CN105021761A - Method for determination of a variety of organic acids in grape juice and authenticity of grape juice - Google Patents

Method for determination of a variety of organic acids in grape juice and authenticity of grape juice Download PDF

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CN105021761A
CN105021761A CN201510387717.0A CN201510387717A CN105021761A CN 105021761 A CN105021761 A CN 105021761A CN 201510387717 A CN201510387717 A CN 201510387717A CN 105021761 A CN105021761 A CN 105021761A
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acid
grape juice
dihydrogen phosphate
content
adp
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CN105021761B (en
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刘睿
夏珺
周密
陈梁
李宝丽
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Huazhong Agricultural University
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Abstract

The present invention discloses a method for simultaneous determination of a variety of organic acids in grape juice. The method is as below: extracting a sample to be detected, and detecting by high performance liquid chromatography containing a DAD detector. the DAD detector has set detection wavelength of 210nm, the HPLC employs a reverse phase chromatographic column, a mobile phase of methanol and ammonium dihydrogen phosphate buffer solution with pH value 2.8 in the volume ratio of 1:99; and the reverse phase column has temperature of 30 DEG C, and a flow rate of 0.8mL / min. The invention also discloses a method for determination of authenticity of grape juice. The method can simultaneously determine the content of eight organic acids in grape juice, has high detection accuracy, good precision and small error, and reaches correct rate of 90% in the identification of authenticity of grape juice.

Description

A kind of method detecting multiple organic acid content and the true and false thereof in grape juice
Technical field
The invention belongs to analytical chemistry and food false distinguishing field, be specifically related to multiple organic acid content assaying method and grape juice authenticity identification method in grape juice.
Background technology
Grape nutritive value is very high; except containing except multiple organic acid, mineral matter, vitamin and essential amino acid; also containing various bioactivators such as resveratrol, procyanidin, anthocyanidin, catechin and flavone compounds; there is the different physiological roles such as scavenging free radicals and antioxidation activity, cardioprotection, raising immunity; therefore, grape juice and grape juice goods have become drink popular in the world in recent years.Under the huge market demand stimulates, the grape juice mixing other fruit juice is pretended to be during the phenomenon of pure grape juice and is found, and therefore, how quick and precisely to differentiate that the true and false of pure grape juice is the active demand of consumer, the producer and quality inspection management organization.
Organic acid is the product of secondary metabolism in fruit growing process, its composition and content are the intrinsic metric attributes of variety classes pure juice, utilize the otherness of organic acid kind and content in pure juice, the finger-print of pure juice Determination of Organic Acids can be set up, in conjunction with chemometrics method adulterated sample analyzed and judge.At present organic acid detection method is mainly contained and comprise reversed-phase liquid chromatography (RP-HPLC) method, ion-exchange chromatography (IEC), Capillary Electrophoresis (HPCE) and gas chromatography.Wherein ion-exchange chromatography has pre-treatment and analyzes multiple organic acid advantage simply, simultaneously, but sometimes occurs the problem of reproducibility of analysis results instability, needs to be solved by the method for multi-detector coupling.Capillary Electrophoresis exists because small molecular organic acid uv absorption is extremely low, and the sensitivity that Conventional UV detects (UV) is not high.And pretreatment process is more complicated during gas Chromatographic Determination organic acid.Reversed-phased high performace liquid chromatographic (RP-HPLC) has the advantage such as quick, sensitive, applicability is wide and is widely deployed and uses.Organic acid is soluble in water, can by the better analysis and identification of RP-HPLC, and in fruit, fruit juice and fruit wine, in organic acid detection, RP-HPLC is widely used at present.But multiple organic acid content in Simultaneously test grape juice, and identify that the method for the grape juice true and false there is not yet bibliographical information based on this.
Summary of the invention
An object of the present invention is to provide the HPLC method of multiple organic acid content in a kind of Simultaneously test grape juice, another object of the present invention is to provide a kind of distinguishing method between true and false of grape juice.
Above-mentioned purpose is achieved through the following technical solutions:
A kind of method of multiple organic acid content in Simultaneously test grape juice, simultaneously it detect tartrate with high performance liquid chromatograph after being extracted by testing sample, malic acid, ascorbic acid, lactic acid, citric acid, acetic acid, fumaric acid, the content of succinic acid, described high performance liquid chromatograph contains DAD detecting device, the setting determined wavelength of described DAD detecting device is 210nm, the chromatographic column of described high performance liquid chromatograph is reversed-phase column, mobile phase to be methyl alcohol and pH be 2.8 ammonium dihydrogen phosphate (ADP) buffer solution, the volume ratio of described methyl alcohol and ammonium dihydrogen phosphate (ADP) buffer solution is 1: 99, the column temperature of described reversed-phase column is 30 DEG C, flow velocity is 0.8mL/min.
Preferably, said method comprising the steps of:
1) precision takes the tartrate, malic acid, ascorbic acid, lactic acid, citric acid, acetic acid, fumaric acid and each 10mg of succinic acid reference substance that are dried to constant weight, 10mL is settled to after dissolving with ultrapure water, with 0.22 μm of filtering with microporous membrane, in contrast product solution;
2) get testing sample 5mL, add methyl alcohol and pH be 2.8 ammonium dihydrogen phosphate (ADP) buffer solution be diluted to 25mL, the volume ratio of described methyl alcohol and ammonium dihydrogen phosphate (ADP) buffer solution is 1: 99, after ultrasonic mixing, centrifugal, get supernatant 1mL through 0.22 μm of filtering with microporous membrane, as sample solution;
3) chromatographic condition and system flexibility: chromatographic column is reversed-phase column, Stationary liquid is polar embedded C18 bonded silica gel; Methyl alcohol and pH be 2.8 ammonium dihydrogen phosphate (ADP) buffer solution be mobile phase, the volume ratio of methyl alcohol and ammonium dihydrogen phosphate (ADP) buffer solution is 1: 99; DAD detecting device, determined wavelength is 210nm; Column temperature 30 DEG C; Flow velocity is 0.8mL/min;
4) accurate absorption reference substance solution and each 10 μ l injection liquid chromatographies of sample solution detect respectively.
A kind of grape juice authenticity identification method, it comprises the following steps:
1) multiple organic acid content in grape juice is measured according to the method described above;
2) content selecting the seven kinds of organic acids (tartrate, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, succinic acid) determined in true grape juice is modeling index, Logistic homing method is adopted to process modeling index, the π value of true grape juice is denoted as 1, the π value of pseudo-grape juice is denoted as 0, can obtain the parameter fitting of Logistic model:
π = exp ( 2.612 + 29.815 x 1 + 3.255 x 2 - 121.056 x 3 - 2.822 x 4 - 14.899 x 5 - 63.457 x 6 + 1.252 x 7 ) 1 + exp ( 2.612 + 29.815 x 1 + 3.255 x 2 - 121.056 x 3 - 2.822 x 4 - 14.899 x 5 - 63.457 x 6 + 1.252 x 7 )
By step 1) content of seven kinds of organic acids (tartrate, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, succinic acid) that records substitutes into above formula, calculate π value, π value be 1 be true grape juice, π value be 0 be pseudo-grape juice, in formula, x 1, x 2, x 3, x 4, x 5, x 6, x 7represent the content of tartrate, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, succinic acid respectively, unit is mg/mL.
The invention has the beneficial effects as follows:
1) the present invention adopts organic acid content in rp-hplc determination grape juice, can measure eight kinds of organic acid content simultaneously, accuracy in detection is high, precision good, error is little, be applicable to organic acid fast qualitative and quantitative test in grape juice.
2) the present invention adopts seven kinds of organic acid content to be modeling index, Logistic homing method is adopted to obtain the parameter fitting of Logistic model, by this model and the organic acid content recorded, can identify the true and false of grape juice exactly, the accuracy of True-false distinguish reaches more than 90%.
Accompanying drawing explanation
Fig. 1 is that the HPLC of reference substance solution detects collection of illustrative plates, in figure: 1. tartrate; 2. malic acid; 3. ascorbic acid; 4. lactic acid; 5. acetic acid; 6. citric acid; 7. fumaric acid; 8. succinic acid.
Fig. 2 is that the HPLC of sample solution detects collection of illustrative plates, in figure: 1. tartrate; 2. malic acid; 3. ascorbic acid; 4. lactic acid; 5. citric acid; 6. fumaric acid; 7. succinic acid.
Embodiment
Below in conjunction with embodiment, the present invention is described in detail.
Embodiment 1
1. detect multiple organic acid content in grape juice simultaneously
A, precision take drying under reduced pressure to the tartrate of permanent quality, malic acid, ascorbic acid, lactic acid, acetic acid, citric acid, fumaric acid and each 10.0mg of succinic acid reference substance, after dissolving with ultrapure water, constant volume is in 10mL volumetric flask, with 0.22 μm of filtering with microporous membrane.
B, get grape juice sample 5mL and add mobile phase and be diluted to 25mL, after ultrasonic mixing, centrifugal (9000rpm/min, 10min), gets supernatant 1mL and obtains sample through 0.22 μm of filtering with microporous membrane.
C, sample obtained to a, b to be analyzed under the following conditions: Thermo Hypersil Gold aQ C18 post (250 × 4.6mm, 5 μm); Mobile phase: methyl alcohol: ammonium dihydrogen phosphate (ADP) buffer solution (0.02mol/L, pH are 2.8)=1: 99 (V/V); Sample size: 10 μ L; Flow velocity: 0.8mL/min; Chromatogram column temperature: 30 DEG C; Detecting device: DAD; Determined wavelength: 210nm.Adopt Gradient elution.
Must peak area be corresponded to, by following formulae discovery organic acid content:
Content (cx)=cr × Ax/Ar
In formula:
Cx----sample concentration mg/mL;
Cr----contrasts concentration mg/mL;
Ax----sample peak area;
Ar----contrasts peak area.
Fig. 1 is shown in by the detection collection of illustrative plates of reference substance solution (containing eight kinds of organic acid standard items).
Fig. 2 is shown in by the detection collection of illustrative plates of sample solution.
2. methodological study
2.1 regretional analyses, detection limit.The results are shown in Table 1.
Table 18 kinds of organic acid regretional analyses, detection limit
2.2 the recovery
Get same two parts, pure grape juice sample, a copy of it is as blank, another part adds a certain amount of standard organic acid mixing reference substance solution, RP-HPLC-DAD analysis is carried out under the chromatographic condition determined, repetitive operation 3 times, calculate recovery of standard addition and relative standard deviation (RSD), the results are shown in Table 2.
Table 28 kinds of organic acid recovery, relative standard deviation (n=3)
8 kinds of organic acid recovery are 95.60%-104.48%, and the recovery is high, and relative standard deviation is all less than 5%, meet the requirement that in grape juice sample, organic acid compound is analyzed.
2.3 precision
Under the liquid phase chromatogram condition determined, carry out RP-HPLC analysis to 8 kinds of organic acid mixing reference substance solution, parallel METHOD FOR CONTINUOUS DETERMINATION 6 times, measures its retention time and peak area, carries out statistical study, calculates relative standard deviation, to investigate precision.The results are shown in Table 3.
Table 38 kinds of organic acid precision (n=6)
The retention time of 8 kinds of organic acid reference substances and the RSD value of integrating peak areas are all less than 5%, and result, without significant change, shows that sample precision is good, is applicable to quantitative test.
The above results shows that set up RP-HPLC-DAD analytical approach is quick, accurate, sensitive, is applicable to organic acid qualitative and quantitative analysis in grape juice.
Embodiment 2
1. the foundation of grape juice false distinguishing model
The organic acid content existed in pure grape juice is designated as X1, X2, X3 respectively ... Xn, then Logistic regression function is:
π = P ( Y = 1 | X 1 = x 1 , ... X n = x n ) = exp ( β 0 + β 1 x 1 + ... + β n x n ) 1 + exp ( β 0 + β 1 x 1 + ... + β n x n ) , The π value of pure grape juice is denoted as 1, and the π value of the pure grape juice of non-pre-packaged is denoted as 0, obtains the parameter fitting of Logistic model: π = exp ( 2.612 + 29.815 x 1 + 3.255 x 2 - 121.056 x 3 - 2.822 x 4 - 14.899 x 5 - 63.457 x 6 + 1.252 x 7 ) 1 + exp ( 2.612 + 29.815 x 1 + 3.255 x 2 - 121.056 x 3 - 2.822 x 4 - 14.899 x 5 - 63.457 x 6 + 1.252 x 7 ) , Wherein, x 1, x 2, x 3, x 4, x 5, x 6, x 7represent the content (unit: mg/mL) of tartrate, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, succinic acid respectively.
The checking of 2.Logistic model
The grape juice of different volumes ratio (basic, normal, high) cider is mixed as checking sample using 10 of market stochastic buying parts of blind samples (comprising pure grape juice and grape juice beverage) and self-control, after carrying out HPLC detection, related data is obtained to wherein organic acid, to calculate its π value, Logistic model is verified.
To 13 verification sample random number.Pure grape juice and the blind sample of grape juice beverage and make adulterated sample by oneself and be numbered 59-71.Wherein the blind sample of pure grape juice is 59,64,68,70,71, and the blind sample of pre-packaged grape juice beverage is 60,61,63,66,67, and the grape juice sample number into spectrum mixing different volumes ratio (basic, normal, high) cider is 62,65,69.Analyzed the content drawing sample mesotartaric acid, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, succinic acid by HPLC, bring above-mentioned equation into, calculate its π value, to model preliminary test, the results are shown in Table 4.Result shows, except the erroneous results of No. 68 samples, the identification result of all the other 12 samples is all correct, and the accuracy of the grape juice true and false is 92.31% to adopt this method to differentiate.
Seven kinds of organic acid content detection data and model inspection table in table 4 13 samples
Numbering x 1 x 2 x 3 x 4 x 5 x 6 x 7 π value
59 1.01 2.57 0.05 0.76 0.41 0.03 2.66 1.00
60 0.98 1.87 0.38 1.17 0.22 0.28 1.17 0.00
61 0.61 1.97 0.24 0.12 0.51 0.71 0.05 0.00
62 0.97 1.69 0.28 0.29 0.20 0.29 0.04 0.00
63 0 0.34 0.05 0 1.29 0 0.27 0.00
64 0.99 2.41 0.09 1.05 0.66 0.18 0.73 1.00
65 0.82 1.76 0.29 0.18 0.29 0.41 0.12 0.00
66 0.64 0.65 0.54 0.02 1.20 0.04 0.12 0.00
67 0.49 0.69 0.65 0.08 2.60 0.05 0.02 0.00
68 1.36 0.35 0.14 0.01 2.50 0.01 0.10 0.00
69 0.59 1.85 0.30 0.23 0.32 0.24 0.06 0.00
70 1.61 1.47 0.14 0.12 0.61 0.21 0.05 1.00
71 0.77 2.52 0.05 0.77 0.36 0.02 0.15 1.00

Claims (3)

1. the method for multiple organic acid content in a Simultaneously test grape juice, it is characterized in that simultaneously it detect tartrate with high performance liquid chromatograph after being extracted by testing sample, malic acid, ascorbic acid, lactic acid, citric acid, acetic acid, fumaric acid, the content of succinic acid, described high performance liquid chromatograph contains DAD detecting device, the setting determined wavelength of described DAD detecting device is 210nm, the chromatographic column of described high performance liquid chromatograph is reversed-phase column, mobile phase to be methyl alcohol and pH be 2.8 ammonium dihydrogen phosphate (ADP) buffer solution, the volume ratio of described methyl alcohol and ammonium dihydrogen phosphate (ADP) buffer solution is 1: 99, the column temperature of described reversed-phase column is 30 DEG C, flow velocity is 0.8mL/min.
2. the method for multiple organic acid content in Simultaneously test grape juice as claimed in claim 1, is characterized in that comprising the following steps:
1) precision takes the tartrate, malic acid, ascorbic acid, lactic acid, citric acid, acetic acid, fumaric acid and each 10mg of succinic acid reference substance that are dried to constant weight, 10mL is settled to after dissolving with ultrapure water, with 0.22 μm of filtering with microporous membrane, in contrast product solution;
2) get testing sample 5mL, add methyl alcohol and pH be 2.8 ammonium dihydrogen phosphate (ADP) buffer solution be diluted to 25mL, the volume ratio of described methyl alcohol and ammonium dihydrogen phosphate (ADP) buffer solution is 1: 99, after ultrasonic mixing, centrifugal, get supernatant 1mL through 0.22 μm of filtering with microporous membrane, as sample solution;
3) chromatographic condition and system flexibility: chromatographic column is reversed-phase column, Stationary liquid is polar embedded C18 bonded silica gel; Methyl alcohol and pH be 2.8 ammonium dihydrogen phosphate (ADP) buffer solution be mobile phase, the volume ratio of methyl alcohol and ammonium dihydrogen phosphate (ADP) buffer solution is 1: 99; DAD detecting device, determined wavelength is 210nm; Column temperature 30 DEG C; Flow velocity is 0.8mL/min;
4) accurate absorption reference substance solution and each 10 μ l injection liquid chromatographies of sample solution detect respectively.
3. a grape juice authenticity identification method, is characterized in that comprising the following steps:
1) multiple organic acid content in grape juice is measured according to the method described in claim 1 or 2;
2) with determine in true grape juice seven kinds of organic acid contents for modeling index, Logistic homing method is adopted to process modeling index, the π value of true grape juice is denoted as 1, and the π value of pseudo-grape juice is denoted as 0, can obtain the parameter fitting of Logistic model:
π = exp ( 2.612 + 29.815 x 1 + 3.255 x 2 - 121.056 x 3 - 2.822 x 4 - 14.899 x 5 - 63.457 x 6 + 1.252 x 7 ) 1 + exp ( 2.612 + 29.815 x 1 + 3.255 x 2 - 121.056 x 3 - 2.822 x 4 - 14.899 x 5 - 63.457 x 6 + 1.252 x 7 )
By step 1) seven kinds of organic acid content recording substitute into above formulas, calculate π value, π value be 1 be true grape juice, π value be 0 be pseudo-grape juice, in formula, x 1, x 2, x 3, x 4, x 5, x 6, x 7represent the content of tartrate, malic acid, ascorbic acid, lactic acid, citric acid, fumaric acid, succinic acid respectively, unit is mg/mL.
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CN106248847A (en) * 2016-07-08 2016-12-21 江苏今世缘酒业股份有限公司 A kind of method of seven kinds of organic acid contents in wine brewing material of mensuration simultaneously
CN107505407A (en) * 2017-07-19 2017-12-22 贵州茅台酒股份有限公司 It is a kind of while determine lactic acid in white wine fermented grain, the method for acetic acid content
CN107328937A (en) * 2017-08-24 2017-11-07 璞晞（广州）生物免疫技术有限公司 For the construction method for the practicality model for evaluating hepatitis B hepatopathy NK cell functions
CN109765309A (en) * 2018-12-27 2019-05-17 辽宁三生科技发展有限公司 A kind of method of effective component in separation detection citric acid disinfectant
CN110672766A (en) * 2019-10-17 2020-01-10 中国科学院山西煤炭化学研究所 C in Fischer-Tropsch synthetic oil1-C4Method for detecting content of organic acid

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