Extraccion QuEChERS

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QuEChERS Sample Preparation for LC-MS/MS Determination of Avermectins in Meat and MilkMasayo Yabu, Mia Summers, and Michael S. Young Waters Corporation, 34 Maple Street, Milford, MA, USA

A P P L I C AT IO N B E N E F I T S ■■ Simple, high throughput preparation

of meat and milk samples using

QuEChERS methodology

■■ Sensitive LC-MS/MS analysis of avermectins

in complex sample matrices using fast and

easy DisQuE sample preparation

■■ Achieve part-per-billion (ppb) level detection

of avermectins to meet country-specific

maximum residue limits (MRL)

■■ Sample preparation using DisQuE products

conforms to CEN method 15662

WAT E r S SO Lu T IO NS

Xevo® TQ-S

ACQUITY UPLC® System

XSelect® CSH™ C18

eXtended Performance [XP ],

2.5 µm Column

DisQuE™ Sample Preparation

for QuEChERS

k E y W O r d S

Avermectins, Abamectin, Ivermectin,

Doramectin, Eprinomectin, Moxidectin,

Dispersive sample preparation,

SPE, LC-MS/MS, milk, meat,

livestock, QuEChERS

IN T rO du C T IO N

Avermectins are sixteen-membered ring macrolide drugs that are used as

veterinary antihelmintics. The lowest allowable limit for these compounds in

food products is set based on worldwide safety evaluations. Regulatory MRLs

for avermectins can vary worldwide but are generally in the ppb concentration

range. The sensitive analysis of avermectins in food products such as milk and

meat can be challenging due to their complex sample matrices.

QuEChERS is a simple and straightforward sample preparation technique that

involves a salting-out liquid extraction followed by optional dispersive solid-

phase extraction (d-SPE). Sample preparation using QuEChERS allows for fast

throughput and high sensitivity analysis of food products. Although QuEChERS

is commonly used for multi-residue pesticide analysis in fruits and vegetables,

it is also applicable in the analysis of veterinary drugs in livestock products.

In this application note, milk and ground beef are prepared and analyzed for

avermectins at the ppb level using QuEChERS methodology and LC-MS/MS.

2QuEChERS Sample Preparation for LC-MS/MS Determination of Avermectins in Meat and Milk

E X P E r IM E N TA L

UPLC conditions

System: ACQUITY UPLC System

Column: XSelect CSH C18 XP, 130 Å,

2.5 µm, 2.1 x 100 mm

(p/n 186006103)

Injection volume: 5 µL

Temperature: 50 °C

Mobile phase: A: 5 mM ammonium

acetate in water

B: 5 mM ammonium

acetate in methanol

Flow rate: 0.40 mL/min

Gradient: 70% B initial, linear

gradient to 97% B in

5 minutes, hold until

8 minutes, back to

70% B at 8.1 minutes.

Hold and re-equilibrate

until 10 minutes

Sample vials: Maximum Recovery Vial

(p/n 600000670CV)

MS conditions

System: Xevo TQ-S

Mass Spectrometer

Ionization mode: Electrospray positive

(ESI+)

Sample Preparation:

Initial Extraction (QuEChERS)

Place 10 mL whole milk (pasteurized) into a 50 mL centrifuge tube, or for meat,

place 8 g ground beef (80% lean) and 2 mL water into a 50 mL centrifuge

tube. Add 10 mL acetonitrile and shake the tube vigorously for 1 minute. Add

the contents of DisQuE pouch salts for European Committee for Standardization

(CEN) QuEChERS (part number: 186006813) and shake vigorously for 1 minute.

Centrifuge for 15 minutes at 4000 rpm and take a 1 mL aliquot of the supernatant

(top layer) for d-SPE cleanup.

d-SPE Cleanup

Transfer the 1 mL aliquot of supernatant to a 2 mL d-SPE cleanup tube that contains

150 mg magnesium sulfate and 50 mg C18 sorbent and shake vigorously for

1 minute. Centrifuge for 5 minutes at 12000 rpm and take a 0.5 mL aliquot a

a sample for LC-MS/MS analysis.

RESULTS

Recovery was measured for each steroid hormone at both low and high concentration

levels (Table 2). Recovery was calculated by comparing the MRM peak area for

samples spiked prior to QuEChERS extraction (pre-spiked samples) with the MRM

peak area for samples spiked after QuEChERS extraction (post-spiked samples).

Table 3 shows the calculated matrix effect for each compound. Matrix effects were

calculated by comparing the MRM peak area of post-spiked samples with the MRM

peak area for equivalent standards prepared in acetonitrile. Figures 1 and 2 show

LC-MS/MS chromatograms obtained from the analysis of meat and milk samples,

respectively, spiked with the low concentration level of avermectins.

Compound NameMrM Transition

(m/z)Cone (V) Collision (eV)

Abamectin 890.5 > 305.5 35 17

Ivermectin 892.5 > 307.5 30 20Doramectin 916.5 > 331.2 10 20Eprinomectin 914.5 > 186.2 20 17Moxidectin 640.3 > 528.3 10 5

Table 1 MRM transitions.

The MRM transitions, cone voltages, and collision cell energies optimized for

avermectins in this study are presented below.

http://www.waters.com/waters/partDetail.htm?partNumber=186006103

http://www.waters.com/waters/partDetail.htm?partNumber=186006813

http://www.waters.com/waters/partDetail.htm?partNumber=600000670CV

3QuEChERS Sample Preparation for LC-MS/MS Determination of Avermectins in Meat and Milk

(a) (b) (c)

(d) (e)

3.0 3.5 4.0 4.5 min 3.5 4.0 4.5 5.0 4.54.03.55.5 min 5.0 min

4.54.03.5 5.0 min3.5 4.0 4.5 5.0 5.5 min3.0 3.5 4.0 4.5 min

3.02.5 3.5 4.0 4.54.03.54.5 min 5.0 min

4.54.03.5 5.0 min3.02.5 3.5 4.0 4.5 min

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

00

100

%

0

100

%

100

%

0

100

%

0

100

%

07132012_075 07132012_075 07132012_075

07132012_06707132012_067 07132012_067

07132012_075 07132012_075

07132012_067 07132012_067

3.77 4.52

4.87 4.99

4.22

4.17

5.36

4.514.66 5.00 5.03 5.11

3.90

3.83

3.56 4.25

3.92 3.99

4.02

2: MRM of 1 Channel ES+TIC (Abamectin)

5.11e3

3: MRM of 1 Channel ES+TIC (Ivermectin)

9.93e3


5.11e3


9.93e3

5: MRM of 1 Channel ES+TIC (Dramectin)

3.42e4


3.42e4

4: MRM of 1 Channel ES+TIC (Epinomectin)

1.71e5

1: MRM of 1 Channel ES+TIC (Moxidectin)

4.85e4


1.71e5


4.85e4

3.0 4.0 4.5 5.0 5.5 min 3.5 4.0 4.5 5.0 min3.5 4.0 4.5 min

3.0 4.0 4.5 5.0 5.5 min 3.5 4.0 4.5 5.0 min3.5 4.0 4.5 min

3.0 4.03.5 4.5 5.0 min3.5 4.0 4.5 min

3.0 4.03.5 4.5 5.0 min3.5 4.0 4.5 min

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

0

100

%

07132012_028

07132012_019

07132012_028

07132012_019 07132012_019

07132012_028

07132012_019 07132012_019

07132012_028 07132012_0283.89

4.31

4.31

4.44

4.44

4.384.66

3.814.01

4.514.66 4.97

4.67 4.29

4.29

4.894.97


1.76e5


1.76e5


7.03e4


7.03e4


1.28e4


1.28e4


7.40e4


7.40e4


5.45e3


5.45e3

(a) (b) (c)

(d) (e)

Figure 1. LC-MS/MS chromatograms of avermectins obtained from ground beef samples; the top trace is the low level spiked sample, the bottom trace is a ground beef blank. (a) abamectin (b) ivermectin (c) doramectin (d) eprinomectin (e) moxidectin.

Figure 2. LC-MS/MS chromatograms of avermectins obtained from whole milk samples; the top trace is the low level spiked sample, the bottom trace is a whole milk blank. (a) abamectin (b) ivermectin (c) doramectin (d) eprinomectin (e) moxidectin.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Waters, ACQUITY UPLC, Xevo, and XSelect are registered trademarks of Waters Corporation. CSH, DisQuE, and The Science of What’s Possible are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

©2013 Waters Corporation. Produced in the U.S.A.June 2013 720004440EN IH-PDF

dISCuSSION

For the analysis of abamectin, ivermectin, doramectin, eprinomectin,

and moxidectin in challenging matrices like whole milk and

ground beef, good recoveries were obtained using DisQuE sample

preparation sorbent pouches. QuEChERS extraction is a simple and

straightforward method for sample preparation and has been shown

to be effective for the extraction of avermectins from milk and

meat. The incorporation of a d-SPE clean-up step prior to LC-MS/MS

analysis aids in the reliable quantitation of avermectins at low ppb

concentrations in complex matrices.

CONCLuSIONS■■ The DisQuE pouch (QuEChERS) protocol is an easy and effective

method for the extraction of avermectins from livestock products.

■■ DisQuE sample preparation provides excellent sample cleanup

prior to LC-MS/MS analysis, with high analyte recoveries and

minimal matrix effects.

■■ DisQuE extraction combined with LC-MS/MS analysis using

eXtended Performance [XP ] 2.5 µm columns provides low ppb

quantitation of avermectins in complex matrices, enabling

reliable testing.

Concentration range (ppb)Average % recovery (%rSd) n=5

Ground Beef Whole MilkConc. Level Low Level High Level Low Level High Level Low Level High Level

Abamectin 1 10 94(3.6) 88(3.6) 86(14.0) 89(3.7)

Ivermectin 1 10 98(17.7) 85(3.1) 84(5.3) 83(14.8)

Doramectin 10 100 89(4.8) 85(4.2) 101(11.7) 90(5.0)

Epinomectin 10 100 99(2.9) 91(1.5) 94(3.9) 93(3.0)

Moxidectin 10 100 90(4.2) 87(1.8) 100(2.4) 90(5.6)

Concentration range (ppb)Matrix Effect (%)* n=5

Ground Beef Whole MilkConc. Level Low Level High Level Low Level High Level Low Level High Level

Abamectin 1 10 105 96 109 93

Ivermectin 1 10 73 95 83 95

Doramectin 10 100 96 92 109 105

Epinomectin 10 100 127 131 92 100

Moxidectin 10 100 101 94 71 85

Table 2. Recoveries of avermectins from ground beef and whole milk samples.

Table 3. Matrix effects of avermectins from ground beef and whole milk samples.

* Matrix effect (%) = peak area (post-spiked sample)/peak area (standard) x 100

A value > 100% indicates ionization enhancement

A value < 100% indicates ionization suppression

Extraccion QuEChERS

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