Using a L-5-MTHF-Ca reference standard, Quantitate other folates and related substances by HPLC. The suitability of the applied HPLC system is checked daily by a "system suitability test" (see below).
Reference standard solution: Accurately weigh 50 mg of L-5-MTHF-Ca (L-5- methyltetrahydrofolic acid, calcium salt (Merck Eprova AG, CH-8200
Schaffhausen, Switzerland) into a 100-ml volumetric flask. Dissolve in a small quantity of water and dilute to volume.
Sample solution: Prepare as for the reference standard using 50 mg of the sample.
Mobile phase solutions
A: Dissolve 7.80 g of NaH2PO4 · 2H2O (0.05 mol) in 1000 ml of water and adjust the pH to 6.5 with 32% NaOH. Filter and degas the solution.
B: Dissolve 5.07 g of NaH2PO4 · 2H2O (0.03 mol) in 650 ml of water and 350 ml of methanol (chromatography grade) and adjust the pH to 8.0 with 32%
NaOH. Filter and degas the solution.
Column: Hypersil-ODS, 5 µm; 250 x 4 mm (Thermo Hypersil Keystone or equivalent)
Flow rate: 1.1 ml/min
% Mobile phase
% Mobile phase
0 - 14
100 – 45
0 – 55
14 – 17
45 – 0
17 – 22
22 – 31
Temperature: Room temperature
Injection volume: 10 µl Detection: UV (280 nm) Run time: 22 min
Retention times given below are approximate:
Folates and related substances
Retention time (min)
4-Aminobenzoylglutamic acid (ABGA)
D-Pyrazino-s-triazine derivative (D-Mefox)
L-Pyrazino-s-triazine derivative (L-Mefox)
Tetrahydrofolic acid (THFA)
7,8-Dihydrofolic acid (DHFA)
Folic acid (FA)
5,10-Methylenetetrahydrofolic acid (CH2THFA)
5-Methyltetrahydrofolic acid (5-MTHF)
5-Methyltetrahydropteroic acid (MeTHPA)
Sample analysis: Inject the reference standard solution and the sample solutions immediately after preparation, using the conditions described above.
(Note: After analysis, flush the column with methanol/water 85:15 and store it under the same conditions.)
Calculate the content of each folate (other than 5-MTHF) and related substance, Xi (%), listed in the above table according to the following formula:
Xi (%) = Ai ×WS × S × (RF)i/AS × W
Ai = the peak area for each folate (other than 5-MTHF) and related substance
AS = the peak area for the L-5-MTHF-Ca Standard
WS = the weight (mg) of L-5-MTHF-Ca Standard
W = the weight (mg) of the sample
S= the percent of L-5-MTHF in the L-5-MTHF-Ca Standard, calculated as
(RF)i = Response Factor for the i-th substance (absorbance at 280 nm in the applied eluent system relative to that of L-5-MTHF)
Other folates and related substances
L-Mefox and D-Mefox
If there are any unidentified impurities, apply a RF of 1.00.
Calculate the total amount of "Other folates and related substances" by summing the Xi for all impurities.
System suitability test procedure
Mixed folates solution: Weigh 25 mg each of ABGA, HOMeTHFA, L-Mefox, DHFA, FA and MeTHPA (all available from Merck Eprova AG) into a 100-ml volumetric flask. Add a small quantity of water to dissolve the mixture; add some sodium hydrogen carbonate and sodium carbonate to aid the dissolution, and fill to the mark with water.
System suitability test solution (SST solution): Weigh accurately 50 mg of a L-
5-MTHF-Ca sample containing DiMeTHFA into a 100-ml volumetric flask. (Available from Merck Eprova AG). Add 1 ml of the Mixed folates solution and a small quantity of water to dissolve, mix and dilute to volume with water.
System suitability test: Inject 10 µl of the SST solution immediately. The resolution between L-5-MTHF and MeTHPA must be at least 5.
D-5-MethylfolateD-5-Methylfolate is quantitated by HPLC using a chromatographic system which allows separation of the D- from the L-stereoisomer. The suitability of the
applied HPLC system is checked daily by a "system suitability test" (see below).
Sample preparation: Accurately weigh 50 mg of the sample into a 100 ml volumetric flask. Dissolve in water and dilute to volume with water.
Mobile phase: Mix 970 ml of 0.03 M NaH2PO4 (obtained by dissolving 4.68 g of NaH2PO4 · 2H2O in water and diluting with water to 1000 ml) with 30 ml of acetonitrile (chromatography grade) and adjust the pH to 6.8 with 32% NaOH. Filter and degas the solution.
Our Factory & Lab
Our factory covers 30000 square meter with a production capacity exceeding 1000 tons per year. At present, we have equipped with a complete set of advanced pretreatment, extraction, separation, concentration, drying, crushing, granulation and packaging equipment. There are six 4.5T multifunction extractors, two drying towers, three 1.0T inspissators, two 2.0T alcohol sink towers and 12 chromatography columns. The test equipments include ultraviolet spectrophotometer, high performance liquid chromatography, gas chromatography and thin-layer scanner.