Alpha GPC test method
Alpha GPC, (also known as Choline alfoscerate) is L-a-glyceryl phosphorylcholine (GPC), it is an important neurotransmitter and phospholipid precursor providing support for cognitive function, memory, cerebrovascular health and boosting HGH
Nowadays, Alpha GPC is very popular in nutrition supplement market, we can find many brands companies use alpha GPC in their brain health and sports nutrition formulations, which makes more and more suppliers of raw material of alpha GPC involved in the global market, and then, the fierce price battle caused a big difference in quality on raw material of alpha GPC.
Therefore, to ensure that alpha GPC we got is qualified, it is necessary to make people know the quality/test standard and method of alpha GPC.
Currently, in Europe alpha GPC is a prescription medication for the treatment of Alzheimer’s disease. It is available in two forms; one is taken by mouth, and the other is given as an injection. In the United States alpha GPC is only available as a dietary supplement, mostly in products promoted to improve memory. Besides, Choline alfoscerate is identified as dietary supplement in most of other countries, so we can’t find any quality standard of alpha GPC in any of Major pharmacopoeia, such as BP, USP, CP etc. At present, Choline alfoscerate is included in Korean Pharmacopoeia (KPC) only , which means alpha GPC test method in KPC is the only quality standard. Now, we’d like to show the details of test method as below :
Alpha GPC test method in KPC
1 Appearance
Clear Viscous Liquid
2 Identification
Dissove 0.2g of Choline Alfoscerate in 1 mL of methanol and use this solution as the test solution. Separately, dissolve about 0.2g of Choline Alfoscerate standard in 1mL of methanol, and use this solution as standard solution. Pipet 0.5 ml of standard solution and add methanol to make exactly 100 mL and use this solution as the control solution. Perform the test with the test solution and the control solution as directed under the thin-layer chromatography. Spot 5uL each of the test solution and the control solution a plate of silica gel with fluorescence indicator for thin-layer chromatography. Develop the plate with a mixture of acetone, methanol, water and glacial acetic acid (30:24:12:12) to a distance of about 10 cm and air-dry the plate. Dissolve about 1.25 g of potassium permanganate and 12 g of sodium hydroxide in 300 mL of water, and spray evenly this solution on the plate: the principal spots obtained from the test solution and the standard solution has the same Rf value and color.
3 Specific optical rotation
-2.40 ~ -2.80 (anhydrous substance).
Dissolve 2.50g in water and dilute to 25ml with the same solvent.
4 PH
5.0 to 7.0
Dissolve 0.250g of carbon dioxide-free water and dilute to 25.0ml with the same solvent.
5 Purity Test
1) Chloride
Proceed with 1.0g of Choline Alfoscerate according to chloride limit test (EP 2,4.4) and perform the test. Prepare the control solution with 0.53mL of 0.01mol/L hydrochoric acid Vs(not more than 0.02 per cent).
2) Sulphate
Proceed with 1.0g of Choline Alfoscerate according to sulphate limit test (EP 2. 4.13) and perform the test. Prepare the control solution with 0.4 mL of 0.01 mol/L sulfuric acid VS (not more than 0.02 per cent).
3) Heavy metals
Proceed with 2.0g Choline Alfoscerate according to Heavy Metals Limit Test Method C and perform the test. Prepare the control solution with 2.0 mL of standard Lead Solution (not more than 10 ppm).
4) Phosphate
Dissolve about 1.0g of Choline Alfoscerate in 10 mL of water and add 5 mL of water and add 5 mL of Ammonium molybdate-sulfuric acid TS and allow to stand for 5 minutes. Then compare the colors of both solutions. The test solution has no more color than the control solution. Pipet 10 mL of 5 ppm Standard Phosphoric Acid Solution and add 5 mL of sulfuric acid and use this solution as the control solution.
5) Residual solvents
Standard solution:
Dissolve 250 mg of EtOH in water and dilute to 100 mL with the same solvent. Dilute 1.0 mL of this solution and 2.omL of internal standard solution to 20.0 mL with water. Introduce 5.0 mL of this solution into an injection vial.
Test solution:
Dissolve 1.0 g of the substance to be examined and 2.0 mL of internal standard solution in water and dilute to 20.0 mL with the same solvent. Introduce 5.0 mLof this solution into an injection vial.
*Internal standard solution: Dissolve 0.1 mL of Acetone in water and dilute to 100 Ml with the same solvent.
Gas Chromatographic Conditions
– Column : Rtx-624 (6 per cent cyanopropyl phenyl – 94 per cent Dimethylpolysiloxane), 0.53mm ×30m
– Carrier gas: He
– Oven temp.: 50℃(5 min) →100℃(10℃/min)
– Injector tem.: 220℃
– Detector temp.: 230℃
- Detector : FID (Flame Ionisation Detector)
The following static head-space injection conditions are used:
- Headspace : Oven Temp. 80℃
Loop Temp. 90℃
Transfer line Temp. 140℃
GC Cycle Time 18min
Vial Eq. Time 10min
Pressuriz Time 0.08min
Loop Fill Time 0.5min
Loop Eq. Time 0.05min
Injection Time 1.0min
Calculation
Calculate the amount of residual solvents in Hanseochem’s Choline Afoscerate taken by the following formula:
Residual solvent in ppm= (Ws*Ru/Wt*Rs)×20,000
Ws: Weight of standard sample(mg).
Wt: Weight of test sample(mg).
Ru: Ratio of peak area obtained from the test solution against peak obtained from the Internal standard solution.
Rs: Ratio of peak area obtained from the standard solution against peak obtained from the Internal standard solution.
6) Related substances
Dissolve 1.0 g of Choline Alfoscerate in 10 mL of methanol and use this solution as the test solution. Separately, dissolve about 1.0g of Choline Alfoscerate standard in 10mL of methanol, and use this solution as standard solution. Pipet 0.5 ml of standard solution and add methanol to make exactly 100 mL and use this solution as the control solution. Perform the test with the test solution and the control solution as dirested under the thin-layer chromatography. Spot 10 uL each of the test solution and the control solution a plate of silica gel with fluprescence indicator for thin-layer chromatography. Develop the plate with a mixture of acetone, methanol, water and glacial acetic acid (30:24:12:12) to a distance of about 10 cm and air-dry the plate. Dissolve about 1.25 g of potassium permanganate and 12 g of sodium hydroxide in 300 mL of water, and spray evenly this solution on the plate: the area of any spot other than the principal spot from the test solution is not more than the area of the principal spot from control solution(0.5 per cent), total area of all spots from the test solution is not more than 4 times the area of the principal spot from the control solution (2.0 per cent).
6 Assay test
The assay should be 98.0~102.0% on anhydrous substance
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)Reagents and materials
Ice Acetic acid; Acetic Anhydride; Mercuric Acetate; Crystal Violet Indicating Reagent; Perchloric acid ; All of them are analytically pure standard.
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)Reagent Solutions prepared
A: 5% Mercuric acetate solution
Weigh 5g Mercuric acetate, put into 100ml Volume flask, add appropriate warm Ice acetic acid and make it fully dissolved. Add Ice Acetic Acid to make the total volume to 100ml scale.
B: 0.2% Crystal Violet Indicating Reagent
Weigh 0.2 Crystal Violet, put into 100ml Volume flask, add appropriate Ice Acetic Acid to fully dissolved, add Ice Acetic Acid to make the total volume to 100ml scale.
C: 0.1 mol/L Standard Perchloric Acid solution
Weigh anhydrous Ice acetic acid (Calculated on the basis of water content, add 5.22ml Acetic Anhydride to each gram water)750ml, add perchloric acid(70%~72%)8.5ml, shake fully, drop acetic anhydride 23ml slowly with shaking at room temperature, after dropping, shake fully and keep it cold at room temperature. Than add appropriate anhydrous Ice Acetic acid to make the total volume to 1000ml scale. Shake fully, keep it at rest for 24 hours.
3.) Testing procedure
3.1 Demarcating the concentration of Standard of Perchloric Acid Solution
Weigh potassium acid phthalate (Which has been dried drastically) 0.4~0.5g ( accurate to 0.0001g), put it into a 250ml triangular flask, Add 50ml Acetic Anhydride to dissolve it fully. Then add 2 to 3 drops crystal violte indicating solution, take perchloric acid solution to titrate till the color of solution from purple to blue, then do the emendation for blank.
C=M/[(V-V0)x0.2042]
In which:
M— weight of Potassium Acid Phthalate ( g)
V—- consumption volume of Perchloric Acid solution of sample titration (ml )
V0—- consumption volume of Perchloric Acid solution of blank titration (ml)
3.2. Assay of Choline Alfoscerate
Accurately weigh 0.3g sample of Choline Alfoscerate (accurate to 0.001g), Add 50ml Acetic Anhydride to make it dissolve fully, add 5ml above mentioned 5% Mercuric acetate solution, covered on the flask orifice with a filter paper, Stir about 20min.
Add 2or 3 drop crystal violet indicating reagent solution, use Perchloric Acid Solution to titrate the solution and make it change the color from purple to blue, then do the emendation for blank.
Choline Alfoscerate % =[(V-V0)xCx257.22×10-3/M]x100%
In which:
V—– consumption volume of Perchloric Acid solution of sample titration (ml )
V0—– consumption volume of Perchloric Acid solution of blank titration (ml)
C—Concentration of the standard Perchloric Acid solution (g/ml)
M—sample weight of Choline Alfoscerate (g)
