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Determination of Thiamphenicol in milk using Venusil XBP C18 (L)

Application Introduction

Thiamphenicol is the congener of chloramphenicol, enjoying the same antibacterial spectrum and antibacterial action with chloramphenicol.

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Introduction

Thiamphenicol is the congener of chloramphenicol, enjoying the same antibacterial spectrum and antibacterial action with chloramphenicol. With the antimicrobial function, it mainly applies to cure the livestock bacteriosis, especially colibacillus, salmonella, pasteurella infection. With the ban on the use of chloramphenicol, thiamphenicol is widely applied into the animal feed and veterinary drug, which leads to a small amount of thiamphenicol residue in the animal foodstuff, such as milk and livestock products, causing harm to the human health. At present, it is commonly recognized that thiamphenicol has a prominent effect in the prevention of livestock disease. Given the phenomenon that the animal feed producers and farmers have been overusing the thiamphenicol at their will, and for the sake of monitoring the residue of the thiamphenicol in the animal foodstuff, European Community and China have regulated that the maximum of the thiamphenicol residue is 0.05 mg/kg.

Experimental

Material and Reagents

HPLC purity of ethyl acetate, acetonitrile, n-hexane; thiamphenicol standard of purity 99%, and the ultrapure water;

Cleanert AQ C18, 500 mg/3 mL;

Cleanert PEP, 200 mg/6 mL.

Sample Preparation

Transfer 5 g milk into a 50 mL centrifuge tube, add 20 mL ethyl acetate, vortex for 10 min. Centrifuge at 4000 r/min for 5 min, and transfer the supernatant into a heart-shaped bottle. Re-extract residue with 20 mL ethyl acetate and combine the supernatants together. Rotary evaporate at 45 °C to near dry, add 5 mL water into the heart-shaped bottle and ultrasonic extraction for 5 min to sufficiently dissolve. Decant the solution into a 50 mL centrifuge tube, add another 5 mL water into the heart-shaped bottle and transfer it into the same centrifuge tube.

Add 20 mL n-hexane, vibrate for 5 min, centrifuge at 4000 r/min for 2 min, and take the subnatant for SPE extraction.

SPE Extraction

Condition: 5 mL acetonitrile and 5 mL water;

Apply extraction, dispense into waste tray and dry the cartridge;

Analytes elution: 5 mL acetonitrile;

Dry with nitrogen flow below 45 °C and reconstitute into 1 mL water. Filter through 0.45 µm PTFE-Q syringe filter followed by HPLC analysis.

SPE procedure was operated on Qdaura Automated SPE system.

Instrumentation

HPLC column: Venusil XBP C18 (L); 5 μm, 150 Å, 4.6 × 250 mm;

Mobile phase: acetonitrile : water = 20:80;

Detection: 225 nm;

Flow rate: 1.0 mL/min;

Column temperature: 35 °C;

Injection: 20 mL.

Results and Discussion

Two kinds of SPE cartridges, Cleanert AQ C18 and Cleanert PEP, were adopted to extract the thiamphenicol from milk sample. HPLC analytical method was also developed and the overall recoveries were above 80% at two concentration levels.

Table 1 Accuracy and reproducibility of spiked milk sample using Cleanert AQ C18

Cartridge Applied

Spiked Level /(mg/kg)

Average Recoveries

/%

CVs

/%

Cleanert AQ C18

2.0

95.7

2.6

Cleanert AQ C18

0.1

83.2

0.6

Table 2 Accuracy and reproducibility of spiked milk sample using Cleanert PEP

Cartridge Applied

Spiked Level /(mg/kg)

Average Recoveries

/%

CVs

/%

Cleanert PEP

2.0

97.8

1.3

Figure 1 Chromatogram of 0.01 mg/mL Thiamphenicol standard solution

Figure 2 Chromatogram of 0.5 mg/mL Thiamphenicol standard solution

Figure 3 Chromatogram of blank milk sample using Cleanert AQ C18

Figure 4 Chromatogram of 2.0 mg/kg spiked milk sample using Cleanert AQ C18

Figure 5 Chromatogram of 0.1 mg/kg spiked milk sample using Cleanert AQ C18

Figure 6 Chromatogram of blank milk sample using Cleanert PEP

Figure 7 Chromatogram of 2.0 mg/kg spiked milk sample using Cleanert PEP

Conclusion

This application note describes the analysis of thiamphenicol residue in milk using simple extraction procedure coupled to HPLC detection. Two different SPE cartridges, Cleanert AQ C18 and Cleanert PEP, were applied in this experiment and good recoveries were gained at two concentration levels. Extract obtained with Cleanert AQ C18/PEP cartridges from milk can be introduced into HPLC system without fear of contaminations.

Ordering Information

Products

Specification

Cat.No

Venusil XBP C18   (L)

5 μm, 150 Å, 4.6   × 250 mm

VX952505-L

Cleanert AQ C18

500 mg/3 mL

S185003-AQ

Cleanert PEP

200 mg/6 mL

PE2006

Qdaura Automated   SPE system

4 channels, 24   samples

SPE-40

Nitrogen Evaporator

Maximal 15   samples

NV15-M

Direct-connection   Guard Cartridge Holder

Compatible with 4.6   × 10 mm and 2.1 × 10 mm

SH-100

1.5 mL vials

Screw neck vials,   12 × 32 mm

AV1001-6

Caps and Septa

Screw neck cap,   center hole; red silicone/ white PTEE septa, slitted

AV2200-0

Syringe   Filter

Monofilm,   13 mm, 0.45 μm

AS081345

Disposable   Needle-Free injection systems

2 mL, 100/pk

LZSQ-2ML





 

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