Liquid chromatography-tandem mass spectrometry for the determination of six macrolides in milk and milk powder

6.
2.
5.
1 Scope of application

It is suitable for the high performance liquid chromatography-tandem mass spectrometry determination of spiramycin, pyrithromycin, elixiromycin, tilmicosin, erythromycin and tylosin residues in milk and milk powder
.

6.
2.
5.
2 Principle of the method

Milk and milk powder spiramycin , pirlimycin , oleandomycin , tilmicosin , erythromycin , tylosin residue with acetonitrile extraction, solid-phase extraction (SPE), liquid chromatography - tandem mass spectrometry Determination and quantification by external standard method
.

6.
2.
5.
3 Reagents and materials

Acetonitrile, methanol, acetic acid, ammonium formate: pure liquid chromatography: disodium hydrogen phosphate , sodium hydroxide : analytical pure
.

Methanol-aqueous solution (3+7, v/v): 300mL methanol mixed with 700mL water; 5mol/L sodium hydroxide solution: Weigh 20g sodium hydroxide, dissolve in water, and dilute to 100mL; 0.
1mol/L phosphate buffer Solution: Dissolve 6g disodium hydrogen phosphate in 450mL water, adjust pH=8 with sodium hydroxide solution, add water to 500mL, prepare before use; 0.
1mol/L ammonium formate solution: 0.
63g ammonium formate in water to dissolve to 1000mL
.

The purity of spiramycin, pyrithromycin, oleandomycin, tilmicosin, erythromycin, and tylosin standard products are all ≥98%
.

Standard stock solution: Weigh the appropriate amount of standard substance accurately and prepare a 100μg/mL standard stock solution with methanol
.

Mixed standard intermediate working solution: Take each 1mL to 100mL volumetric flask of the standard stock solution, dilute to the mark with methanol, and prepare a mixed standard working solution with a concentration of 1μg/mL
.

HLB solid phase extraction column or equivalent: 500mg, 6mL
.

6.
2.
5.
4 Instruments and equipment

High performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source (ESI); centrifuge: speed greater than 3000r/min; vortex mixer; rotary evaporator; nitrogen blowing instrument; solid phase extraction device
.

6.
2.
5.
5 Sample pretreatment

(1) Sample preparation

a.
Milk: Take a uniform sample of about 250g into a clean container as a sample, seal it and store it at 4°C, and mark it with a mark
.

b.
Milk powder: Take a uniform sample of about 250g into a clean container as a sample, seal it, and mark it
.

(2) Extraction

a.
Milk: Weigh 4g, accurate to 0.
01g, and place the milk sample in a 50mL centrifuge tube with stopper.
Add 20mL acetonitrile and shake for 2min.
Centrifuge at 3000r/min for 10min.
Pipette the supernatant and filter it into a chicken heart bottle
.

b.
Milk powder: Weigh 0.
5g, accurate to 0.
01g, milk powder sample, add 4mL water to a 50mL centrifuge tube with stopper, and mix well
.

(3) Purification

Rotate the extract to about 4 mL at 45°C, add 2 mL of phosphate buffer, and after mixing, transfer to the conditioned HLB solid phase extraction column, and then wash the heart bottle twice with 2 mL of phosphate buffer , The washing liquid is transferred to the column together and dropped at a flow rate of less than 2mL/min
.

(4) Preparation of blank matrix solution

Take 4g of negative milk sample, 0.

Related Links: Recovery and precision of 8 kinds of macrolides and lincosamide drug residues in puffer fish and eels