Determination of phenolic compounds in kiwifruit juice by UPLC

Abstract:【Objective】The similar chemical structures and properties of phenolic compounds make it difficult to separate phenolic compounds. At the same time, there are many problems in the detection of phenolics, such as low separation degree and being a very time-consuming process. In order to frerquently, rapidly and simultaneously analyze the phenolic compounds in kiwifruit juice, a method was developed for the qualitative and quantitative analysis of 22 phenolic compounds (gallic acid, coumalic acid, protocatechuic acid, neochlorogenic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, caffeic acid, syringic acid, (-) -epigallocatechin gallate, (-) -epicatechin, 1, 3-dicaffeoylquinic acid, p-coumaric acid, ferulic acid, sinapic acid, rutin, quercitrin, resveratrol, phlorizin, quercetin, cinnamic acid and phloretin) by ultra high performance liquid chromatography (UPLC) , and the phenolic compounds in kiwifruit juice of 4 cultivars were determined in order to provide technical support for the development of phenolic substances in kiwifruit juice.【Methods】The UPLC experiments were conducted using an ACQUITY UPLC BEH C18 phase column (2.1 mm×100 mm, 1.7 μm) . Samples were maintained at 35 ℃ using a flow rate of 0.2 m L · min-1 and a sample injection volume of 0.6 μL. A detector wave length of 280 nm was used. The mobile phase consisted of 2% (V/V) acetic acid in water (A) and acetonitrile (B) . The gradient elution conditions were as follows: isocratic elution 95% A, 0-2 min; linear gradient from 95% A to 90% A, 2-4 min; isocratic elution 90% A, 4-6 min; linear gradient from 90% A to 85% A, 6-8 min;isocratic elution 85% A, 8-12 min; linear gradient from 85% A to 83% A, 12-16 min; linear gradient from 83% A to 80% A, 16-18 min; linear gradient from 80% A to 70% A, 18-19 min; isocratic elution70% A, 19-23 min; linear gradient from 70% A to 25% A, 23-26 min; linear gradient from 25% A to 0%A, 26-29 min; isocratic elution 0% A, 29-31 min; linear gradient from 0% A to 70% A, 31-32 min; and linear gradient from 70% A to 95% A, 32-33 min.【Results】This study took into account the optimiza-tion of the analysis conditions, including comparison of the effects of the mobile phase, detection wavelength, sample injection volume, column temperature, flow rate and choice of elution gradient. The detection wavelength chosen was 280 nm. The mixture of phenolic compounds had maximum peaks and maximum response values of 280 nm. The excess or too little volumes of sample injections can affect the shape of the peaks, separation degrees and the baseline. 0.6 μL was considered the proper sample injection volume. 35 ℃ was used as the column temperature. When the flow rate increases, the time of the sample analysis can be shortened, but the separation results of the sample components will be worse when the velocity of the flow is too fast. 0.2 m L · min-1 was considered the proper flow rate. The retention time, peak shape and recovery tests were used to verify the presence of the targeted analytes in the samples. Limits of detection (LODs) ranged from 0.000 34 μg·m L^-1 to 0.16 μg·m L^-1 and the recovery rate ranged from 84.11% to 117.15%. The mean accuracy of the relative peak area was 1.68% (ranging from1.05% to 4.07%) . The stability of the method was evaluated by using Xuxiang juice. The stability, and the RSD of peak area was not more than 7.52%, which indicated good stability. The repeatability of this method was evaluated by using‘Xuxiang'juice. The repeatability, and the mean RSD of peak area was less than 4.88%, which showed a good repeatability. The applicability of this analytical approach was confirmed by the successful analysis of real samples of kiwifruit juices. The kiwifruit juice consisted of13 kinds of phenolic compounds, with ferulic acid, (-) -epigallocatechin, sinapic acid, rutin resveratrol, phlorizin quercetin, cinnamic acid and phloretin not being found in any of the cultivars of the kiwifruit juice. Gallic acid, neochlorogenic acid, p-hydroxybenzoic acid, catechin, chlorogenic acid, syringic acid, 1, 3-dicaffeoyiquinic acid, quercitrin were detected in all the cultivars of the kiwifruit juice. Among the 4 cultivars of kiwifruit juice, ‘Huangjinguo'‘Huangyang'and‘Xuxiang'kiwifruit juice had the most content of catechin (4.35, 4.52 and 9.32 μg · m L^-1 respectively) . The most content of phenolic compounds in the‘Hayward'kiwifruit juice was chlorogenic acid (2.25 μg·m L^-1) . Protocatechic acid was only detected in‘Hayward'kiwifruit juice, caffeic acid was not detected in‘Huangyang'kiwifruit juice, and (-) -epicatechin was not detected in‘Xuxiang'kiwifruit juice. The chromatogram of kiwifruit juice showed that this UPLC method can be used in the analysis of phenolic in kiwifruit juice and most of the chromatographic peaks were totally separated. The richness, variety and complexity of the phenolic compounds of kiwifruit juice present an extensive applicability for analyzing the constitution and content of phenolic.【Conclusion】By using this method, 22 kinds of phenolic compounds can be completely separated within 33 min with separating degrees of greater than 1.5. This method showed excellent accuracy, repeatability and stability. It was also confirmed that this method can be used for the analysis of phenolic composition in kiwifruit juice.