Determination of Methylene Blue Residues in Aquatic Products

1. Application range

This standard prescribes a HPLC method for the determination of methylene blue residue in aquatic products.

This standard is applicable to the determination and confirmation of methylene blue residues in fish, shrimp and their products. (The sample of this experiment is Longli Fish)

Determination of Methyl Blue Residue in SN 1974-2007 Import and Export Aquatic Products by LC-MS / MS-HPLC

2. Extraction steps

Sample pretreatment: crush the sample by mixer, then store it in refrigerator.

Weigh 5g fish sample (accurate to 0.01 g) in 50 mL centrifuge tube. Add 5 g of neutral alumina, 1 mL of p-toluenesulfonic acid solution and 4 mL of sodium acetate buffer solution in turn, vortex for 1 min. Add 8 mL acetonitrile, homogenize at 10000 r/min for 2 min, then centrifuge at 4000 r/min for 5 min. Transfer the supernatant to another 50 mL centrifuge tube, add 4 mL acetonitrile to extract residue again and combine the supernatant. Add 8 mL diethylene glycol solution and 10 mL dichloromethane to the supernatant, vortex for 1 min at 4000 r/min and centrifuge for 5 min. Collect the lower organic layer in the concentration flask at 40℃, rotate and evaporate to near dry, then add 6 mL acetonitrile to dissolve the residue to be purified.

3. Purification steps

SPE column: Welchrom®Alumina-N column, specification :500 mg/3mL

Activation :3 mL acetonitrile, discard

Injection: collect after injecting all the purified liquid

Flushing :2 mL acetonitrile, collect

Elution :4 mL methanol, collect

Concentration: blow with nitrogen to near dry at 40℃, add 10% methanol solution to have a constant volume of 1 mL, then filter through 0.22μm microporous membrane for test

4. Chromatographic conditions

Column: Ultisil®XB-C18 4.6×150 mm,5μm

Elution procedure: gradient elution. At 0 min, methanol solution/0.1% formic acid= 10:90; At 7 min, methanol solution/0.1% formic acid= 95:5; At 9 min, methanol solution/0.1% formic acid=10:90; At 15 min, methanol solution/0.1% formic acid=10:90.

Flow rate :0.5 mL/min

Column temperature : 30℃

Injection : 50μL

Detection wavelength : 655 nm

5. Data of chromatogram or spiked recovery rate

Figure 1. Standard chromatogram of Atlas methylene blue at 0.025 ug/ml

Name	Retention	Area	Height	Relative area	Tailing factor	Resolution	Plates
_	min	mAU*min	mAU	%	_	(EP)	(USP)
Methylthionine chloride	8.672	0.1039	1.40	100.00	1.74	n.a.	108040

Table 1. Peak situation

Figure 2. Blank chromatogram of fish sample

Figure 3. Chromatogram of fish sample spiked at 0.005 ug/ml

Material	Spike level	Recovery rate	RSD（n=2）
Methylthionine chloride	0.005mg/kg	103.08%	1.78%

Table 2、Spike recovery rate table

6. Related products

P/N	Product	Specification
00518-20005	SPE column	Welchrom® Alumina-N,500mg/3mL, 50pk
00837-05006	50mL Centrifugal tube with screw tip bottom	Welchrom® centrifuge tube, disposable centrifuge tube, flat cap, conical bottom , RCF12000xg,without sterilization, 50mL, 50/pk
00824-31001	SPE instrument	Welch instrument, 12 ports square cylinder
00821-32291	Caps and septa	Pre-slit red PTFE/white Silicone septa, 9mm blue short screw-thread polypropylene cap, 6mm centre hole, 100/pk
00821-40927	Sample vial	2ml wide opening short screw-thread vial with write-on spot, clear, 11.6*32mm, 100/pk
00201-31041	HPLC column	Ultisil® XB-C18，4.6×150mm, 5μm
00802-02201	Syringe filter	Welchrom® NY, 13mm*0.22μm, 100pk