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 |