with ISO 9648 (Table 4).We could seen from Table 4, the recovery rate of the optimized method was 84.30%-104.40%,while that of the ISO 9648 method was 92.60%-99.50%.In the optimized method,when the concentration of the PA-ML standard added was 0.1 mg/mL,the recovery rate for the GZ-WS sorghum sample was 84.25%,which was lower than the suggested standard of AOAC International.Other values basically met the 85%-110% standard recommended by AOAC International.In comparison with ISO 9648, the larger recovery range of the optimized method might be due to the destruction of PA structure during the extraction of spiked samples.And the structure of TA was more stable.Overall,the recovery values were higher than 84%,suggesting that the accuracy of the optimized method was appropriate.

3.4.3.Repetition rate

The repetition rate of the optimized method was compared with ISO 9648.The results were expressed as the relative standard deviation(RSD%)of the measured values.The RSDs of the three representative sample analyses were less than 1.0%for intraday analysis and 10%for interday analysis.In the GZ-WS samples with lower tannin content,the low concentration caused a large coefficient of variation during the experiment (Table 5), the repetition rate was 5.26%(optimized method)and 9.09%(ISO 9648).The possible reason was the sample dilutions could cause great errors and uncertainties.Overall, the optimized method could also meet the precision requirements.

Table 2 Tannin content(W)of sorghum seeds by Ferric ammonium citrate assay.%,dry weight

Fig.3.Regression curves of PA-ML under different extraction solvents in the determination of the Ferric ammonium citrate assay.

4.Conclusion

In this study,sorghum tannins with higher content was extracted and purified as a reference for the standard in the Ferric ammonium citrate assay and the standard was optimized to improve the accuracy of this method.Meanwhile, the extraction solvent was optimized to enhance the safety of this method,and single-lab validations,including repetition rate and recovery, were conducted per criteria set by AOAC International.The study found that PA was superior to TA and EA,as its structure and reaction properties were similar to ST,which could improve the accuracy of quantification of sorghum tannins.Moreover,70%acetone solution was used as a low toxicity substitute of 75% DMF solution to extract sorghum tannins, which could improve the safety of the Ferric ammonium citrate assay.What's more,both the repetition rate and recovery rate of the optimized method met with the standards AOAC International.In summary,the optimization methodcould quantify the content of sorghum tannins more accurately and could be widely used in multi-laboratory.

Table 3 Tannin content(W)of sorghum seeds by Ferric ammonium citrate assay.%,dry weight

Table 4 The recovery rate by Ferric ammonium citrate assay.

Table 5 Interday repeatability experiment of Ferric ammonium citrate assay.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work is a revision of the ISO 9648:1988“Sorghumtannin content determination”,and the authors are grateful for the funding of the ISO/TC34/SC4 and the Construction of Wheat Technology System in Henan Province (S2017-01-G06)special fund.