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6.
2.
3.
1 Scope of application
Suitable for the liquid phase of lincomycin , erythromycin , tilmicosin , tylosin , clindamycin , spiramycin , kitasamycin and josamycin residues in royal jelly and royal jelly freeze-dried powder Chromatography-tandem mass spectrometry determination
.
Detection limit of method: Lincomycin, erythromycin, tilmicosin, tylosin, josamycin, spiramycin, clindamycin, and kitasamycin in royal jelly are all 2.
6.
2.
3.
2 Principle of the method
Residues of lincomycin, erythromycin, tilmicosin, tylosin, clindamycin, spiramycin, kitasamycin and josamycin in royal jelly and royal jelly freeze-dried powder are extracted with Tris extract The extract is purified by OasisHLB solid-phase extraction column, eluted with methanol, and the eluate is concentrated to a constant volume and then subjected to liquid chromatography-tandem mass spectrometry
.
Quantification by external standard method
6.
2.
3.
3 Reagents and materials
Methanol, acetonitrile: chromatographically pure; ammonium acetate , hydrochloric acid, tris (Tris, C 4 H 11 NO 3 ), calcium chloride (CaCl 2 ·2H 2 O): analytically pure
.
Methanol solution: (2+3, v/v)
.
400mL methanol mixed with 600mL water; 0.
Standard materials: Lincomycin (CAS7179-49-9), Erythromycin (CAS59319-72-1), Tilmicosin (CAS108050-54-0), Tylosin (CAS74610-55-2), The purity of spiramycin (CAS8025-81-8), clindamycin (CAS21462-39-5), kitasamycin (CAS1392-21-8) and josamycin (CAS16846-24-5) is ≥95%
.
Standard stock solution I: 1.
0mg/mL
.
Accurately weigh the appropriate amount of each standard substance, put them into the corresponding 10mL volumetric flask, dissolve it with methanol and dilute to the mark, and mix
Standard stock solution II: 10.
0μg/mL
.
Draw 0.
Standard working solution: 2.
0μg/mL
.
Draw 2.
OasisHLB solid phase extraction column or equivalent: 500mg, 6mL
.
Before use, activate with 10 mL methanol and 10 mL water in sequence, and keep the column moist in each step before vacuuming
6.
2.
3.
4 Instruments and equipment
Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source; balance: sensitivity 0.
01g, 0.
0001g; solid phase extraction device; nitrogen concentrator; polypropylene centrifuge tube with stopper: 50mL; centrifuge; ultrasonic cleaner; pH meter; oscillator
.
6.
2.
3.
5 Sample pretreatment
(1) Sample preparation
Take out about 200g representative samples from all samples, mix them thoroughly, divide them into two parts, and put them into clean containers
.
After being sealed, it is used as a sample and marked with a mark
(2) Extraction
Weigh 2g sample of royal jelly (1g of lyophilized royal jelly powder), accurate to 0.
01g, place it in a 50mL centrifuge tube, add 10.
0mL tris extract, and shake vigorously on a shaker for 10 minutes
.
Centrifuge at 18000g centrifugal force for 10 minutes, and take the supernatant through the OasisHLB solid phase extraction column at a flow rate of less than 1.
0mL/min
.
Then add 10.
0mL Tris extract, shake vigorously on a shaker for 10min
.
Centrifuge at 18000g centrifugal force for 10 minutes, and take the supernatant through the OasisHLB solid phase extraction column at a flow rate of less than 1.
0 mL/min
.
(3) Purification
After all the sample solution flows out, wash the column with 10 mL water and 10 mL methanol solution in sequence, discard all the effluent, and drain the solid phase extraction column under reduced pressure with a vacuum pump for 1 hour
.
Then eluted with 10mL methanol in a 15mL nitrogen torch, and used a nitrogen concentrator to blow to near dryness in a water bath at 50°C.
After accurately adding 1.
0mL of constant volume solution, dissolve the residue in an ultrasonic cleaner
.
After passing through the 0.
2um filter membrane, it is used for liquid chromatography-tandem mass spectrometer determination
.
(4) Preparation of matrix standard mixed working solution for royal jelly
Draw 1.
0μL, 2.
0μL, 5.
0μL, 25μL of lincomycin, erythromycin, tilmicosin, tylosin, clindamycin, spiramycin, kitasamycin and josamycin standards respectively The working solution is sequentially added to the corresponding negative samples, and lincomycin, erythromycin, tilmicosin, tylosin, clindamycin, spiramycin, kititamycin and Josamycin is a series of matrix standard mixed working solutions with four concentration levels of 2.
0ng/mL, 5.
0ng/mL, 10.
0ng/mL, and 50.
0ng/mL
.
Preparation of matrix standard mixed working solution for royal jelly lyophilized powder: draw 1.
0μL, 2.
0μL, 5.
0μL, 25μL of lincomycin, erythromycin, clindamycin, tylosin, josamycin standard work respectively The solution and 2.
5μL, 5.
0μL, 10.
0uL, 25μL standard working solutions of Tilmicosin, Spiramycin and Kitatamycin were added to the corresponding negative samples in sequence, and lincomycin and red were prepared according to the above extraction and purification steps.
2.
0ng/mL, 4.
0ng/mL, 10.
0ng/mL, 50.
0ng/mL and tilmicosin, spiramycin, tylosin and josamycin respectively It is a series of matrix standard mixed working solutions with four concentration levels of 5.
0ng/mL, 10.
0ng/mL, 20.
0ng/mL, and 50.
0ng/mL
.
Related links: Applicability of the method for the determination of 8 macrolides and lincosamide residues in honey by liquid chromatography-tandem mass spectrometry
