A Modified QuEChERS Approach to the Isolation and ... in Animal Food -Stuff Food products SPE sample...

PittCon 2010March 3rd, (2000-7)

A Modified QuEChERS Approach to the Isolation and Determination of Drugs in

Food Products

Joan Stevens, Ph.D., Sample Preparation Applications Chemist, Agilent Technologies

Introduction

QuEChERS What, when, why, and how Basic procedure Detection approaches

Advancements in QuEChERS Extractions areas of investigation research and procedure analysis and data


QuEChERS

Quick, Easy, Cheap, Effective, Robust and Safe

Developed by the US FDA and EU Food Regulatory Agencies

Procedure was validated in 2003, “toddler stage” Extraction and analysis of pesticides in food product


QuEChERS

Majority of current method for pesticides in food use SPE SPE requires multiple methods for specific classes of

compounds A single QuEChERS method can extract 250+ pesticides Amenable to GC/MS and LC/QQQ analysis


Why is QuEChERS?• Reduced solvent, reduced labor, increased lab

productivity

Page 5

Standard SPE Methods QuEChERS Method

Sample Processing: 120 min. 30 min. 25% of the time

Solvent usage: 60-90 ml Solvent usage: 10-15 ml

Chlorinated Solvents: 20-30 ml Chlorinated Solvents: None

PittCon 2010

March 3rd, (2000-7)

First Step: QuEChERS Extraction


Second Step: Dispersive-SPE


Advantages of Dispersive SPE Over Standard SPE

Dispersive SPE =

No SPE Apparatus No Flow Control

No SPE Cartridges No Elution Solvent

No Vacuum No Dilution of Extract

No pretreatment No Solvent Evaporation

No Channeling Less Sorbent

No Drying Out Less Time

No Collection Less Cost


Detection Methods

Past detection methods LC, GC possibly LC/MS Limited selectivity and sensitivity Required exhaustive sample preparation

Modern detection methods: LC/QQQ, GC/MS, GC/QQQ Exponential increase in selectivity and sensitivity Advancements in detector technology GC compound selective libraries Minimal sample preparation “JUST ENOUGH”


Advancements in QuEChERS Extractions

“Toddler Stage” Designed for pesticide residue analysis Why stop there? Food SafetyAntibiotics in fish and animal productionSteroids in animal and soilMycotoxins, fungicides in grains and dried foodPCB, PAH in fish

Pharmaceuticals in biologicals


Antibiotics in Animal Food-Stuff

Food products SPE sample preparation, difficult Extraction (LLE) and SPE

Implementing QuEChERS Quick, easy and less solvent consumption Quinolones and sulfonamides in liver Antibiotics used in animal husbandry Established limits within countries


Weigh 2 g homogenized liver sample ( 0.05 g) in 50 mL centrifuge tube

Spike 50 µL of IS spike solution, 50 µL of QC spike solution if necessary, vortex 30 sec

Centrifuge @ 4000rpm for 5 min at 4 C

Transfer 1 mL of ACN layer to SampliQ QuEChERS dispersive-SPE 2 mL tube, drug residues in meat

Vortex 1 min, centrifuge @ 13000rpm for 3 min with micro-centrifuge

Add 10 mL of 5% FA in ACN, and shake vigorously for 30 sec

Add SampliQ EN QuEChERS extraction kit, and shake vigorously for 1 min

Transfer 800 µL extract to another tube, blow down @ 40 C with N2

Sample are ready for LC/MS/MS analysis

Add 8 mL of 30 mM KH2PO4, pH 7.0, vortex.

Reconstitute into 800 µL 1:9 MeOH/H2O w/ 0.1% FA, vortex and sonicate 10 min

Filter samples w/ 0.22µm Cellulose Acetate Spin Filter

Figure 1. Flow chart of QuEChERS procedure for the determination of quinolones in bovine liver


Neat extracts by EN extraction kit onlyNeat extracts by AOACextraction kit onlyNeat extracts by Originalextraction kit only

Figure 2. Feasibility test results 1: chromatograms comparison of the neat extracts (no dispersive SPE) obtained by SampliQ QuEChERS EN extraction kit , AOAC extraction kit, and Original extraction kit.


Figure 3. Feasibility test 2. Analytes peak area comparison for the neat extract processed by different Procedures. Comparisons include pure ACN and acidified ACN, with and without PSA dispersive SPE.

0

5000

10000

15000

20000

25000

30000 A) 5% FA ACN, No Dispersive SPEB) 5% FA ACN, C18 Dispersive SPEC) 5% FA ACN, C18 + PSA Dispersive SPED) ACN, No dispersiveE) ACN, C18 + PSA Dispersive SPE


Pipemidic acid Ofloxacin Ciprofloxacin Danofloxacin

EnrofloxacinLomefloxacin Sarafloxacin Cinoxacin

Oxolinic acid Nalidixic acid FlumequineNorfloxacin (IS)

Figure 4. Chemical structures of the quinolone antibiotics


Figure 5. LC/MS/MS Chromatograms of A) liver blank extract, and B) 5 ng/g fortified liver extract (LOQ). Peaks identification: 1. Pipemidic acid, 2. Ofloxacin, 3. Ciprofloxacin, 4. Danofloxacin, 5. Lomefloxacin, 6. Enrofloxacin, 7. Sarafloxacin, 8. Cinoxacin, 9., Oxolinic acid, 10. Nalidixic acid, 11. Flumequine,

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10 11

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B


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Recovery and ReproducibilityR

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Figure 6:Recoveries for the 11 Quinolones from Bovine Liver


Table 1: Recovery and Repeatability of Quinolones in Fortified Liver 2 mL Dispersive-SPE tube


Resulting New Dispersive-SPE Kits for Quinolones in Food-stuffs


2 mL tubes: 25 mg C18, 150 mg MgSO4

15 mL tubes: 150 mg C18, 900 mg MgSO4

Sulfadizine Sulfathiazole Sulfamerazine

SulfamethazineSulfamethizole Sulfamethoxypyridazine

Sulfachloropyridazine Sulfamethoxazole Sulfadimethoxin Sulfapyridine (IS)

Figure 7. Chemical structures of the sulfonamide antibiotics investigated in this study.


Weigh 2g homogenized liver sample ( 0.05g) in 50mL centrifuge tube

Spike 50 µL of IS spike solution, 50µL of QC spike solution if necessary vortex 30 s

Add 8mL of water, vortex.

Add 10mL of 1% AA in ACN, and shake vigorously for 30s

Centrifuge @ 4000 rpm for 5 min

Transfer 6 mL of upper ACN layer to SampliQ EN QuEChERS fatty dispersive-SPE 2 mL tube

Vortex 2 min, centrifuge @ 4000 rpm for 5 min

Add SampliQ EN QuEChERS extraction kit, and shake vigorously for 1min

Transfer 4 mL extract to another tube, blow down @ 40 C with N2

Sample are ready for LC/MS/MS analysis

Reconstitute into 800 µL 1:9 MeOH/H2O w/ 0.1% FA, vortex and sonicate

Filter samples w/ 0.22 µm Cellulose Acetate Spin Filter

Figure 8. QuEChERS procedure for the determination of sulfonamides in bovine liver.


A

B

1 2

IS

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4

5 6

78

9

Figure 9. LC/MS/MS Chromatograms of A) liver blank extract, and B) 100 ng/g fortified liver extract. Peaks identification: 1.Sulfadizine, 2. Sulfathiazole, 3. Sulfamerazine, 4. Sulfamethizole, 5. Sulfamethazine, 6. Sulfamethoxypyridazine, 7. Sulfachloropyridazine, 8. Sulfamethoxazole, 9., Sulfadimethoxin, IS (internal standard). Sulfapyridine.


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Sulfa

dizi

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Sulfa

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Recovery and ReproducibilityR

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Figure 10: Recoveries for the 9 Sulfonamides in Bovine Liver


Table 2: Recovery and repeatability of sulfonamides in fortified bovine liver, 2 mL dispersive-SPE tube


Conclusion: QuEChERS Advancements/Modifications

Data presented shows that the QuEChERS methodology can be used for other residues besides pesticides

Very large scope of possibilities: food, pharma, enviro

Very simple sample preparation technique, “Just Enough” sample preparation”

Relatively green and very cost effective technique when compared to Liquid-Liquid Extraction and Solid Phase Extraction


Thank You!


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A Modified QuEChERS Approach to the Isolation and ... in Animal Food -Stuff Food products SPE sample...

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