CN102156174A - Detection of phosphatidylcholine in yolk lecithin by using high performance liquid chromatography - Google Patents
Detection of phosphatidylcholine in yolk lecithin by using high performance liquid chromatography Download PDFInfo
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- CN102156174A CN102156174A CN2011101161416A CN201110116141A CN102156174A CN 102156174 A CN102156174 A CN 102156174A CN 2011101161416 A CN2011101161416 A CN 2011101161416A CN 201110116141 A CN201110116141 A CN 201110116141A CN 102156174 A CN102156174 A CN 102156174A
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Abstract
The invention provides a method for detecting phosphatidylcholine, which is used for determining the chemical composition and quantifying the phosphatidylcholine in yolk lecithin by using a method of using both high performance liquid chromatography and an ultraviolet detector. The method is characterized in that a silicagel column ZORBAX RX-SIL (4.6 mm X 250 mm, 5 microns) manufactured by Agilent is adopted as an analytical column and methanol/acetic acid is used as a mobile phase to carry out isocratic elution, and other elution conditions are an ultraviolet detection wavelength of 200 nm, a column temperature of 30 DEG C, a sample feeding quantity of 15 microliters and a flowing speed of 0.5 ml/min. The mobile phase of the method has the characteristics of binary system, simpler composition, lower flowing speed of 0.5 ml/min, faster peak time within 14 minutes and linear range of a phosphatidylcholine standard curve of 10-160 microgram/ml; the added 3.75% acetic acid solution improves a peak shape of the phosphatidylcholine and effectively inhibits serious tailing phenomenon; and the use cost of a solvent is saved. It is proved by a methodological replication experiment that the method has the advantages of high precision, good stability and higher accuracy.
Description
Technical field
This method belongs to function factor detection method field, relates to the method that adopts high performance liquid chromatography to detect phosphatid ylcholine in the egg yolk lecithin.
Background technology
Lecithin is a kind of phosphorous polarity lipid material, is often referred to the potpourri of different phospholipid fractions such as containing phosphatidyl choline, phosphatidyl-ethanolamine, phosphatidylinositols.Lecithin has important alimentary health-care function.It is biomembranous important component, has to delay senility, and regulates fat metabolism, and the prevention cardiovascular and cerebrovascular disease improves the brain vigor, improves effects such as immunity, is widely used in fields such as food, medicine, cosmetics, feed, coating, agricultural production.Phosphatid ylcholine is a most important function composition in the lecithin.At present more for the research of soybean lecithin, the lecithin in yolk source is in recent years just owing to its content height, oxidation stability better and gradually cause people's attention.The detection method of egg yolk lecithin mainly contains the acetone insoluble matter method, surveys the phosphorus method, ultraviolet spectrophotometry, but can accurately not detect the content of its major function composition phosphatid ylcholine.
High performance liquid chromatography is each component of present separating phospholipids, detects the phosphatid ylcholine content method of broad research and application the most, is divided into normal phase chromatography and reversed phase chromatography.Moving phase commonly used mainly contains ternary systems such as chloroform/methanol/ammoniacal liquor, normal hexane/isopropanol, acetonitrile/methanol/water, acetonitrile/methanol/phosphoric acid.But because phosphatide is a kind of lipid material; constitute by different types of fatty acid and double bond structure; also comprise different molecular speciess even be both phosphatidylcholine molecules; extension, the conditions of streaking at peak appears in regular meeting when liquid phase analysis; even the phosphatid ylcholine component can be told several peaks, specified rate has brought difficulty.And also more complicated of moving phase system at present commonly used.Flow velocity mostly is 1ml/min greatly.From the range of linearity of phosphatid ylcholine typical curve, its minimum concentration mostly is every milliliter of hundreds of microgram greatly when using UV-detector, and required standard items amount is bigger.
Summary of the invention
The technical problem to be solved in the present invention is: remedy moving phase system composition complexity in the liquid phase analysis method that has phosphatid ylcholine now, flow velocity is bigger, the bigger deficiency of required standard items amount when using UV-detector, the method that the invention provides a kind of high performance liquid chromatography and UV-detector coupling is carried out qualitative and quantitative to the phosphatid ylcholine in the egg yolk lecithin.This method moving phase adopts methyl alcohol/acetate binary system, forms simplyr, and the acetic acid solution of adding has improved the peak shape of phosphatid ylcholine, has effectively suppressed serious conditions of streaking.And flow velocity is lower, is 0.5ml/min, guarantee appearance time faster under the prerequisite (in the 14min) reduced the use amount of mobile phase solvent, reduced cost.The maximum absorption wavelength of having determined phosphatid ylcholine in high performance liquid chromatogram-UV-detector system environments is 200nm.The phosphatid ylcholine typical curve range of linearity is 10 μ g/ml～160 μ g/ml.Through the methodology proving test, prove this method precision, have good stability, accuracy is higher.
The technical solution adopted in the present invention is:
(1), the selection of analytical column and moving phase:
C
18Chromatographic column is as one of liquid phase analysis post that is most widely used, also Ceng Zuowei investigation object of the present invention, as moving phase separation detection phosphatid ylcholine, the phosphatid ylcholine standard items are separated several peaks as a result with methyl alcohol/acetonitrile/water system, are unfavorable for quantitatively.Agilent ZORBAX RX-SIL silicagel column is investigated as analytical column in the back, at first investigates normal hexane-isopropyl alcohol system as moving phase, and standard items do not go out the peak, after change methyl alcohol into as moving phase, standard items go out the peak, but because it has many different molecular speciess, serious conditions of streaking therefore occurs.After in methyl alcohol, attempting adding the acetic acid solution of a certain amount of 0.2mol/L, can improve peak shape.And the amount of acetate too hour, peak shape is improved not obvious, when the amount of acetate is too big, though peak shape is obviously improved, the drift phenomenon but the generation baseline makes progress, therefore the last acetic acid aqueous solution of selecting adding 3.75%, promptly the volume ratio of methyl alcohol and acetic acid solution is 96.25: 3.75, this moment, the effect of peak shape and baseline was the most desirable.As shown in Figure 1.
(2) detect wavelength determination:
According to Documentary Records, the ultraviolet maximum absorption wavelength of phosphatid ylcholine in different solutions is in 210nm.Especially concentrate on 204nm～206nm.For avoiding high performance liquid chromatogram and UV-detector system and independently the UV-detector effect is different, the present invention abandons and uses the independently way of the maximum absorption wavelength of UV-detector scanning phosphatid ylcholine in advance, and directly screens its maximum absorption wavelength according to the size of phosphatid ylcholine standard items response in high performance liquid chromatogram and UV-detector system.In the 206nm scope, measure its peak area and peak height response from 198nm under each nano wave length, the result is presented at that phosphatid ylcholine peak area and peak height response all reach maximum under the 200nm.As Fig. 2, shown in Figure 3, determine that therefore the detection wavelength of phosphatid ylcholine is 200nm.
(3) selection of flow rate of mobile phase:
Investigated flow velocity from 0.4ml/min to the 1.0ml/min scope in the phosphatid ylcholine variation that goes out the peak situation.The result shows that the size of flow velocity do not have influence to peak shape, and only influential to going out the peak speed, it is fast that flow velocity greatly then goes out the peak, and flow velocity is little, and then to go out the peak slow.Select less flow velocity 0.5ml/min as technical parameter, both saved the cost that solvent uses, and guaranteed appearance time very fast (in the 14min).
(4) making of typical curve:
With the peak area is index, adopts phosphatid ylcholine content in the external standard method lecithin sample.Accurately draw phosphatid ylcholine standard solution 0.25,1,2,3, the 4ml of 400 μ g/ml, place the volumetric flask of five 10ml respectively, and use the chromatographically pure methanol constant volume, obtain the phosphatid ylcholine standard serial solution that concentration is respectively 10,40,80,120,160 μ g/ml.With its sample introduction under fixed chromatographic condition, measure peak area respectively.With phosphatid ylcholine standard serial solution concentration is horizontal ordinate, is ordinate with the peak area value that responds, and draws peak area-concentration curve, and as shown in Figure 4, the equation of linear regression that obtains is y=17996x-53820, and the coefficient R value is 0.9986.
(5) calculating of methodology proving test and detection limit and quantitative limit:
The investigation of precision repeats sample introduction 6 times with the phosphatid ylcholine standard solution of 120 μ g/ml, statistical study, and the RSD value is 1.1269%, and is as shown in table 1.The investigation of stability, with the phosphatid ylcholine standard solution of 120 μ g/ml respectively 0,2,4,8, the 12h sample introduction, statistical study, the RSD value is 3.0261%, and is as shown in table 2.The investigation of accuracy, carry out the recovery of standard addition test, accurately draw the lecithin sample liquid that 3 parts of each 0.2ml have recorded phosphatid ylcholine concentration, place the volumetric flask of 6 10ml,, use the chromatographically pure methanol constant volume to wherein adding 80,120,160 each 6ml of μ g/ml phosphatid ylcholine titer respectively, sample detection, the recovery is 93.5701%～104.1224%, and average recovery rate is 97.5989%, and is as shown in table 3.The calculating of detection limit and quantitative limit detects and is limited to 3 times of signal to noise ratio (S/N ratio)s, quantitatively is limited to 10 times of signal to noise ratio (S/N ratio)s.Choose a period of time and calculate noise peak area mean value, measure the peak area of 10 μ g/ml phosphatid ylcholines mark product, by the signal to noise ratio (S/N ratio) of this moment, the phosphatid ylcholine when being converted into 3 times and 10 times of signal to noise ratio (S/N ratio)s is marked product concentration, promptly is respectively detection limit and quantitative limit.Record to detect and be limited to 0.5904 μ g/ml, quantitatively be limited to 1.9680 μ g/ml, as shown in table 4.
Table 1 method precision result
Table 2 method stability result
Table 3 recovery of standard addition test findings
The mensuration of table 4 phosphatid ylcholine detection limit and quantitative limit
The invention has the beneficial effects as follows: provide a kind of high performance liquid chromatography and UV-detector coupling to detect the method for phosphatid ylcholine in the egg yolk lecithin.Moving phase adopts simple more binary system, has effectively improved the peak shape of phosphatid ylcholine.Guarantee out the peak faster under the prerequisite flow velocity lower, the use cost of having saved solvent.Also saved the use amount of phosphatid ylcholine standard items.Precision, have good stability, accuracy is higher.
Description of drawings
Fig. 1 adds the same concentration phosphatid ylcholine mark product peak shape contrast of 3.75% acetate system moving phase for methyl alcohol and methyl alcohol;
Fig. 2 be from 198nm to the 206nm scope in the variation of phosphatid ylcholine mark product peak area response;
Fig. 3 be from 198nm to the 206nm scope in the variation of phosphatid ylcholine mark product peak height response;
Fig. 4 is phosphatid ylcholine mark product typical curve;
Fig. 5 is the blank chromatogram of methyl alcohol;
Fig. 6 is phosphatid ylcholine mark product chromatogram;
Fig. 7 is egg yolk lecithin sample chromatogram figure;
Embodiment
Embodiment is for using the content that this method detects phosphatid ylcholine in the egg yolk lecithin.
Accurately take by weighing the egg yolk lecithin sample 0.005g that goes out through acetone and alcohol extract, each 3 parts,, in the 25ml volumetric flask, shake up with the chromatographically pure methanol constant volume, be mixed with sample solution, to be measured.Accurately take by weighing phosphatid ylcholine standard items 0.01g, in the 25ml volumetric flask, be made into the standard reserving solution that phosphatid ylcholine content is 400 μ g/ml, shake up with the chromatographically pure methanol constant volume, stand-by.Prepare the methyl alcohol blank solution simultaneously.
Liquid phase-ultraviolet chromatographic condition is: high performance liquid chromatograph: Tianjin, island LC-2010.Chromatographic column: ZORBAX RX-SIL (Agilent, 4.6mm * 250mm, 5 μ m); Isocratic elution; Moving phase: methyl alcohol/0.2mol/L acetic acid solution (96.25/3.75); Column temperature: 30 ℃; Detecting device: UV-detector; Detect wavelength: 200nm; Sample size: 15 μ L, flow velocity: 0.5ml/min.
Respectively according to above chromatographic condition sample introduction, the spectrogram that obtains is respectively as Fig. 5, Fig. 6, shown in Figure 7 with methyl alcohol blank solution, phosphatid ylcholine standard solution and sample solution.Can determine that peak 5, the peak among Fig. 78 among Fig. 6 are phosphatid ylcholine.Write down the peak area of 3 duplicate samples solution phosphatid ylcholines, average, calculate sample solution phosphatid ylcholine concentration according to equation of linear regression y=17996x-53820, the result is as shown in table 5.The sample solution phosphatid ylcholine concentration of 25ml is 125.4057 μ g/ml, and then the content of phosphatid ylcholine is 62.7029% in the egg yolk lecithin.
Table 5 sample determination result
Claims (5)
1. the detection method of a phosphatid ylcholine, adopt the method for high performance liquid chromatography and UV-detector coupling the phosphatid ylcholine in the egg yolk lecithin to be carried out qualitative and quantitative, it is characterized in that with methyl alcohol/acetate binary system being that moving phase is carried out isocratic elution, flow velocity is 0.5ml/min, the ultraviolet detection wavelength is 200nm, and the range of linearity of phosphatid ylcholine typical curve is 10 μ g/ml～160 μ g/ml.
2. the detection method of phosphatid ylcholine according to claim 1 is characterized in that: when adopting ZORBAX RX-SIL pure silicon glue chromatographic column as analytical column, moving phase is the acetic acid aqueous solution of methyl alcohol and 0.2mol/L, and the volume ratio of the two is 96.25: 3.75.
3. the detection method of phosphatid ylcholine according to claim 1, it is characterized in that: the flow velocity of moving phase is 0.5ml/min.
4. the detection method of phosphatid ylcholine according to claim 1, it is characterized in that: in high performance liquid chromatogram-UV-detector system, the maximum absorption wavelength of phosphatid ylcholine is 200nm.
5. the detection method of phosphatid ylcholine according to claim 1 is characterized in that: this method adopts external standard method quantitative, and the range of linearity of phosphatid ylcholine typical curve is 10 μ g/ml～160 μ g/ml.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680630A (en) * | 2012-05-21 | 2012-09-19 | 天津大学 | Method for analyzing micro-algae phospholipid group |
| CN104007079A (en) * | 2014-04-18 | 2014-08-27 | 深圳赛保尔生物药业有限公司 | Spectral detection method for recombinant human erythropoietin PEGylation reaction liquid and application thereof |
| CN105092719A (en) * | 2014-05-20 | 2015-11-25 | 重庆药友制药有限责任公司 | Method for measuring content of lysophospholipid in multivitamins for injection |
| CN108020602A (en) * | 2016-11-02 | 2018-05-11 | 广东嘉博制药有限公司 | A kind of method of phosphatide and fatty glyceride in Simultaneous Determination pharmaceutical preparation |
| CN108181387A (en) * | 2017-12-20 | 2018-06-19 | 烟台东诚药业集团股份有限公司 | The new method of phospholipid fraction content detection in a kind of Curosurf bulk pharmaceutical chemicals |
| CN109851631A (en) * | 2017-11-30 | 2019-06-07 | 江苏曼氏生物科技股份有限公司 | A kind of isolation and purification method of lecithin in high purity |
| CN112739441A (en) * | 2018-09-21 | 2021-04-30 | 沃特世科技公司 | Systems and methods for lipid quantification |
| CN114137108A (en) * | 2021-11-22 | 2022-03-04 | 安徽元创科技有限公司 | Method for measuring content of phosphatidylcholine in soft capsule by high performance liquid chromatography |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102680630A (en) * | 2012-05-21 | 2012-09-19 | 天津大学 | Method for analyzing micro-algae phospholipid group |
| CN104007079A (en) * | 2014-04-18 | 2014-08-27 | 深圳赛保尔生物药业有限公司 | Spectral detection method for recombinant human erythropoietin PEGylation reaction liquid and application thereof |
| CN104007079B (en) * | 2014-04-18 | 2016-06-29 | 深圳赛保尔生物药业有限公司 | The spectrum detection method of Recombinant Human Erythropoietin pegylation reaction liquid and application |
| CN105092719A (en) * | 2014-05-20 | 2015-11-25 | 重庆药友制药有限责任公司 | Method for measuring content of lysophospholipid in multivitamins for injection |
| CN108020602A (en) * | 2016-11-02 | 2018-05-11 | 广东嘉博制药有限公司 | A kind of method of phosphatide and fatty glyceride in Simultaneous Determination pharmaceutical preparation |
| CN108020602B (en) * | 2016-11-02 | 2021-01-29 | 广东嘉博制药有限公司 | Method for simultaneously and quantitatively measuring phospholipid and fatty glyceride in pharmaceutical preparation |
| CN109851631A (en) * | 2017-11-30 | 2019-06-07 | 江苏曼氏生物科技股份有限公司 | A kind of isolation and purification method of lecithin in high purity |
| CN109851631B (en) * | 2017-11-30 | 2021-11-30 | 江苏曼氏生物科技股份有限公司 | Separation and purification method of high-purity phosphatidylcholine |
| CN108181387A (en) * | 2017-12-20 | 2018-06-19 | 烟台东诚药业集团股份有限公司 | The new method of phospholipid fraction content detection in a kind of Curosurf bulk pharmaceutical chemicals |
| CN112739441A (en) * | 2018-09-21 | 2021-04-30 | 沃特世科技公司 | Systems and methods for lipid quantification |
| CN114137108A (en) * | 2021-11-22 | 2022-03-04 | 安徽元创科技有限公司 | Method for measuring content of phosphatidylcholine in soft capsule by high performance liquid chromatography |
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Application publication date: 20110817 |