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Simultaneous Detection of 5 Kinds of glucocorticoids in animal-derived food by LC-MS/MS

Application Introduction

With the anti-inflammatory and anti-allergic effect of glucocorticoid, it can provide feed conversion ratio, which will help stimulate the growth of livestock and poultry. Therefore, it is widely used in the animal husbandry industry.

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Introduction

With the anti-inflammatory and anti-allergic effect of glucocorticoid, it can provide feed conversion ratio, which will help stimulate the growth of livestock and poultry. Therefore, it is widely used in the animal husbandry industry. However, the overuse of glucocorticoid in the process of animal growth will lead to the residue of glucocorticoid in the animal-derived food, which means the extreme harm on human health. In this sense, all countries in the world attach higher importance to the monition of glucocorticoid in animal-origin food and stipulate the maximum residual amount.

Figure 1 Information of Analytes

Analytes

Structure

CAS

Molecular   Weigh

MethyLprednisoLone

                                                                                 

83-43-2

374.5

Dexamethasone  

50-02-2

392.5

Hydrocortisone

0-23-7

362.5

Prednisone

53-03-2

358.4

PrednisoLone

50-24-8

360.4

Experimental

Preparation of standard stock solution

Precisely measure certain volume of prednisone, prednisolone, hydrocortisone, methyl-prednisolone, and dexamethasone. Dissolve the sample into methanol and make up to 1000 g/mL stock solution respectively. Reserve the standard solutions in the environment below minus 20℃.

Preparation of mixed standard working solution

 Standard stock solutions of glucocorticoid drugs are accurately measured, 20% acetonitrile aqueous solution is used for preparation the mixed standard working solution of 0.2 μg, 0.5 μg/L, 1 μg/L, 2 μg/L, 5 μg/L respectively.

Sample extraction

Meat or tissue: transfer 2 g homogenized sample (+/- 0.05 g) into 50 mL centrifuge tube, add 15 mL ethyl acetate, vortex, centrifuge at 8000 rpm for 15 min, and transfer the ethyl acetate layer. Add 10 mL of 0.1 moL/L sodium hydroxide solution into the residue and mix it. Add 20 mL ethyl acetate into the tube, vortex well and centrifuge at 8000 rpm for 15 min. Extract the ethyl acetate layer and combine the two extracting solutions. Rotary evaporate at 40 ℃ to near dry, dissolve the residue into constant volume using 1 mL ethyl acetate and 5 mL n-hexane for purification.

Milk or eggs: transfer 2 mL milk sample or 2 g egg sample (+/- 0.05 mL or g)  into 50 mL centrifuge tube, add 20 mL ethyl acetate, vortex well and centrifuge at 8000 rpm for 15 min. Aspirate the ethyl acetate layer. Rotary evaporate at 40℃ to near dry. Reconstitute into 1 mL ethyl acetate and 5 mL n-hexane.

Sample phase extraction

Condition: 6 mL of n-hexane;

Sample addition: apply the extract to the Cleanert® Silica and vacuum the cartridge;

Analytes Elution: 6 mL acetone / n-hexane (6/4, V/V);

Dry under nitrogen below 50 °C and reconstitute into 0.5 mL of 20% acetonitrile in water. Centrifuge at 15000 r/min for 20 min. Filter the supernate through 0.22 membrane followed by LC-MS/MS analysis.

Instrumentation

LC columns: Venusil ASB C18, 5 μm, 150 Å, 2.1 × 150 mm;

Mobile phases: A: Water of 0.1% formic acid; B: Acetonitrile;

Flow rate: 0.2 mL/min;

Column temperature: 30 °C;

Injection: 10 µL;

Ion source: ESI (-);

IS: -4500 V;

GS1: 50 Psi;

GS2: 45 Psi;

TEM: 550 ℃;

Collecting mode: MRM.

Figure 2 LC Gradient

Time/min

A/%

B/%

0.00

62

38

2.00

62

38

8.00

55

45

8.01

10

90

10.00

10

90

10.01

62

38

15.00

62

38

Figure 3 MS parameters

Analytes

Retention(min)

Ion   pair monitoring

DP

EP

CE

CXP

Prednisone

4.70

403.0/327.0

-45

-10

-18

-9

403.0/457.0

-40

-10

-18

-9

PrednisoLone

4.55

405.1/329.1

-50

-10

-22

-13

405.1/359.1

-50

-10

-22

-13

Hydrocortisone

4.65

407.1/331.1

-45

-10

-24

-9

407.1/361.1

-45

-10

-24

-9

MethyLprednisoLone

6.44

419.2/343.1

-45

-10

-22

-9

419.2/373.1

-40

-10

-22

-9

Dexamethasone

7.23

437.1/361.1

-55

-10

-22

-9

437.1/391.1

-60

-10

-22

-9

Results and Discussion

Calibration Curve

5-point calibration curve was plotted from 0.2 to 5 mg/L. Excellent method linearity is demonstrated for each analyte from 0.2 to 5 mg/L at five concentration levels.

Figure 4 Regression equations and correlation coefficients of calibration curve

Analytes

Regression   equation

R2

Prednisone

Y=30225x+731.14

R2=0.9999

PrednisoLone

Y=25436x+706.91

R2=0.9999

Hydrocortisone

Y=69494x+1167.0

R2=0.9999

MethyLprednisoLone

Y=56491x+2313.4

R2=0.9999

Dexamethasone

Y=33437x+952.7

R2=0.9999

Method stability

Inject the 5 mg/L standard solution 6 times continuously and calculate the RSDs of the peak areas and retention times to evaluate the method stability. Excellent stability of the method was obtained with RSD range of 1.58-2.96% and 1.76-3.19% for retention times and peak areas.

Figure 5 RSDs of peak areas and retention times of 5 kinds of glucocorticoids working solution

Analytes

RSD of retention   time

RSD of peak area

Prednisone

1.85%

1.85%

PrednisoLone

1.70%

2.06%

Hydrocortisone

1.58%

1.76%

MethyLprednisoLone

2.65%

2.11%

Dexamethasone

2.96%

3.19%

Sensitivity

Figure 6 LODs of 5 kinds of glucocorticoids in animal-derived food matrix

Analytes

LOD

Milk matrix, μg/Kg

S/N≥10

LOD

Eggs matrix, μg/Kg

S/N≥10

LOD

Pork liver matrix,   μg/Kg

S/N≥10

Prednisone

0.1

0.1

0.2

PrednisoLone

0.2

0.2

0.5

Hydrocortisone

0.1

0.1

0.2

MethyLprednisoLone

0.1

0.1

0.2

Dexamethasone

0.1

0.1

0.2

Accuracy

Figure 7 Recoveries and accuracies of 1.0 μg/kg spiked pork liver samples

Analytes

Sample 1

Sample 2

Sample 3

Average   recoveries

RSD

Prednisone

71.00%

65.49%

69.35%

68.61%

4.12%

PrednisoLone

66.80%

68.82%

70.23%

68.62%

2.51%

Hydrocortisone

71.16%

74.29%

79.51%

74.99%

5.63%

MethyLprednisoLone

62.38%

64.44%

63.38%

63.40%

1.62%

Dexamethasone

74.81%

79.38%

79.66%

77.95%

3.49%

Figure 8 Recoveries and accuracies of 1.0 μg/kg spiked eggs samples

Analytes

Sample 1

Sample 2

Sample 3

Average   recoveries

RSD

Prednisone

57.40%

61.00%

64.58%

60.99%

5.89%

PrednisoLone

43.72%

39.43%

45.99%

43.05%

7.74%

Hydrocortisone

57.06%

52.56%

56.45%

55.36%

4.41%

MethyLprednisoLone

47.60%

45.73%

47.78%

47.04%

2.41%

Dexamethasone

65.05%

57.45%

66.75%

63.08%

7.85%

Figure 9 Recoveries and accuracies of 1.0 μg/kg spiked milk samples

Analytes

Sample 1

Sample 2

Sample 3

Average   recoveries

RSD

Prednisone

65.02%

71.03%

58.90%

64.98%

9.33%

PrednisoLone

53.86%

50.97%

58.39%

54.41%

6.87%

Hydrocortisone

55.09%

61.38%

52.67%

56.38%

7.97%

MethyLprednisoLone

51.39%

55.28%

53.91%

53.53%

3.69%

Dexamethasone

59.03%

58.75%

62.02%

59.93%

3.02%

Figure 10 Recoveries and accuracies of 1.0 μg/kg spiked pork samples

Analytes

Sample 1

Sample 2

Sample 3

Average   recoveries

RSD

Prednisone

90.47%

88.03%

90.71%

89.74%

1.65%

PrednisoLone

76.09%

76.35%

80.38%

77.61%

3.10%

Hydrocortisone

84.34%

81.45%

85.76%

83.85%

2.62%

MethyLprednisoLone

83.77%

86.08%

88.24%

86.03%

2.60%

Dexamethasone

85.52%

88.40%

84.37%

86.10%

2.41%

Conclusion

This application note describes the analysis of glucocorticoids residues in animal-derived food using sample phase extraction coupled to LC-MS/MS detection. SPE for simultaneous extraction of all five analytes from animal-derived food is shown with excellent recoveries and accuracies.

Order Information

Products

Specification

Cat.No

Venusil®   ASB C18

5 μm, 150 Å, 2.1   × 150 mm

VS951502-0

Cleanert®   Silica

500 mg/ 6 mL

SI5006

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 (Nylon)  

Monofilm, 13 mm,   0.22 μm

AS021320

Disposable   Needle-Free injection systems

2 mL, 100/pk

LZSQ-2ML


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