CN104458942B - The detection method of impurity in Choline Glycerophosphate - Google Patents

The detection method of impurity in Choline Glycerophosphate Download PDF

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CN104458942B
CN104458942B CN201410698223.XA CN201410698223A CN104458942B CN 104458942 B CN104458942 B CN 104458942B CN 201410698223 A CN201410698223 A CN 201410698223A CN 104458942 B CN104458942 B CN 104458942B
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choline glycerophosphate
solution
liquid
present
choline
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CN104458942A (en
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傅超婷
云冲
刘五生
李锦云
朱峰
王玉娟
李富志
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HAINAN GENERAL SANYANG PHARMACEUTICAL CO Ltd
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HAINAN GENERAL SANYANG PHARMACEUTICAL CO Ltd
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Abstract

The present invention is that the mixed solution of the ammonium formate solution of 0.0005 ~ 0.002mol/L and acetonitrile is as mobile phase A using volumetric molar concentration, be that the mixed solution of the ammonium formate solution of 0.03 ~ 0.05mol/L and acetonitrile is as Mobile phase B using volumetric molar concentration, adopt binary gradient elution program, first liquid chromatographic detection is carried out to Choline Glycerophosphate testing sample solution, obtains the content of impurity glycerine, Choline Chloride and glycerophosphatide acyl inositol; Adopt acetonitrile and water as mobile phase, the detection of second liquid phase chromatogram is carried out to Choline Glycerophosphate testing sample, obtains the content of impurity L-ALPHA-GPE.Detection method provided by the invention can establish different chromatographic conditions and detect for different impurity, thus obtain the content of whole known impurities, avoid and only measure the one-sidedness that partial impurities just judges Choline Glycerophosphate quality, achieve comprehensively, effectively monitor the object of Choline Glycerophosphate quality.

Description

The detection method of impurity in Choline Glycerophosphate
Technical field
The invention belongs to Pharmaceutical Analysis field, particularly relate to the detection method of impurity in Choline Glycerophosphate.
Background technology
Senile dementia is a kind of progressive neurodegenerative deficiency disorder, and its main manifestations is memory function decay and recognition capability obstacle, simultaneously with various nervous symptoms and behavior disorder.Research finds, China's senile dementia incidence of disease remains high, and wherein in 65 years old old man, obvious dement accounts for 2% ~ 5%, and in the old man of more than 85 years old, almost half suffers from senile dementia in various degree.Along with the quickening of world population ages's speed, senile dementia has become the fourth-largest cause of disease jeopardizing the elderly's life, has had a strong impact on the healthy of the elderly and quality of life.Therefore, the research and development of anti-dementia medicine cause the great attention of countries in the world the world of medicine.
Research shows, the pathogenesis of senile dementia and the shortage of neurotransmitter acetylcholine closely related, if the level of acetylcholine in brain can be improved, just can improve the symptom of senile dementia.Choline Glycerophosphate can pass blood-brain barrier, synthesis for acetylcholine provides necessary choline source, impel the synthesis of acetylcholine, add the content of the acetylcholine in brain between nerve synapse, thus hippocampus, Cerebral cortex and amygdaloid body are associated with basal forebrain areas, extend the effect of cholinergic recepter, reach the object for the treatment of senile dementia.Choline Glycerophosphate is evident in efficacy in treatment senile dementia, few side effects, and has good tolerance and security, is a kind of medicine very with market outlook.
But, Choline Glycerophosphate is in the process of producing and store, due to the impact of different technological factors and environmental factor (as temperature, oxidation and illumination etc.), likely can produce some impurity, directly can affect the quality of medicine, so, need a kind of analytical approach, can detect the contamination of impurity various in Choline Glycerophosphate, thus reach the object controlling Choline Glycerophosphate quality.Also there is no the method that effectively can detect impurity in Choline Glycerophosphate at present.
Summary of the invention
The object of the invention is to provide the detection method of impurity in Choline Glycerophosphate, and in Choline Glycerophosphate provided by the invention, the detection method of impurity effectively can detect impurity in Choline Glycerophosphate and content thereof, realizes the object controlling Choline Glycerophosphate quality.
The invention provides the detection method of impurity in Choline Glycerophosphate, comprise the following steps:
Choline Glycerophosphate testing sample solution is carried out the first liquid chromatographic detection, obtains the first liquid chromatogram of Choline Glycerophosphate testing sample solution;
The mobile phase of described first liquid chromatographic detection comprises mobile phase A and Mobile phase B; Described mobile phase A comprises the first ammonium formate solution and acetonitrile, and the volumetric molar concentration of described first ammonium formate solution is 0.0005 ~ 0.002mol/L, and the volume ratio of described first ammonium formate solution and acetonitrile is (1 ~ 10): (99 ~ 90);
Described Mobile phase B comprises the second ammonium formate solution and acetonitrile, and the volumetric molar concentration of described second ammonium formate solution is 0.03 ~ 0.05mol/L, and the volume ratio of described second ammonium formate solution and acetonitrile is (35 ~ 45): (65 ~ 55);
According to the first typical curve of described first liquid chromatogram and predetermined standard model solution, obtain the content of the first impurity in Choline Glycerophosphate testing sample, described first impurity comprises glycerine, Choline Chloride and glycerophosphatide acyl inositol;
Choline Glycerophosphate testing sample solution is carried out the detection of second liquid phase chromatogram, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution, the mobile phase of described second liquid phase chromatogram comprises acetonitrile and water, and the volume ratio of described acetonitrile and water is (72 ~ 78): (28 ~ 22);
According to the second typical curve of described second liquid phase chromatogram and predetermined standard model solution, obtain the content of the second impurity in Choline Glycerophosphate testing sample, described second impurity comprises L-ALPHA-GPE.
Preferably, the chromatographic column in described first liquid chromatographic detection is cation exchange column;
The column temperature of described first liquid chromatographic detection chromatographic column is 20 ~ 30 DEG C;
Preferably, in described first liquid chromatographic detection, the flow velocity of mobile phase A is 0.95 ~ 1.05mL/min;
The flow velocity of described Mobile phase B is 0.95 ~ 1.05mL/min.
Preferably, described first liquid chromatographic detection adopts gradient elution program;
Described gradient elution program is:
In < 35min, the volume ratio of mobile phase A and Mobile phase B is (93 ~ 97): (7 ~ 3);
>=35 and in < 65min, the volume ratio of mobile phase A and Mobile phase B is (45 ~ 55): (55 ~ 45);
>=65 and in < 70min, the volume ratio of mobile phase A and Mobile phase B is (35 ~ 44): (65 ~ 56);
>=70 and in < 120min, the volume ratio of mobile phase A and Mobile phase B is (0 ~ 5): (100 ~ 95).
Preferably, the pH value of described mobile phase A is 2.0 ~ 4.0;
The pH value of described Mobile phase B is 4.0 ~ 6.0.
Preferably, in described second liquid phase chromatogram detection, the flow velocity of mobile phase is 0.95 ~ 1.05mL/min.
Preferably, in described second liquid phase chromatogram detection, the pH value of mobile phase is 2.0 ~ 4.0.
Preferably, the chromatographic column in described second liquid phase chromatogram detection is amino bonded silica gel chromatographic column;
During described second liquid phase chromatogram detects, the column temperature of chromatographic column is 20 ~ 40 DEG C.
Preferably, the detecting device of described first liquid chromatographic detection is evaporative light-scattering detector;
In described first liquid chromatographic detection, the sprayer temperature of evaporative light-scattering detector is 70 ~ 90 DEG C;
In described first liquid chromatographic detection, the nebulizer gas pressure of evaporative light-scattering detector is 0.1 ~ 0.2MPa.
Preferably, the detecting device that described second liquid phase chromatogram detects is evaporative light-scattering detector;
During described second liquid phase chromatogram detects, the sprayer temperature of evaporative light-scattering detector is 70 ~ 90 DEG C;
During described second liquid phase chromatogram detects, the nebulizer gas pressure of evaporative light-scattering detector is 0.1 ~ 0.2MPa.
The present invention is that the mixed solution of the ammonium formate solution of 0.0005 ~ 0.002mol/L and acetonitrile is as mobile phase A using volumetric molar concentration, be that the mixed solution of the ammonium formate solution of 0.03 ~ 0.05mol/L and acetonitrile is as Mobile phase B using volumetric molar concentration, adopt binary gradient elution program, first liquid chromatographic detection is carried out to Choline Glycerophosphate testing sample solution, obtains the content of glycerine in Choline Glycerophosphate, Choline Chloride and glycerophosphatide acyl inositol; Adopt acetonitrile and water as mobile phase, the detection of second liquid phase chromatogram is carried out to Choline Glycerophosphate testing sample solution, obtains the content of L-ALPHA-GPE in Choline Glycerophosphate.Detection method provided by the invention can establish different chromatographic conditions and detect for different impurity, thus obtain the content of whole known impurities, avoid and only measure the one-sidedness that partial impurities just judges Choline Glycerophosphate quality, achieve the object of comprehensive, effective monitoring Choline Glycerophosphate quality, meanwhile, the quality monitoring standard of Choline Glycerophosphate is also improved.
Experimental result shows, detection method provided by the invention effectively can detect the content of whole known impurities, and after repeated detection, the retention time of same substance is substantially constant, illustrates that detection method provided by the invention has good repeatability and stability.
Accompanying drawing explanation
Fig. 1 is Choline Glycerophosphate mixed solution first liquid chromatogram that the embodiment of the present invention 1 obtains;
Fig. 2 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 2 obtains;
Fig. 3 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 3 obtains;
Fig. 4 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 4 obtains;
Fig. 5 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 5 obtains;
Fig. 6 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 6 obtains;
Fig. 7 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 7 obtains;
Fig. 8 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 8 obtains;
Fig. 9 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 9 obtains;
Figure 10 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 10 obtains;
Figure 11 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 11 obtains;
Figure 12 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 12 obtains;
First liquid chromatogram of Figure 13 to be signal to noise ratio (S/N ratio) that the embodiment of the present invention 13 obtains be Choline Glycerophosphate testing sample solution first time sample introduction of 3:1;
First liquid chromatogram of Figure 14 to be signal to noise ratio (S/N ratio) that the embodiment of the present invention 13 obtains be Choline Glycerophosphate testing sample solution second time sample introduction of 3:1;
First liquid chromatogram of Figure 15 to be signal to noise ratio (S/N ratio) that the embodiment of the present invention 13 obtains be Choline Glycerophosphate testing sample solution third time sample introduction of 3:1;
Figure 16 is first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 20 DEG C that the embodiment of the present invention 14 obtains;
Figure 17 is first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 25 DEG C that the embodiment of the present invention 15 obtains;
Figure 18 is first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 30 DEG C that the embodiment of the present invention 16 obtains;
The first liquid chromatogram that Figure 19 is the Choline Glycerophosphate mixed solution that obtains of the embodiment of the present invention 17 when the flow velocity of mobile phase A and Mobile phase B is 0.95mL/min;
The first liquid chromatogram that Figure 20 is the Choline Glycerophosphate mixed solution that obtains of the embodiment of the present invention 18 when the flow velocity of mobile phase A and Mobile phase B is 1.05mL/min;
Figure 21 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution of placement after 0 hour that the embodiment of the present invention 19 obtains;
Figure 22 is the first liquid chromatogram of placement Choline Glycerophosphate testing sample solution after 2 hours that the embodiment of the present invention 20 obtains;
Figure 23 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution of placement after 4 hours that the embodiment of the present invention 21 obtains;
Figure 24 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution of placement after 8 hours that the embodiment of the present invention 22 obtains;
Figure 25 is the second standard liquid chromatograph figure of the Choline Glycerophosphate standard model solution that the embodiment of the present invention 23 obtains;
Figure 26 is the second standard liquid chromatograph figure of the Choline Chloride standard model solution that the embodiment of the present invention 24 obtains;
Figure 27 is the second standard liquid chromatograph figure of the glycerophosphatide acyl inositol standard model solution that the embodiment of the present invention 25 obtains;
Figure 28 is the second standard liquid chromatograph figure of the L-ALPHA-GPE standard model solution that the embodiment of the present invention 26 obtains;
Figure 29 is the second standard liquid chromatograph figure of the auxiliary material standard model solution that the embodiment of the present invention 27 obtains;
Figure 30 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution a that the embodiment of the present invention 28 obtains;
Figure 31 is the second liquid phase chromatogram of the blank solvent that the embodiment of the present invention 29 obtains;
Figure 32 is the second liquid phase chromatogram of the L-ALPHA-GPE standard model solution that the embodiment of the present invention 30 obtains;
Figure 33 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution that comparative example 1 of the present invention obtains;
Figure 34 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution that comparative example 2 of the present invention obtains;
Figure 35 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution that comparative example 3 of the present invention obtains;
Figure 36 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution that comparative example 4 of the present invention obtains;
Figure 37 is the second liquid phase chromatogram of the Choline Chloride sample solution that comparative example 5 of the present invention obtains;
Figure 38 is the second liquid phase chromatogram of the glycerophosphatide acyl inositol sample solution that comparative example 6 of the present invention obtains;
Figure 39 is the second liquid phase chromatogram of the L-ALPHA-GPE sample solution that comparative example 7 of the present invention obtains;
Figure 40 is the second liquid phase chromatogram of the auxiliary material sample solution that comparative example 8 of the present invention obtains;
Figure 41 is the second liquid phase chromatogram that Choline Glycerophosphate that comparative example 9 of the present invention obtains mixes sample solution;
Figure 42 is the second liquid phase chromatogram of placement Choline Glycerophosphate testing sample solution after 0 hour that the embodiment of the present invention 31 obtains;
Figure 43 is the second liquid phase chromatogram of placement Choline Glycerophosphate testing sample solution after 2 hours that the embodiment of the present invention 32 obtains;
Figure 44 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 4 hours that the embodiment of the present invention 33 obtains;
Figure 45 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 6 hours that the embodiment of the present invention 34 obtains;
Figure 46 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 8 hours that the embodiment of the present invention 35 obtains;
Figure 47 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 12 hours that the embodiment of the present invention 36 obtains;
Figure 48 is the second liquid phase chromatogram of the blank solvent that the embodiment of the present invention 37 obtains;
Figure 49 is the second liquid phase chromatogram of the auxiliary material sample solution that the embodiment of the present invention 38 obtains;
Figure 50 is the first liquid chromatogram of the blank solvent that the embodiment of the present invention 39 obtains;
Figure 51 is first liquid chromatogram of the Choline Glycerophosphate contrast solution a that the embodiment of the present invention 40 obtains;
Figure 52 is first liquid chromatogram of the Choline Glycerophosphate testing sample solution a that the embodiment of the present invention 40 obtains;
Figure 53 is the second liquid phase chromatogram of the Choline Glycerophosphate contrast solution a that the embodiment of the present invention 40 obtains;
Figure 54 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution a that the embodiment of the present invention 40 obtains;
Figure 55 is first liquid chromatogram of the Choline Glycerophosphate contrast solution b that the embodiment of the present invention 41 obtains;
Figure 56 is first liquid chromatogram of the Choline Glycerophosphate testing sample solution b that the embodiment of the present invention 41 obtains;
Figure 57 is the second liquid phase chromatogram of the Choline Glycerophosphate contrast solution b that the embodiment of the present invention 41 obtains;
Figure 58 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution b that the embodiment of the present invention 41 obtains;
Figure 59 is first liquid chromatogram of the Choline Glycerophosphate contrast solution c that the embodiment of the present invention 42 obtains;
Figure 60 is first liquid chromatogram of the Choline Glycerophosphate testing sample solution c that the embodiment of the present invention 42 obtains;
Figure 61 is the second liquid phase chromatogram of the Choline Glycerophosphate contrast solution c that the embodiment of the present invention 42 obtains;
Figure 62 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution c that the embodiment of the present invention 42 obtains;
Figure 63 is the first liquid chromatogram of the commercially available Choline Glycerophosphate contrast solution that the embodiment of the present invention 43 obtains;
Figure 64 is the first liquid chromatogram of the commercially available Choline Glycerophosphate testing sample solution that the embodiment of the present invention 43 obtains
Figure 65 is the second liquid phase chromatogram of the commercially available Choline Glycerophosphate contrast solution that the embodiment of the present invention 43 obtains;
Figure 66 is the second liquid phase chromatogram of the commercially available Choline Glycerophosphate testing sample solution that the embodiment of the present invention 43 obtains.
Embodiment
The invention provides the detection method of impurity in Choline Glycerophosphate, comprise the following steps:
Choline Glycerophosphate testing sample solution is carried out the first liquid chromatographic detection, obtains the first liquid chromatogram of Choline Glycerophosphate testing sample solution;
The mobile phase of described first liquid chromatographic detection comprises mobile phase A and Mobile phase B; Described mobile phase A comprises the first ammonium formate solution and acetonitrile, and the volumetric molar concentration of described first ammonium formate solution is 0.0005 ~ 0.002mol/L, and the volume ratio of described first ammonium formate solution and acetonitrile is (1 ~ 10): (99 ~ 90);
Described Mobile phase B comprises the second ammonium formate solution and acetonitrile, and the volumetric molar concentration of described second ammonium formate solution is 0.03 ~ 0.05mol/L, and the volume ratio of described second ammonium formate solution and acetonitrile is (35 ~ 45): (65 ~ 55);
According to the first typical curve of described first liquid chromatogram and predetermined standard model solution, obtain the content of the first impurity in Choline Glycerophosphate testing sample, described first impurity comprises glycerine, Choline Chloride and glycerophosphatide acyl inositol;
Choline Glycerophosphate testing sample solution is carried out the detection of second liquid phase chromatogram, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution, the mobile phase of described second liquid phase chromatogram comprises acetonitrile and water, and the volume ratio of described acetonitrile and water is (72 ~ 78): (28 ~ 22);
According to the second typical curve of described second liquid phase chromatogram and predetermined standard model solution, obtain the content of the second impurity in Choline Glycerophosphate testing sample, described second impurity comprises L-ALPHA-GPE.
Choline Glycerophosphate testing sample solution is carried out the first liquid chromatographic detection by the present invention, obtains the first liquid chromatogram of Choline Glycerophosphate testing sample solution.Choline Glycerophosphate testing sample preferably mixes with water by the present invention, obtains Choline Glycerophosphate testing sample solution.In the present invention, the mass concentration of described Choline Glycerophosphate testing sample solution is preferably 40 ~ 60g/L, is more preferably 50g/L.
The sample size of described first liquid chromatographic detection is preferably 15 ~ 25 μ L, is more preferably 17 ~ 23 μ L, most preferably is 20 μ L.
In the present invention, the chromatographic column of described first liquid chromatographic detection is preferably cation exchange column, and the filler of described chromatographic column is preferably silica gel, concrete, can use Shiseido CAPCELLPAKSCX liquid-phase chromatographic column; The chromatographic column column temperature of described first liquid chromatographic detection is preferably 20 ~ 30 DEG C, is more preferably 22 ~ 37 DEG C, most preferably is 25 DEG C;
In the present invention, the mobile phase of described first liquid chromatographic detection comprises mobile phase A and Mobile phase B.In the present invention, described mobile phase A comprises the first ammonium formate solution and acetonitrile, and the concentration of described first ammonium formate solution is 0.0005 ~ 0.002mol/L, is preferably 0.0008 ~ 0.0015mol/L, is more preferably 0.001mol/L; The volume ratio of described first ammonium formate solution and acetonitrile is (1 ~ 10): (99 ~ 90), is preferably (1 ~ 5): (99 ~ 95), are more preferably 1:99; The pH value of described mobile phase A is preferably 2.0 ~ 4.0, is more preferably 2.5 ~ 3.5, most preferably is 3.0; The present invention preferably adopts the pH value of acid medium to described mobile phase A to regulate, and described acid medium is preferably formic acid; The present invention preferably adopts acid solution as acid medium, and the present invention does not have special restriction to the concentration of described acid solution and consumption, the pH value of described mobile phase A can be adjusted to above-mentioned scope.
In the present invention, the flow velocity of described mobile phase A is preferably 0.95 ~ 1.05mL/min, is more preferably 0.98 ~ 1.02mL/min, most preferably is 1mL/min.
In the present invention, described Mobile phase B comprises the second ammonium formate solution and acetonitrile, and the concentration of described second ammonium formate solution is 0.03 ~ 0.05mol/L, is preferably 0.035 ~ 0.045mol/L, is more preferably 0.04mol/L; The volume ratio of described second ammonium formate solution and acetonitrile is (35 ~ 45): (65 ~ 55), is preferably (38 ~ 43): (62 ~ 57), are more preferably 40:60; The pH value of described Mobile phase B is preferably 4.0 ~ 6.0, is more preferably 4.5 ~ 5.5, most preferably is 5.0; The present invention preferably adopts the pH value of acid medium to described Mobile phase B to regulate, and described acid medium is preferably formic acid; The present invention preferably adopts acid solution as acid medium, and the present invention does not have special restriction to the concentration of described acid solution and consumption, the pH value of described Mobile phase B can be adjusted to above-mentioned scope.
In the present invention, the flow velocity of described Mobile phase B is preferably 0.95 ~ 1.05mL/min, is more preferably 0.98 ~ 1.02mL/min, most preferably is 1mL/min.
In the present invention, described first liquid chromatographic detection preferably adopts gradient elution program, and described gradient elution program is preferably:
In < 35min, the volume ratio of mobile phase A and Mobile phase B is (93 ~ 97): (7 ~ 3);
>=35 and in < 65min, the volume ratio of mobile phase A and Mobile phase B is (45 ~ 55): (55 ~ 45);
>=65 and in < 70min, the volume ratio of mobile phase A and Mobile phase B is (35 ~ 44): (65 ~ 56);
>=70 and in < 120min, the volume ratio of mobile phase A and Mobile phase B is (0 ~ 5): (100 ~ 95).
Be more preferably:
In < 35min, the volume ratio of mobile phase A and Mobile phase B is (94 ~ 96): (6 ~ 4);
>=35 and in < 65min, the volume ratio of mobile phase A and Mobile phase B is (47 ~ 53): (53 ~ 47);
>=65 and in < 70min, the volume ratio of mobile phase A and Mobile phase B is (47 ~ 53): (53 ~ 47);
>=70 and in < 120min, the volume ratio of mobile phase A and Mobile phase B is (0 ~ 3): (100 ~ 97);
Most preferably be:
In < 35min, the volume ratio of mobile phase A and Mobile phase B is 95:5;
>=35 and in < 65min, the volume ratio of mobile phase A and Mobile phase B is 50:50;
>=65 and in < 70min, the volume ratio of mobile phase A and Mobile phase B is 40:60;
>=70 and in < 120min, the volume ratio of mobile phase A and Mobile phase B is 0:100.
The detecting device of described first liquid chromatographic detection is preferably evaporative light-scattering detector, and in described first liquid chromatographic detection, the drift tube temperature of evaporative light-scattering detector is preferably 60 ~ 100 DEG C, is more preferably 70 ~ 90 DEG C, most preferably is 80 DEG C; In described first liquid chromatographic detection, evaporative light-scattering detector preferred nitrogen is as atomization gas; The pressure of described atomization gas is preferably 0.1 ~ 0.2MPa, is more preferably 0.13 ~ 0.18MPa, most preferably is 0.15MPa; The flow velocity of described atomization gas is preferably 2 ~ 3L/min, is more preferably 2.5L/min; In described first liquid chromatographic detection, the sprayer temperature of evaporative light-scattering detector is preferably 70 ~ 90 DEG C, is more preferably 80 DEG C; .
After obtaining the first liquid chromatogram of Choline Glycerophosphate testing sample solution, the present invention is according to the first typical curve of the first liquid chromatogram of described Choline Glycerophosphate testing sample solution and predetermined standard model solution, obtain the content of the first impurity in Choline Glycerophosphate testing sample, described first impurity comprises glycerine, Choline Chloride and glycerophosphatide acyl inositol.Before the present invention preferably quantitatively detects described first impurity, carrying out qualitative to the chromatographic peak in described Choline Glycerophosphate testing sample solution first liquid chromatogram, namely positioning going out peak position;
The standard model solution of described first impurity is preferably carried out the first liquid chromatographic detection by the present invention, obtain the first liquid chromatogram of the first impurity, the retention time of chromatographic peak in the retention time of chromatographic peak in the first liquid chromatogram of described first impurity and described Choline Glycerophosphate testing sample solution first liquid chromatogram is compared, realizes the qualitative of chromatographic peak in described Choline Glycerophosphate testing sample solution first liquid chromatogram.
In the present invention, described first typical curve is the linearity curve between the mass concentration of the first impurity chromatographic peak area corresponding with it, and described first typical curve preferably obtains in accordance with the following methods:
First standard model solution of preparation series concentration, described first standard model is the standard model of the first impurity;
First standard model solution of described series concentration is carried out the first liquid chromatographic detection, obtains the first standard liquid chromatograph figure of series;
The concentration of the first corresponding with it for the chromatographic peak area of the first impurity in the first standard liquid chromatograph figure of described series contamination levels sample is carried out linear fit, obtains the first typical curve.
In the present invention, described first standard model solution comprises glycerine standard model solution, Choline Chloride standard model solution and glycerophosphatide acyl inositol standard model solution.In the present invention, described first standard model solution can be each component independently standard model solution, also can be the hybrid standard sample solution of each component.The present invention is preferably by water-soluble for the sterling of glycerine, and preparation obtains the glycerine standard model solution of series concentration.The source of the present invention to described glycerine sterling does not have special restriction, adopts the commercial goods of glycerine sterling.In the present invention, the mass concentration of described glycerine standard model solution is preferably 1 ~ 5mg/mL, is more preferably 2 ~ 4mg/mL, most preferably is 3mg/mL.
After obtaining the glycerine standard model solution of described series concentration, the glycerine standard model solution of the series concentration obtained preferably is carried out the first liquid chromatographic detection by the present invention, obtains the series first standard liquid chromatograph figure of glycerine standard model solution.
After obtaining the series first standard liquid chromatograph figure of described glycerine standard model solution, the present invention preferably with the peak area in the series first standard liquid chromatograph figure of described glycerine standard model solution for ordinate, with the mass concentration of the glycerine standard model solution of its correspondence for horizontal ordinate, obtain the curve of peak area and mass concentration, described curve is carried out linear fit, obtains the first typical curve of glycerine standard model solution.In the present invention, described peak area preferably adopts integration to obtain, and the method for the present invention to described integration does not have special restriction, adopts point system well known to those skilled in the art.The method of the present invention to described linear fit does not have special restriction, adopts the method for linear fit well known to those skilled in the art.
The present invention is preferably according to technique scheme, obtain the first typical curve of the standard model solution of other known impurities in described first standard model solution except glycerine, comprise the first typical curve of Choline Chloride standard model solution and the first typical curve of glycerophosphatide acyl inositol standard model solution, unlike, in the present invention, the mass concentration of described Choline Chloride standard model solution is preferably 0.05 ~ 0.45g/L, and the mass concentration of described glycerophosphatide acyl inositol standard model solution is preferably 0.05 ~ 0.45g/L.
After obtaining the first typical curve of described first standard model solution, the present invention, according to described first typical curve, obtains the mass concentration of the first impurity in described Choline Glycerophosphate testing sample solution; According to the quality of the quality of described Choline Glycerophosphate testing sample, described Choline Glycerophosphate testing sample solution, the mass concentration with the first impurity in described Choline Glycerophosphate testing sample solution, obtain the content of the first impurity in described Choline Glycerophosphate testing sample, described first impurity comprises glycerine.Choline Chloride and glycerophosphatide acyl inositol.
Except known glycerine, Choline Chloride and glycerophosphatide acyl inositol, in described first impurity, preferably also comprise unknown impuritie.The impurity that described unknown impuritie produces under being included in the conditions such as acid, alkali and illumination.Described Choline Glycerophosphate sample is preferably carried out the detection of first principal component own control by the present invention, to obtain the content of unknown impuritie in described Choline Glycerophosphate sample, comprises the following steps:
1) Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution are provided;
2) by described step 1) in Choline Glycerophosphate contrast solution injection liquid chromatography, regulate chromatograph sensitivity;
3) by described step 1) in Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution carry out the first liquid chromatographic detection respectively, obtain the first liquid chromatogram of described Choline Glycerophosphate testing sample solution and the first liquid chromatogram of Choline Glycerophosphate contrast solution;
4) according to the first liquid chromatogram of described Choline Glycerophosphate testing sample solution and the first liquid chromatogram of Choline Glycerophosphate contrast solution, the content of unknown impuritie in Choline Glycerophosphate testing sample solution is obtained.
In the present invention, the source of described Choline Glycerophosphate testing sample solution is consistent with the source of Choline Glycerophosphate testing sample solution in mass concentration and technique scheme and mass concentration, does not repeat them here.Described Choline Glycerophosphate testing sample solution preferably dilutes by the present invention, obtains Choline Glycerophosphate contrast solution.In the present invention, described Choline Glycerophosphate contrast solution is preferably 1:100 with the ratio of the mass concentration of described Choline Glycerophosphate testing sample solution.
After obtaining described Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution, the present invention is by described Choline Glycerophosphate contrast solution injection liquid chromatography, regulate sensitivity, make the peak height of major component chromatographic peak be 10 ~ 25% of full scale, be preferably 20%.In the present invention, the sample size of described Choline Glycerophosphate contrast solution is preferably 20 μ L.
After adjustment of sensitivity, the present invention is by described step 1) in Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution carry out the first liquid chromatographic detection respectively, obtain the first liquid chromatogram of described Choline Glycerophosphate testing sample solution and the first liquid chromatogram of Choline Glycerophosphate contrast solution.In first principal component own control of the present invention detects, be preferably 2 times of major component chromatographic peak retention time the writing time of the first liquid chromatogram of described Choline Glycerophosphate testing sample solution; If any glycerine, glycerophosphatide acyl inositol and Choline Chloride impurity peaks in first liquid chromatogram of described Choline Glycerophosphate testing sample solution, its peak area must not be greater than the half of main peak area in the first liquid chromatogram of described Choline Glycerophosphate contrast solution, namely must not be greater than 0.5% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution; Other arbitrary impurity peaks peak areas must not be greater than 1/5 of main peak area in the first liquid chromatogram of described Choline Glycerophosphate contrast solution, namely must not be greater than 0.2% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution; Each impurity peak area sum must not be greater than the twice of main peak area in the first liquid chromatogram of described Choline Glycerophosphate contrast solution, namely must not be greater than 2.0% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution.
The present invention preferably according to the technical scheme of the first typical curve of above-mentioned acquisition glycerine standard model solution, obtains the first typical curve of Choline Glycerophosphate standard model solution.The peak area that the present invention is corresponding according to Choline Glycerophosphate in the first typical curve of the Choline Glycerophosphate standard model solution obtained and the first liquid chromatogram of described Choline Glycerophosphate testing sample solution, obtains the content of Choline Glycerophosphate in described Choline Glycerophosphate testing sample.
After obtaining the first liquid chromatogram of described Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution, the present invention is according to the content of Choline Glycerophosphate in described Choline Glycerophosphate testing sample, the ratio of described Choline Glycerophosphate contrast solution and the mass concentration of described Choline Glycerophosphate testing sample solution, the peak area of Choline Glycerophosphate in the peak area of unknown impuritie and described Choline Glycerophosphate contrast solution first liquid chromatogram in first liquid chromatogram of described Choline Glycerophosphate testing sample solution, obtain the content of unknown impuritie in described Choline Glycerophosphate testing sample.
Choline Glycerophosphate testing sample solution is carried out the detection of second liquid phase chromatogram by the present invention, obtains the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution.Choline Glycerophosphate testing sample preferably mixes with water by the present invention, obtains Choline Glycerophosphate testing sample solution.In the present invention, the mass concentration of described Choline Glycerophosphate testing sample solution is preferably 40 ~ 60g/L, is more preferably 50g/L.
In the present invention, the sample size that described second liquid phase chromatogram detects is preferably 15 ~ 25 μ L, is more preferably 17 ~ 23 μ L, most preferably is 20 μ L.
In the present invention, the chromatographic column that described second liquid phase chromatogram detects is preferably amino bonded silica gel chromatographic column; The chromatographic column column temperature that described second liquid phase chromatogram detects is preferably 20 ~ 40 DEG C, is more preferably 30 DEG C.
In the present invention, the mobile phase that described second liquid phase chromatogram detects comprises acetonitrile and water, the volume ratio of described acetonitrile and water is (72 ~ 78): (28 ~ 22), is preferably (73 ~ 77): (27 ~ 23), are more preferably 75:25; During described second liquid phase chromatogram detects, the pH value of mobile phase is preferably 2.0 ~ 4.0, is more preferably 2.5 ~ 3.5, most preferably is 3.0; The present invention preferably adopts the pH value of acid medium to the mobile phase that described second liquid phase chromatogram detects to regulate, and described acid medium is preferably glacial acetic acid; The present invention preferably adopts acid solution as acid medium, and the present invention does not have special restriction to the concentration of described acid solution and consumption, and in described second liquid phase chromatogram can being detected, the pH value of mobile phase is adjusted to above-mentioned scope.
In the present invention, during described second liquid phase chromatogram detects, the flow velocity of mobile phase is preferably 0.95 ~ 1.05mL/min, is more preferably 0.98 ~ 1.03mL/min, most preferably is 1mL/min.
In the present invention, the detecting device that described second liquid phase chromatogram detects is preferably evaporative light-scattering detector, and during described second liquid phase chromatogram detects, the drift tube temperature of evaporative light-scattering detector is preferably 60 ~ 100 DEG C, is more preferably 70 ~ 90 DEG C, most preferably is 80 DEG C; Evaporative light-scattering detector preferred nitrogen during described second liquid phase chromatogram detects is as atomization gas; The pressure of described atomization gas is preferably 0.1 ~ 0.2MPa, is more preferably 0.13 ~ 0.18MPa, most preferably is 0.15MPa; The flow velocity of described atomization gas is preferably 2 ~ 3L/min, is more preferably 2.5L/min; During described second liquid phase chromatogram detects, the sprayer temperature of evaporative light-scattering detector is preferably 70 ~ 90 DEG C, is more preferably 80 DEG C;
After obtaining the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution, the present invention is according to the second typical curve of the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution and the second predetermined standard model solution, obtain the content of the second impurity in Choline Glycerophosphate testing sample solution, described second impurity comprises L-ALPHA-GPE.Before the present invention preferably quantitatively detects described second impurity, carrying out qualitative to the chromatographic peak in described Choline Glycerophosphate testing sample solution second liquid phase chromatogram, namely positioning going out peak position;
The standard model solution of described second impurity is preferably carried out the detection of second liquid phase chromatogram by the present invention, obtain the second liquid phase chromatogram of the second impurity, the retention time of chromatographic peak in the retention time of chromatographic peak in the second liquid phase chromatogram of described second impurity and described Choline Glycerophosphate testing sample solution second liquid phase chromatogram is compared, realizes the qualitative of chromatographic peak in described Choline Glycerophosphate testing sample solution second liquid phase chromatogram.
In the present invention, described second typical curve is the linearity curve between the mass concentration of the second impurity chromatographic peak area corresponding with it, and described second typical curve preferably obtains in accordance with the following methods:
Second standard model solution of preparation series concentration, described second standard model is the standard model of the second impurity;
Second standard model solution of described series concentration is carried out the detection of second liquid phase chromatogram, obtains the serial second liquid phase chromatogram of the second standard model solution;
The concentration of the second corresponding with it for the chromatographic peak area of the second impurity in the series second standard liquid chromatograph figure of described second standard model solution contamination levels sample is carried out linear fit, obtains the second typical curve.
In the present invention, described second standard model solution comprises L-ALPHA-GPE standard model solution.In the present invention, source and the mass concentration of the source of described L-ALPHA-GPE standard model solution and mass concentration and L-ALPHA-GPE standard model solution in technique scheme are consistent, do not repeat them here.
After obtaining the second standard model solution of described series concentration, the series concentration L-ALPHA-GPE sample solution obtained preferably is carried out the detection of second liquid phase chromatogram by the present invention, obtains the series second standard liquid chromatograph figure of L-ALPHA-GPE sample solution.
After obtaining the series second standard liquid chromatograph figure of described L-ALPHA-GPE, the present invention preferably with the peak area in the series second standard liquid chromatograph figure of described L-ALPHA-GPE standard model solution for ordinate, with the mass concentration of the L-ALPHA-GPE standard model solution of its correspondence for horizontal ordinate, obtain the curve of peak area and mass concentration, described curve is carried out linear fit, obtains the typical curve of L-ALPHA-GPE.In the present invention, described peak area preferably adopts integration to obtain, and the method for the present invention to described integration does not have special restriction, adopts point system well known to those skilled in the art.The method of the present invention to described linear fit does not have special restriction, adopts the method for linear fit well known to those skilled in the art.
After obtaining the typical curve of described L-ALPHA-GPE, the present invention can obtain the mass concentration of L-ALPHA-GPE in described Choline Glycerophosphate testing sample solution according to the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution and the typical curve of described L-ALPHA-GPE; According to the mass concentration of L-ALPHA-GPE in the quality of described Choline Glycerophosphate testing sample, the quality of described Choline Glycerophosphate testing sample solution and described Choline Glycerophosphate testing sample solution, obtain the content of the second impurity in described Choline Glycerophosphate testing sample, described second impurity comprises L-ALPHA-GPE.
Except known impurities L-ALPHA-GPE, described second impurity also comprises unknown impuritie.The impurity that described unknown impuritie produces under being included in the conditions such as acid, alkali and illumination.Described Choline Glycerophosphate sample is preferably carried out the detection of Second principal component, own control by the present invention, to obtain the content of unknown impuritie in described Choline Glycerophosphate sample, comprises the following steps:
I) Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution are provided;
II) by described step I) in Choline Glycerophosphate contrast solution injection liquid chromatography, regulate chromatograph sensitivity;
III) by described step I) in Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution carry out the detection of second liquid phase chromatogram respectively, obtain the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution;
IV) according to the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution and the second liquid phase chromatogram of Choline Glycerophosphate contrast solution, the content of unknown impuritie in Choline Glycerophosphate testing sample solution is obtained.
The present invention obtains Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution according to technique scheme, and according to technique scheme by described Choline Glycerophosphate contrast solution injection liquid chromatography, regulates sensitivity.
After adjustment of sensitivity, the present invention is by described step I) in Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution carry out the detection of second liquid phase chromatogram respectively, obtain the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution and the second liquid phase chromatogram of Choline Glycerophosphate contrast solution.In described Second principal component, own control detects, if any L-ALPHA-GPE impurity peaks in the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution, its peak area must not be greater than 0.5 times of the second liquid phase chromatogram main peak area of described Choline Glycerophosphate contrast solution, namely must not be greater than 0.5% of the second liquid phase chromatogram main peak area of described Choline Glycerophosphate testing sample solution.
The present invention preferably according to the technical scheme of above-mentioned acquisition second typical curve, obtains the second typical curve of Choline Glycerophosphate standard model solution.The peak area that the present invention is corresponding according to Choline Glycerophosphate in the second typical curve of the Choline Glycerophosphate standard model solution obtained and the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution, obtains the content of Choline Glycerophosphate in described Choline Glycerophosphate testing sample.
After obtaining the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution and Choline Glycerophosphate contrast solution, the present invention is according to the content of Choline Glycerophosphate in described Choline Glycerophosphate testing sample, the ratio of described Choline Glycerophosphate contrast solution and the mass concentration of described Choline Glycerophosphate testing sample solution, the peak area of Choline Glycerophosphate in the peak area of unknown impuritie and described Choline Glycerophosphate contrast solution second liquid phase chromatogram in the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution, obtain the content of unknown impuritie in described Choline Glycerophosphate testing sample.
Detection method provided by the invention can not only detect the content of known impurities glycerine in Choline Glycerophosphate, Choline Chloride, glycerophosphatide acyl inositol and L-ALPHA-GPE, the content of issuable unknown impuritie under the conditions such as acid, alkali and illumination can also be detected, avoid and only measure the one-sidedness that partial impurities just judges Choline Glycerophosphate quality, achieve comprehensively, effectively monitor the object of Choline Glycerophosphate quality.
In order to further illustrate the present invention, below in conjunction with embodiment, the detection method of impurity in Choline Glycerophosphate provided by the invention being described in detail, but they can not being interpreted as limiting the scope of the present invention.
In the following embodiments, acetonitrile used is purchased from MREDATECHNOLOGYINC company, and purity is chromatographically pure, ammonium formate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group, and purity is pure for analyzing, and formic acid is purchased from Beijing Chemical Plant, purity is pure for analyzing, and acetic acid is purchased from Beijing Chemical Plant, and purity is pure for analyzing.
Embodiment 1
Choline Glycerophosphate, Choline Chloride, glycerophosphatide acyl inositol, hungry area softgel shell and glycerine are mixed with water, filter after mixing, obtain Choline Glycerophosphate mixed solution A, containing Choline Glycerophosphate 50mg, Choline Chloride 0.25mg, glycerophosphatide acyl inositol 0.25mg, hungry area softgel shell 50mg, glycerine 3mg in every 1mL Choline Glycerophosphate mixed solution.
Get Choline Glycerophosphate mixed solution A described in 20 μ L, inject Shimadzu liquid chromatograph, adopt Alltech2000ES type evaporative light-scattering detector to detect.
Shimadzu liquid chromatograph adopts LC-10ATvp type pump, CBM-20A type controller, CLASS-VP type chromatographic work station;
The drift tube temperature of Alltech2000ES type evaporative light-scattering detector is, sprayer temperature is 80 DEG C, and atomization gas is nitrogen, and atomization gas flow velocity is 2.5L/min, and nebulizer gas pressure is 0.15MPa;
Chromatographic condition is: Shiseido CAPCELLPAKSCX chromatographic column, chromatographic column filler grain size 5 μm, and column size is 4.6 × 250mm; Adopt binary gradient elution program, as shown in table 1, table 1 is the binary gradient elution program that the embodiment of the present invention 1 adopts.Acetonitrile and the volumetric molar concentration of mobile phase A to be volume ratio be 99:1 are 0.001mol/L ammonium formate solution, and flow velocity is 1.0mL/min, is 3.0 by formic acid adjust ph; Acetonitrile and the volumetric molar concentration of Mobile phase B to be volume ratio be 60:40 are 0.04mol/L ammonium formate solution, and flow velocity is 1.0mL/min, is 5.0 by formic acid adjust ph;
The binary gradient elution program that table 1 embodiment of the present invention 1 adopts
Time (min) Mobile phase A (%) Mobile phase B (%)
0.01 95 5
7.0 95 5
35.0 50 50
65.0 40 60
70.0 0 100
120.0 0 100
Obtain the first liquid chromatogram of Choline Glycerophosphate mixed solution A, as shown in Figure 1, Fig. 1 is Choline Glycerophosphate mixed solution A first liquid chromatogram that the embodiment of the present invention 1 obtains.As seen from Figure 1, it is good that Choline Glycerophosphate is adjacent magazins' layout degree, and it is good that known impurities is adjacent peak degree of separation.
The present invention calculates according to the first liquid chromatogram of the Choline Glycerophosphate mixed solution A that the present embodiment obtains, obtain the data such as the retention time of impurity in Choline Glycerophosphate, result is as shown in table 2, and table 2 is the retention time of each component in the Choline Glycerophosphate mixed solution A that obtains of the embodiment of the present invention 1.
The retention time of each component in the Choline Glycerophosphate mixed solution A that table 2 embodiment of the present invention 1 obtains
Prepare respectively according to technique scheme and obtain Choline Glycerophosphate mixed solution B, Choline Glycerophosphate mixed solution C, Choline Glycerophosphate mixed solution D and Choline Glycerophosphate mixed solution E.Containing Choline Glycerophosphate 30mg, Choline Chloride 0.05mg, glycerophosphatide acyl inositol 0.05mg, hungry area softgel shell 30mg, glycerine 1mg in every 1mL Choline Glycerophosphate mixed solution B; Containing Choline Glycerophosphate 40mg, Choline Chloride 0.15mg, glycerophosphatide acyl inositol 0.15mg, hungry area softgel shell 40mg, glycerine 2mg in every 1mL Choline Glycerophosphate mixed solution C; Containing Choline Glycerophosphate 60mg, Choline Chloride 0.35mg, glycerophosphatide acyl inositol 0.35mg, hungry area softgel shell 60mg, glycerine 4mg in every 1mL Choline Glycerophosphate mixed solution D; Containing Choline Glycerophosphate 70mg, Choline Chloride 0.45mg, glycerophosphatide acyl inositol 0.45mg, hungry area softgel shell 70mg, glycerine 5mg in every 1mL Choline Glycerophosphate mixed solution E.
According to the detection method in technique scheme, liquid chromatographic detection is carried out to the Choline Glycerophosphate mixed solution B obtained, Choline Glycerophosphate mixed solution C, Choline Glycerophosphate mixed solution D and Choline Glycerophosphate mixed solution E, obtains first liquid chromatogram of Choline Glycerophosphate mixed solution B, Choline Glycerophosphate mixed solution C, Choline Glycerophosphate mixed solution D and Choline Glycerophosphate mixed solution E respectively.
The present invention, according to the peak area of each component and the mass concentration of corresponding component in first liquid chromatogram of the Choline Glycerophosphate mixed solution A obtained, Choline Glycerophosphate mixed solution B, Choline Glycerophosphate mixed solution C, Choline Glycerophosphate mixed solution D and Choline Glycerophosphate mixed solution E, obtains the typical curve of each component in Choline Glycerophosphate mixed solution.
Embodiment 2
Get Choline Glycerophosphate sample 500mg, be placed in 10ml volumetric flask, be dissolved in water and constant volume, rock evenly, obtain Choline Glycerophosphate testing sample solution.
Measure 20 μ L Choline Glycerophosphate testing sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, obtain the first liquid chromatogram of Choline Glycerophosphate testing sample solution, as shown in Figure 2, Fig. 2 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 2 obtains.Can be obtained the retention time of major impurity in Choline Glycerophosphate sample by Fig. 2, result is as shown in table 3, and table 3 is the retention time of major impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 2,3,5,7,9 and 11.
Embodiment 3
Get Choline Glycerophosphate sample 500mg, be placed in 10ml volumetric flask, add the hydrochloric acid solution 2ml of 1mol/L, place in the water-bath of 40 DEG C after 1 hour, solution in volumetric flask is neutralized to neutrality by the sodium hydroxide solution adding 1mol/L, is then dissolved in water and constant volume, rocks evenly and filters, get subsequent filtrate, obtain Choline Glycerophosphate testing sample solution.
Measure 20 μ L Choline Glycerophosphate testing sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, the Mobile phase B acetonitrile in the present embodiment and the volume ratio of ammonium formate solution adopt 35:65.Obtain the first liquid chromatogram of Choline Glycerophosphate testing sample solution, as shown in Figure 3, Fig. 3 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 3 obtains.Can be obtained the retention time of major impurity in Choline Glycerophosphate sample by Fig. 3, result is as shown in table 3, and table 3 is the retention time of major impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 2,3,5,7,9 and 11.
Embodiment 4
Get glycerine 250mg, be placed in 10ml volumetric flask, add the hydrochloric acid solution 2ml of 1mol/L, place in the water-bath of 40 DEG C after 1 hour, solution in volumetric flask is neutralized to neutrality by the sodium hydroxide solution adding 1mol/L, is then dissolved in water and constant volume, rocks evenly and filters, get subsequent filtrate, obtain auxiliary material sample solution.
Measure 20 μ L auxiliary material sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the Mobile phase B in the present embodiment, the volume ratio of acetonitrile and ammonium formate solution adopts 45:55.Obtain the first liquid chromatogram of auxiliary material sample solution, as shown in Figure 4, Fig. 4 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 4 obtains.
Embodiment 5
Get Choline Glycerophosphate sample 500mg, be placed in 10ml volumetric flask, add the sodium hydroxide solution 2ml of 1mol/L, place in the water-bath of 40 DEG C after 30 minutes, solution in volumetric flask is neutralized to neutrality by the hydrochloric acid solution adding 1mol/L, is then dissolved in water and constant volume, rocks evenly and filters, get subsequent filtrate, obtain Choline Glycerophosphate testing sample solution.
Measure 20 μ L Choline Glycerophosphate testing sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the pH value of mobile phase A adopts 2.0.Obtain the first liquid chromatogram of Choline Glycerophosphate testing sample solution, as shown in Figure 5, Fig. 5 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 5 obtains.Can be obtained the retention time of major impurity in Choline Glycerophosphate sample by Fig. 5, result is as shown in table 3, and table 3 is the retention time of major impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 2,3,5,7,9 and 11.
Embodiment 6
Get glycerine 500mg, be placed in 10ml volumetric flask, add the sodium hydroxide solution 2ml of 1mol/L, place in the water-bath of 40 DEG C after 30 minutes, solution in volumetric flask is neutralized to neutrality by the hydrochloric acid solution adding 1mol/L, is then dissolved in water and constant volume, rocks evenly and filters, get subsequent filtrate, obtain auxiliary material sample solution.
Measure 20 μ L auxiliary material sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the pH value of mobile phase A adopts 4.0.Obtain the first liquid chromatogram of auxiliary material sample solution, as shown in Figure 6, Fig. 6 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 6 obtains.
Embodiment 7
Get Choline Glycerophosphate sample 500mg, be placed in 10ml volumetric flask, add the hydrogen peroxide 4ml that massfraction is 30%, 60 DEG C of water-baths place 4 hours, are dissolved in water and constant volume.Rock evenly and filter, get subsequent filtrate, obtain Choline Glycerophosphate testing sample solution.
Measure 20 μ L Choline Glycerophosphate testing sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the pH value of Mobile phase B adopts 4.0.Obtain the first liquid chromatogram of Choline Glycerophosphate testing sample solution, as shown in Figure 7, Fig. 7 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 7 obtains.Can be obtained the retention time of major impurity in Choline Glycerophosphate sample by Fig. 7, result is as shown in table 3, and table 3 is the retention time of major impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 2,3,5,7,9 and 11.
Embodiment 8
Get glycerine sample 500mg, be placed in 10ml volumetric flask, add the hydrogen peroxide 4ml that massfraction is 30%, 60 DEG C of water-baths place 4 hours, are dissolved in water and constant volume.Rock evenly and filter, get subsequent filtrate, obtain auxiliary material sample solution.
Measure 20 μ L auxiliary material sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the pH value of Mobile phase B adopts 6.0.Obtain the first liquid chromatogram of auxiliary material sample solution, as shown in Figure 8, Fig. 8 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 8 obtains.
Embodiment 9
Get Choline Glycerophosphate sample 500mg, be placed in 10ml volumetric flask, place 36 hours under 105 DEG C of conditions, be dissolved in water and constant volume.Shake up rear filtration, get subsequent filtrate, obtain Choline Glycerophosphate testing sample solution.
Measure 20 μ L Choline Glycerophosphate testing sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the sprayer temperature of evaporative light-scattering detector adopts 70 DEG C.Obtain the first liquid chromatogram of Choline Glycerophosphate testing sample solution, as shown in Figure 9, Fig. 9 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 9 obtains.Can be obtained the retention time of major impurity in Choline Glycerophosphate sample by Fig. 9, result is as shown in table 3, and table 3 is the retention time of major impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 2,3,5,7,9 and 11.
Embodiment 10
Get glycerine 500mg, be placed in 10ml volumetric flask, place 36 hours under 105 DEG C of conditions, be dissolved in water and constant volume.Shake up rear filtration, get subsequent filtrate, obtain auxiliary material sample solution.
Measure 20 μ L auxiliary material sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the sprayer temperature of evaporative light-scattering detector adopts 90 DEG C.Obtain the first liquid chromatogram of auxiliary material sample solution, as shown in Figure 10, Figure 10 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 10 obtains.
Embodiment 11
Get Choline Glycerophosphate sample 500mg, be placed in 10ml volumetric flask, place 12 hours under 4500LX light-illuminating, be dissolved in water and constant volume.Shake up rear filtration, get subsequent filtrate, obtain Choline Glycerophosphate testing sample solution.
Measure 20 μ L Choline Glycerophosphate testing sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the nebulizer gas pressure of evaporative light-scattering detector adopts 0.1MPa.Obtain the first liquid chromatogram of Choline Glycerophosphate testing sample solution, as shown in figure 11, Figure 11 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 11 obtains.Can be obtained the retention time of major impurity in Choline Glycerophosphate sample by Figure 11, result is as shown in table 3, and table 3 is the retention time of major impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 2,3,5,7,9 and 11.
Embodiment 12
Get glycerine sample 500mg, be placed in 10ml volumetric flask, place 12 hours under 4500LX light-illuminating, be dissolved in water and constant volume.Shake up rear filtration, get subsequent filtrate, obtain auxiliary material sample solution.
Measure 20 μ L auxiliary material sample solutions, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the nebulizer gas pressure of evaporative light-scattering detector adopts 0.2MPa.Obtain the first liquid chromatogram of auxiliary material sample solution, as shown in figure 12, Figure 12 is the first liquid chromatogram of the auxiliary material sample solution that the embodiment of the present invention 12 obtains.
The retention time of major impurity in the Choline Glycerophosphate testing sample solution that table 3 embodiment of the present invention 2,3,5,7,9 and 11 obtains
From table 3 result, this product all has degradation product to produce under highly basic, strong acid, oxidation, high temperature, illumination condition, and this product is relatively more responsive to highly basic, strong acid, illumination.Highly basic, strong acid, high temperature, oxidation and illumination destroy the impurity of degrading out in sample chromatogram figure, are all separated well with main peak.
Embodiment 13
Choline Glycerophosphate sample is mixed with water, obtains the Choline Glycerophosphate testing sample solution that mass concentration is 0.01mg/mL respectively;
The Choline Glycerophosphate testing sample solution obtained is carried out the detection of signal to noise ratio (S/N ratio), be Choline Glycerophosphate testing sample solution continuous sample introduction three pin of 3:1 by signal to noise ratio (S/N ratio), the sample size of three pins is 20 μ L.According to the liquid chromatography detecting method in the embodiment of the present invention 1, the Choline Glycerophosphate testing sample solution that signal to noise ratio (S/N ratio) is 3:1 is detected, unlike, in the present embodiment, mobile phase A adopts volumetric molar concentration to be the ammonium formate solution of 0.0005mol/L.Obtain the first liquid chromatogram that signal to noise ratio (S/N ratio) is the Choline Glycerophosphate testing sample solution of 3:1, as shown in Figure 13 ~ 15, the first liquid chromatogram of Figure 13 to be signal to noise ratio (S/N ratio) that the embodiment of the present invention 13 obtains be Choline Glycerophosphate testing sample solution first time sample introduction of 3:1; First liquid chromatogram of Figure 14 to be signal to noise ratio (S/N ratio) that the embodiment of the present invention 13 obtains be Choline Glycerophosphate testing sample solution second time sample introduction of 3:1; First liquid chromatogram of Figure 15 to be signal to noise ratio (S/N ratio) that the embodiment of the present invention 13 obtains be Choline Glycerophosphate testing sample solution third time sample introduction of 3:1.The minimum detectable quantity being obtained the Choline Glycerophosphate of the first liquid chromatographic detection by Figure 13 ~ 15 is 0.2 μ g.
Embodiment 14 ~ 16
Measure the Choline Glycerophosphate mixed solution that the 20 μ L embodiment of the present invention 1 obtain respectively, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, the chromatographic column column temperature of the embodiment of the present invention 14 ~ 16 is respectively 20 DEG C, 25 DEG C and 30 DEG C, obtains first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 20 DEG C, 25 DEG C and 30 DEG C.As shown in Figure 16 ~ 18, Figure 16 is first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 20 DEG C that the embodiment of the present invention 14 obtains, Figure 17 is first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 25 DEG C that the embodiment of the present invention 15 obtains, and Figure 18 is first liquid chromatogram of Choline Glycerophosphate mixed solution at column temperature 30 DEG C that the embodiment of the present invention 16 obtains.As can be seen from Figure 16 ~ 18, the Choline Glycerophosphate mixed solution that the embodiment of the present invention 14 ~ 16 obtains is under the column temperature of 20 ~ 30 DEG C, and main peak and impurity peaks, the degree of separation of auxiliary material peak and impurity peaks all conforms with the regulations.
Embodiment 17 ~ 18
Measure the Choline Glycerophosphate mixed solution that the 20 μ L embodiment of the present invention 1 obtain respectively, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the embodiment of the present invention 17, the flow velocity of mobile phase A and Mobile phase B is 0.95ml/min, in embodiment 18, the flow velocity of mobile phase A and Mobile phase B is 1.05ml/min, obtains first liquid chromatogram of Choline Glycerophosphate mixed solution when the flow velocity of mobile phase is respectively 0.95ml/min and 1.05ml/min.As shown in Figure 19 ~ 20, Figure 19 is first liquid chromatogram of Choline Glycerophosphate mixed solution when flow rate of mobile phase is 0.95ml/min that the embodiment of the present invention 17 obtains, Figure 20 is first liquid chromatogram of Choline Glycerophosphate mixed solution when flow rate of mobile phase is 1.05ml/min that the embodiment of the present invention 18 obtains, as can be seen from Figure 19 ~ 20, the Choline Glycerophosphate mixed solution that the embodiment of the present invention 17 ~ 18 obtains is in the flow velocity 0.95 ~ 1.05ml/min of mobile phase, main peak and impurity peaks, the degree of separation of auxiliary material peak and impurity peaks all conforms with the regulations.
Embodiment 19 ~ 22
Choline Glycerophosphate sample is mixed with water, preparation obtains the Choline Glycerophosphate testing sample solution that mass concentration is 50g/L, under being positioned over room temperature, respectively when 0h, 2h, 4h and 8h, get Choline Glycerophosphate testing sample solution 20 μ L, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the mobile phase A in embodiment 19, the volume ratio of acetonitrile and ammonium formate solution adopts 90:10; In mobile phase A in embodiment 20, the volume ratio of acetonitrile and ammonium formate solution adopts 95:5; In mobile phase A in embodiment 21, the volumetric molar concentration of ammonium formate solution adopts 0.002mol/L; In Mobile phase B in embodiment 22, the volumetric molar concentration of ammonium formate solution adopts 0.03mol/L.Obtain the first liquid chromatogram after the placement of Choline Glycerophosphate testing sample solution 0h, 2h, 4h and 8h, as shown in Figure 21 ~ 24, Figure 21 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution of placement after 0 hour that the embodiment of the present invention 19 obtains; Figure 22 is the first liquid chromatogram of placement Choline Glycerophosphate testing sample solution after 2 hours that the embodiment of the present invention 20 obtains; Figure 23 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution of placement after 4 hours that the embodiment of the present invention 21 obtains; Figure 24 is the first liquid chromatogram of the Choline Glycerophosphate testing sample solution of placement after 8 hours that the embodiment of the present invention 22 obtains.Obtain the stability test result of Choline Glycerophosphate testing sample solution provided by the invention according to Figure 21 ~ 24, result is as shown in table 4, the stability test result of the Choline Glycerophosphate testing sample solution that table 4 provides for the embodiment of the present invention 19 ~ 22.
The stability test result of the Choline Glycerophosphate testing sample solution that table 4 embodiment of the present invention 19 ~ 22 provides
Embodiment 19 20 21 22
Choline Chloride 0 0 0 0
Glycerophosphatide acyl inositol 0 0 0 0
Single assorted (%) 0.13 0.12 0.12 0.12
Total assorted (%) 0.13 0.12 0.12 0.12
As can be seen from Table 4, the content of Choline Glycerophosphate testing sample solution provided by the invention each impurity after room temperature places 8 hours is almost constant, illustrates that Choline Glycerophosphate testing sample solution stability provided by the invention is better.
Embodiment 23
Mixed with water by Choline Glycerophosphate standard model, preparation obtains the Choline Glycerophosphate standard model solution that mass concentration is 50g/L.
Measure the Choline Glycerophosphate standard model solution that 20 μ L obtain, inject Shimadzu liquid chromatograph, Shimadzu liquid chromatograph adopts LC-10ATvp type pump, SCL-10Avp type controller and LC-solution type chromatographic work station;
Chromatographic condition is: phenomenexLunaNH 2post, is of a size of 50mm × 4.6mm, and chromatographic column filler grain size is 5 μm; Mobile phase is acetonitrile and water, and the volume ratio of acetonitrile and water is 75:25, and use acetic acid that the pH value of mobile phase is adjusted to 3.0, the flow velocity of mobile phase is 1.0mL/min;
Adopt SEDEX75 type evaporative light-scattering detector to detect, the drift tube temperature of SEDEX75 type evaporative light-scattering detector is, sprayer temperature is 80 DEG C, and atomization gas is nitrogen, and atomization gas flow velocity is 2.5L/min, and nebulizer gas pressure is 1.5bar.
Obtain the second standard liquid chromatograph figure of Choline Glycerophosphate standard model solution, as shown in figure 25, Figure 25 is the second standard liquid chromatograph figure of the Choline Glycerophosphate standard model solution that the embodiment of the present invention 23 obtains, and as shown in Figure 25, the retention time of Choline Glycerophosphate is 14.713min.
Embodiment 24
Choline Chloride standard model is mixed with water, filters after mixing, obtain the Choline Chloride standard model solution that mass concentration is 0.25g/L.
According to the liquid chromatography detecting method in the embodiment of the present invention 23, the Choline Chloride standard model solution obtained is detected, obtain the second standard liquid chromatograph figure of Choline Chloride standard model solution.As shown in figure 26, Figure 26 is the second standard liquid chromatograph figure of the Choline Chloride standard model solution that the embodiment of the present invention 24 obtains.As shown in Figure 26, the retention time of described Choline Chloride is 2.067min.
Embodiment 25
Glycerophosphatide acyl inositol is mixed with water, filters after mixing, obtain the glycerophosphatide acyl inositol standard model solution that mass concentration is 0.25g/L.
According to the liquid chromatography detecting method in the embodiment of the present invention 23, the glycerophosphatide acyl inositol standard model solution obtained is detected, obtain the second standard liquid chromatograph figure of glycerophosphatide acyl inositol standard model solution.As shown in figure 27, Figure 27 is the second standard liquid chromatograph figure of the glycerophosphatide acyl inositol standard model solution that the embodiment of the present invention 25 obtains.As shown in Figure 27, the retention time of described Choline Chloride is 2.055min.
Embodiment 26
L-ALPHA-GPE is mixed with water, filters after mixing, obtain the L-ALPHA-GPE standard model solution that mass concentration is 0.25g/L.
According to the liquid chromatography detecting method in the embodiment of the present invention 23, the L-ALPHA-GPE standard model solution obtained is detected, obtain the second standard liquid chromatograph figure of L-ALPHA-GPE standard model solution.As shown in figure 28, Figure 28 is the second standard liquid chromatograph figure of the L-ALPHA-GPE standard model solution that the embodiment of the present invention 26 obtains.As shown in Figure 28, the retention time of described L-ALPHA-GPE is 21.277min.
Embodiment 27
Glycerine and blank capsules shell and water mixed preparing are obtained the auxiliary material standard model solution that mass concentration is respectively 3g/L and 3g/L.
According to the liquid chromatography detecting method in the embodiment of the present invention 23, the auxiliary material standard model solution obtained is detected, obtain the second standard liquid chromatograph figure of auxiliary material standard model solution.As shown in figure 29, Figure 29 is the second standard liquid chromatograph figure of the auxiliary material standard model solution that the embodiment of the present invention 27 obtains.As shown in Figure 29, the retention time of described auxiliary material is 1.796min and 9.043min.
Embodiment 28
Choline Glycerophosphate, Choline Chloride, glycerophosphatide acyl inositol, L-ALPHA-GPE, starch and glycerine are mixed with water, obtain Choline Glycerophosphate mixed solution a, containing Choline Glycerophosphate 50mg, Choline Chloride 0.25mg, glycerophosphatide acyl inositol 0.25mg, L-ALPHA-GPE 50mg, starch 3mg and glycerine 3mg in every 1mL Choline Glycerophosphate mixed solution a.
Get Choline Glycerophosphate mixed solution a described in 20 μ L, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, obtain the second liquid phase chromatogram of Choline Glycerophosphate mixed solution a, as shown in figure 30, Figure 30 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution a that the embodiment of the present invention 28 obtains.As seen from Figure 30, it is good that Choline Glycerophosphate is adjacent magazins' layout degree, and it is good that known impurities is adjacent peak degree of separation.
Prepare respectively according to technique scheme and obtain Choline Glycerophosphate mixed solution b, Choline Glycerophosphate mixed solution c, Choline Glycerophosphate mixed solution d and Choline Glycerophosphate mixed solution e.Containing Choline Glycerophosphate 30mg, Choline Chloride 0.05mg, glycerophosphatide acyl inositol 0.05mg, L-ALPHA-GPE 30mg, starch 1mg and glycerine 1mg in every 1mL Choline Glycerophosphate mixed solution b; Containing Choline Glycerophosphate 40mg, Choline Chloride 0.15mg, glycerophosphatide acyl inositol 0.15mg, L-ALPHA-GPE 40mg, starch 2mg and glycerine 2mg in every 1mL Choline Glycerophosphate mixed solution c; Containing Choline Glycerophosphate 60mg, Choline Chloride 0.35mg, glycerophosphatide acyl inositol 0.35mg, L-ALPHA-GPE 60mg, starch 4mg and glycerine 4mg in every 1mL Choline Glycerophosphate mixed solution d; Containing Choline Glycerophosphate 70mg, Choline Chloride 0.45mg, glycerophosphatide acyl inositol 0.45mg, L-ALPHA-GPE 70mg, starch 5mg and glycerine 5mg in every 1mL Choline Glycerophosphate mixed solution e.
According to the detection method in technique scheme, liquid chromatographic detection is carried out to the Choline Glycerophosphate mixed solution b obtained, Choline Glycerophosphate mixed solution c, Choline Glycerophosphate mixed solution d and Choline Glycerophosphate mixed solution e, obtains the second liquid phase chromatogram of Choline Glycerophosphate mixed solution b, Choline Glycerophosphate mixed solution c, Choline Glycerophosphate mixed solution d and Choline Glycerophosphate mixed solution e respectively.
The present invention, according to the peak area of each component and the mass concentration of corresponding component in the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution a obtained, Choline Glycerophosphate mixed solution b, Choline Glycerophosphate mixed solution c, Choline Glycerophosphate mixed solution d and Choline Glycerophosphate mixed solution e, obtains the typical curve of each component in Choline Glycerophosphate mixed solution.
Embodiment 29
Get 20 μ L water, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, unlike, in the mobile phase of the present embodiment, the volume ratio of acetonitrile and water adopts 72:28.Obtain the second liquid phase chromatogram of blank solvent.As shown in figure 31, Figure 31 is the second liquid phase chromatogram of the blank solvent that the embodiment of the present invention 29 obtains.
Embodiment 30
Preparing mass concentration according to the technical scheme of embodiment 26 is 0.25mg/mL L-ALPHA-GPE standard model solution.
According to the liquid chromatography detecting method in the embodiment of the present invention 23, the L-ALPHA-GPE standard model solution obtained is detected, obtain the second liquid phase chromatogram of L-ALPHA-GPE standard model solution.As shown in figure 32, Figure 32 is the second liquid phase chromatogram of the L-ALPHA-GPE standard model solution that the embodiment of the present invention 30 obtains.
Comparative example 1
Choline Glycerophosphate mixed solution is obtained according to the technical scheme preparation of the embodiment of the present invention 28, get described Choline Glycerophosphate mixed solution 20 μ L, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, obtain the second liquid phase chromatogram of Choline Glycerophosphate mixed solution.Unlike, in the present embodiment, in mobile phase, the volume ratio of acetonitrile and water is 80:20, and the pH of mobile phase is 2.0.As shown in figure 33, Figure 33 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution that comparative example 1 of the present invention obtains.
Comparative example 2
Choline Glycerophosphate mixed solution is obtained according to the technical scheme preparation of the embodiment of the present invention 28, get described Choline Glycerophosphate mixed solution 20 μ L, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, obtain the second liquid phase chromatogram of Choline Glycerophosphate mixed solution.Unlike, in the present embodiment, in mobile phase, the volume ratio of acetonitrile and water is 80:20.As shown in figure 34, Figure 34 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution that comparative example 2 of the present invention obtains.
Comparative example 3
Choline Glycerophosphate mixed solution is obtained according to the technical scheme preparation of the embodiment of the present invention 28, unlike, not containing glycerine and starch in the Choline Glycerophosphate mixed solution obtained in the present embodiment.Get described Choline Glycerophosphate mixed solution 20 μ L, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, obtain the second liquid phase chromatogram of Choline Glycerophosphate mixed solution.As shown in figure 35, Figure 35 is the second liquid phase chromatogram of the Choline Glycerophosphate mixed solution that comparative example 3 of the present invention obtains.
Comparative example 4 ~ 9
Choline Glycerophosphate testing sample solution, Choline Chloride sample solution, glycerophosphatide acyl inositol sample solution, L-ALPHA-GPE sample solution auxiliary material sample solution and Choline Glycerophosphate biased sample solution is obtained respectively according to embodiment 23 ~ 28 preparation.
Detect respectively according to the liquid chromatography detecting method in the embodiment of the present invention 23, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution, Choline Chloride sample solution, glycerophosphatide acyl inositol sample solution, L-ALPHA-GPE sample solution, auxiliary material sample solution and Choline Glycerophosphate biased sample solution.Unlike, in comparative example 4 ~ 9, the volume ratio of mobile phase acetonitrile and water is 70:30.As shown in Figure 36 ~ 41, Figure 36 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution that comparative example 4 of the present invention obtains; Figure 37 is the second liquid phase chromatogram of the Choline Chloride sample solution that comparative example 5 of the present invention obtains; Figure 38 is the second liquid phase chromatogram of the glycerophosphatide acyl inositol sample solution that comparative example 6 of the present invention obtains; Figure 39 is the second liquid phase chromatogram of the L-ALPHA-GPE sample solution that comparative example 7 of the present invention obtains; Figure 40 is the second liquid phase chromatogram of the auxiliary material sample solution that comparative example 8 of the present invention obtains; Figure 41 is the second liquid phase chromatogram that Choline Glycerophosphate that comparative example 9 of the present invention obtains mixes sample solution.
Embodiment 31 ~ 36
Choline Glycerophosphate sample is mixed with water, preparation obtains the Choline Glycerophosphate testing sample solution that mass concentration is 25g/L, under being positioned over room temperature, respectively when 0h, 2h, 4h, 6h, 8h and 12h, get Choline Glycerophosphate testing sample solution 20 μ L, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, unlike, in embodiment 31, the volume ratio of acetonitrile and water adopts 78:22; In embodiment 32, the pH of mobile phase adopts 4.0; In embodiment 33, the sprayer temperature of evaporative light-scattering detector adopts 70 DEG C; In embodiment 34, the sprayer temperature of evaporative light-scattering detector adopts 90 DEG C; In embodiment 35, the nebulizer gas pressure of evaporative light-scattering detector adopts 0.1MPa; In embodiment 36, the nebulizer gas pressure of evaporative light-scattering detector adopts 0.2MPa.Obtain Choline Glycerophosphate testing sample solution and place the second liquid phase chromatogram after 0h, 2h, 4h, 6h, 8h and 12h hour, as shown in Figure 42 ~ 47, Figure 42 is the second liquid phase chromatogram of placement Choline Glycerophosphate testing sample solution after 0 hour that the embodiment of the present invention 31 obtains; Figure 43 is the second liquid phase chromatogram of placement Choline Glycerophosphate testing sample solution after 2 hours that the embodiment of the present invention 32 obtains; Figure 44 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 4 hours that the embodiment of the present invention 33 obtains; Figure 45 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 6 hours that the embodiment of the present invention 34 obtains; Figure 46 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 8 hours that the embodiment of the present invention 35 obtains; Figure 47 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution of placement after 12 hours that the embodiment of the present invention 36 obtains.Obtain the stability test result of Choline Glycerophosphate testing sample solution provided by the invention according to Figure 42 ~ 47, result is as shown in table 5, the stability test result of the Choline Glycerophosphate testing sample solution that table 5 provides for the embodiment of the present invention 31 ~ 36.
The stability test of the Choline Glycerophosphate testing sample solution that table 5 embodiment of the present invention 31 ~ 36 provides
Result
Embodiment 31 32 33 34 35 36 RSD
Choline Glycerophosphate peak area 5988579 5990968 6023653 6003651 5865160 5954557 0.95％
L-ALPHA-GPE % 0 0 0 0 0 0 ——
As can be seen from Table 5, the Choline Glycerophosphate testing sample solution that the embodiment of the present invention 31 ~ 36 provides is little in the peak area change of Choline Glycerophosphate after 12 hours, illustrate that the content of major component Choline Glycerophosphate in Choline Glycerophosphate testing sample solution is little, illustrate that Choline Glycerophosphate testing sample solution stability provided by the invention is better.
Embodiment 37
Get 20 μ L water, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, obtain the second liquid phase chromatogram of blank solvent.As shown in figure 48, Figure 48 is the second liquid phase chromatogram of the blank solvent that the embodiment of the present invention 37 obtains.
Embodiment 38
Glycerine and blank capsules shell and water mixed preparing are obtained the auxiliary material standard model solution that mass concentration is respectively 3g/L and 3g/L.
According to the liquid chromatography detecting method in the embodiment of the present invention 23, the auxiliary material sample solution obtained is detected, obtain the second liquid phase chromatogram of auxiliary material sample solution.As shown in figure 49, Figure 49 is the second liquid phase chromatogram of the auxiliary material sample solution that the embodiment of the present invention 38 obtains.
Embodiment 39
Get 20 μ L water, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, unlike, in the present embodiment, the volumetric molar concentration of the ammonium formate solution of Mobile phase B adopts 0.05mol/L.Obtain the first liquid chromatogram of blank solvent.As shown in figure 50, Figure 50 is the first liquid chromatogram of the blank solvent that the embodiment of the present invention 39 obtains.
Embodiment 40
Get 500mg Choline Glycerophosphate sample a, be placed in 10ml volumetric flask, be dissolved in water and constant volume, shake up and filter, obtaining Choline Glycerophosphate testing sample solution a;
Get Choline Glycerophosphate testing sample solution a described in 1mL, be placed in 100mL volumetric flask, thin up constant volume, rock evenly, obtain Choline Glycerophosphate contrast solution a;
Get 20 μ L Choline Glycerophosphate contrast solution a, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, regulate sensitivity, make the peak height of major component chromatographic peak be about 20% of full scale;
Get 20 μ L Choline Glycerophosphate contrast solution a, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, record chromatogram is to 2 times of main peak retention time, obtain first liquid chromatogram of Choline Glycerophosphate contrast solution a, as shown in figure 51, Figure 51 is first liquid chromatogram of the Choline Glycerophosphate contrast solution a that the embodiment of the present invention 40 obtains; .
Get 20 μ L Choline Glycerophosphate testing sample solution a, detect according to the liquid chromatography detecting method in the embodiment of the present invention 1, record chromatogram is to 2 times of main peak retention time, obtain first liquid chromatogram of Choline Glycerophosphate testing sample solution a, as in figure 52, Figure 52 is first liquid chromatogram of the Choline Glycerophosphate testing sample solution a that the embodiment of the present invention 40 obtains
If any the impurity peaks of glycerophosphatide acyl inositol and Choline Chloride in first liquid chromatogram of described Choline Glycerophosphate testing sample solution a, its each peak area all must not be greater than 0.5 times of described Choline Glycerophosphate contrast solution main peak area, namely must not be greater than 0.5% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution; Except auxiliary material peak, the peak area of other single impurity all must not be greater than 0.2 times of described Choline Glycerophosphate contrast solution main peak area, namely must not be greater than 0.2% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution; Each impurity peak area sum must not be greater than the twice of described Choline Glycerophosphate contrast solution main peak area, namely must not be greater than 2.0% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution.
Get 20 μ L Choline Glycerophosphate contrast solution a, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, regulate sensitivity, make the peak height of major component chromatographic peak be about 20% of full scale;
Get 20 μ L Choline Glycerophosphate contrast solution a, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, record chromatogram is to 2 times of main peak retention time, obtain the second liquid phase chromatogram of Choline Glycerophosphate contrast solution a, as shown in Figure 53, Figure 53 is the second liquid phase chromatogram of the Choline Glycerophosphate contrast solution a that the embodiment of the present invention 40 obtains;
Get 20 μ L Choline Glycerophosphate testing sample solution a, detect according to the liquid chromatography detecting method in the embodiment of the present invention 23, record chromatogram is to 2 times of main peak retention time, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution a, as shown in Figure 54, Figure 54 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution a that the embodiment of the present invention 40 obtains
If any L-ALPHA-GPE impurity peaks in the second liquid phase chromatogram of described Choline Glycerophosphate testing sample solution a, its peak area must not be greater than 0.5 times of contrast solution main peak area, namely must not be greater than 0.5% of main peak area in the first liquid chromatogram of described Choline Glycerophosphate testing sample solution.
Can be obtained retention time and the content of each impurity in Choline Glycerophosphate testing sample solution a by Figure 51 ~ 54, result is as shown in table 6, and table 6 is the retention time of impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 40 ~ 42.
Embodiment 41
Get 500mg Choline Glycerophosphate sample b, preparation obtains Choline Glycerophosphate testing sample solution b and Choline Glycerophosphate contrast solution b, and preparation method is consistent with the preparation method in the embodiment of the present invention 40.
According to the detection method of the embodiment of the present invention 1, Choline Glycerophosphate testing sample solution b and Choline Glycerophosphate contrast solution b is detected, obtain first liquid chromatogram of Choline Glycerophosphate testing sample solution b and first liquid chromatogram of Choline Glycerophosphate contrast solution b, as shown in Figure 55 ~ 56, Figure 55 is first liquid chromatogram of the Choline Glycerophosphate contrast solution b that the embodiment of the present invention 41 obtains; Figure 56 is first liquid chromatogram of the Choline Glycerophosphate testing sample solution b that the embodiment of the present invention 41 obtains.
According to the detection method of the embodiment of the present invention 23, Choline Glycerophosphate testing sample solution b and Choline Glycerophosphate contrast solution b is detected, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution b and the second liquid phase chromatogram of Choline Glycerophosphate contrast solution b, as shown in Figure 57 ~ 58, Figure 57 is the second liquid phase chromatogram of the Choline Glycerophosphate contrast solution b that the embodiment of the present invention 41 obtains; Figure 58 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution b that the embodiment of the present invention 41 obtains.
Can be obtained retention time and the content of each impurity in Choline Glycerophosphate testing sample solution b by Figure 57 ~ 58, result is as shown in table 6, and table 6 is retention time and the content of impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 40 ~ 42.
Embodiment 42
Get 500mg Choline Glycerophosphate sample c, preparation obtains Choline Glycerophosphate testing sample solution c and Choline Glycerophosphate contrast solution c, and preparation method is consistent with the preparation method in the embodiment of the present invention 40.
According to the detection method of the embodiment of the present invention 1, Choline Glycerophosphate testing sample solution c and Choline Glycerophosphate contrast solution c is detected, obtain first liquid chromatogram of Choline Glycerophosphate testing sample solution c and first liquid chromatogram of Choline Glycerophosphate contrast solution c, as shown in Figure 59 ~ 60, Figure 59 is first liquid chromatogram of the Choline Glycerophosphate contrast solution c that the embodiment of the present invention 42 obtains; Figure 60 is first liquid chromatogram of the Choline Glycerophosphate testing sample solution c that the embodiment of the present invention 42 obtains.
According to the detection method of the embodiment of the present invention 23, Choline Glycerophosphate testing sample solution c and Choline Glycerophosphate contrast solution c is detected, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution c and the second liquid phase chromatogram of Choline Glycerophosphate contrast solution c, as shown in Figure 61 ~ 62, Figure 61 is the second liquid phase chromatogram of the Choline Glycerophosphate contrast solution c that the embodiment of the present invention 42 obtains; Figure 62 is the second liquid phase chromatogram of the Choline Glycerophosphate testing sample solution c that the embodiment of the present invention 42 obtains.
Can be obtained retention time and the content of each impurity in Choline Glycerophosphate testing sample solution c by Figure 59 ~ 62, result is as shown in table 6, and table 6 is retention time and the content of impurity in the Choline Glycerophosphate testing sample solution that obtains of the embodiment of the present invention 40 ~ 42.
The retention time of impurity and content in the Choline Glycerophosphate testing sample solution that table 6 embodiment of the present invention 40 ~ 42 obtains
As can be seen from Table 6, detection method provided by the invention can detect the content of known impurities and unknown impuritie in Choline Glycerophosphate testing sample solution.
Embodiment 43
Get the commercially available Choline Glycerophosphate sample of 500mg (purchased from Italian ITALFARMACOS.p.A company), preparation obtains commercially available Choline Glycerophosphate testing sample solution and commercially available Choline Glycerophosphate contrast solution, and preparation method is consistent with the preparation method in the embodiment of the present invention 39.
According to the detection method of the embodiment of the present invention 1, commercially available Choline Glycerophosphate testing sample solution and commercially available Choline Glycerophosphate contrast solution are detected, obtain the first liquid chromatogram of commercially available Choline Glycerophosphate testing sample solution and the first liquid chromatogram of commercially available Choline Glycerophosphate contrast solution, as shown in Figure 63 ~ 64, Figure 63 is the first liquid chromatogram of the commercially available Choline Glycerophosphate contrast solution that the embodiment of the present invention 43 obtains; Figure 64 is the first liquid chromatogram of the commercially available Choline Glycerophosphate testing sample solution that the embodiment of the present invention 43 obtains.The present invention calculates according to Figure 63 ~ 64, and the content obtaining impurity in commercially available Choline Glycerophosphate is 0.14%.
According to the detection method of the embodiment of the present invention 23, commercially available Choline Glycerophosphate testing sample solution and commercially available Choline Glycerophosphate contrast solution are detected, obtain the second liquid phase chromatogram of commercially available Choline Glycerophosphate testing sample solution and the second liquid phase chromatogram of commercially available Choline Glycerophosphate contrast solution, as shown in Figure 65 ~ 66, Figure 65 is the second liquid phase chromatogram of the commercially available Choline Glycerophosphate contrast solution that the embodiment of the present invention 43 obtains; Figure 66 is the second liquid phase chromatogram of the commercially available Choline Glycerophosphate testing sample solution that the embodiment of the present invention 43 obtains.Calculate according to Figure 65 ~ 66, the content obtaining impurity L-ALPHA-GPE in commercially available Choline Glycerophosphate testing sample is 0%.
As can be seen from above embodiment and comparative example, detection method provided by the invention establishes different chromatographic conditions for different impurity and detects, thus obtain the content of whole known impurities, under chromatographic condition in detection method provided by the invention, major component own control also can be adopted to detect the content of unknown impuritie in Choline Glycerophosphate testing sample.Avoid and only measure the one-sidedness that partial impurities just judges Choline Glycerophosphate quality, achieve comprehensively, effectively monitor the object of Choline Glycerophosphate quality.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.

Claims (9)

1. the detection method of impurity in Choline Glycerophosphate, comprises the following steps:
Choline Glycerophosphate testing sample solution is carried out the first liquid chromatographic detection, obtains the first liquid chromatogram of Choline Glycerophosphate testing sample solution;
The mobile phase of described first liquid chromatographic detection comprises mobile phase A and Mobile phase B; Described mobile phase A comprises the first ammonium formate solution and acetonitrile, and the volumetric molar concentration of described first ammonium formate solution is 0.0005 ~ 0.002mol/L, and the volume ratio of described first ammonium formate solution and acetonitrile is (1 ~ 10): (99 ~ 90);
Described Mobile phase B comprises the second ammonium formate solution and acetonitrile, and the volumetric molar concentration of described second ammonium formate solution is 0.03 ~ 0.05mol/L, and the volume ratio of described second ammonium formate solution and acetonitrile is (35 ~ 45): (65 ~ 55);
Described first liquid chromatographic detection adopts gradient elution program;
Described gradient elution program is:
In < 35min, the volume ratio of mobile phase A and Mobile phase B is (93 ~ 97): (7 ~ 3);
>=35 and in < 65min, the volume ratio of mobile phase A and Mobile phase B is (45 ~ 55): (55 ~ 45);
>=65 and in < 70min, the volume ratio of mobile phase A and Mobile phase B is (35 ~ 44): (65 ~ 56);
>=70 and in < 120min, the volume ratio of mobile phase A and Mobile phase B is (0 ~ 5): (100 ~ 95);
According to the first typical curve of described first liquid chromatogram and predetermined standard model solution, obtain the content of the first impurity in Choline Glycerophosphate testing sample, described first impurity comprises glycerine, Choline Chloride and glycerophosphatide acyl inositol;
Chromatographic column in described first liquid chromatographic detection is cation exchange column;
The detecting device of described first liquid chromatographic detection is evaporative light-scattering detector;
Choline Glycerophosphate testing sample solution is carried out the detection of second liquid phase chromatogram, obtain the second liquid phase chromatogram of Choline Glycerophosphate testing sample solution, the mobile phase of described second liquid phase chromatogram comprises acetonitrile and water, and the volume ratio of described acetonitrile and water is (72 ~ 78): (28 ~ 22);
According to the second typical curve of described second liquid phase chromatogram and predetermined standard model solution, obtain the content of the second impurity in Choline Glycerophosphate testing sample, described second impurity comprises L-ALPHA-GPE;
Chromatographic column during described second liquid phase chromatogram detects is amino bonded silica gel chromatographic column;
The detecting device that described second liquid phase chromatogram detects is evaporative light-scattering detector.
2. detection method according to claim 1, is characterized in that, the column temperature of described first liquid chromatographic detection chromatographic column is 20 ~ 30 DEG C.
3. detection method according to claim 1, is characterized in that, in described first liquid chromatographic detection, the flow velocity of mobile phase A is 0.95 ~ 1.05mL/min;
The flow velocity of described Mobile phase B is 0.95 ~ 1.05mL/min.
4. detection method according to claim 1, is characterized in that, the pH value of described mobile phase A is 2.0 ~ 4.0;
The pH value of described Mobile phase B is 4.0 ~ 6.0.
5. detection method according to claim 1, is characterized in that, during described second liquid phase chromatogram detects, the flow velocity of mobile phase is 0.95 ~ 1.05mL/min.
6. detection method according to claim 1, is characterized in that, during described second liquid phase chromatogram detects, the pH value of mobile phase is 2.0 ~ 4.0.
7. detection method according to claim 1, is characterized in that, during described second liquid phase chromatogram detects, the column temperature of chromatographic column is 20 ~ 40 DEG C.
8. detection method according to claim 1, is characterized in that, in described first liquid chromatographic detection, the sprayer temperature of evaporative light-scattering detector is 70 ~ 90 DEG C;
In described first liquid chromatographic detection, the nebulizer gas pressure of evaporative light-scattering detector is 0.1 ~ 0.2MPa.
9. detection method according to claim 1, is characterized in that, during described second liquid phase chromatogram detects, the sprayer temperature of evaporative light-scattering detector is 70 ~ 90 DEG C;
During described second liquid phase chromatogram detects, the nebulizer gas pressure of evaporative light-scattering detector is 0.1 ~ 0.2MPa.
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