tn-0022 applications · 2016. 12. 16. · applications tn-0022 page 2 of 8 for additional technical...

APPLICATIONSTN-0022

Page 1 of 8For additional technical notes, visit www.phenomenex.com

Rapid, High Resolution Analysis of Aflatoxin Extracts from Peanut Butter Using Kinetex™ Core-Shell Technology and Strata® SPESky Countryman, Shahana Huq, Terrell Mathews, et al.Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

A two-stage Solid Phase Extraction (SPE) procedure using Strata® Florisil® and Strata® Silica is effective in removing detector interfering contaminants from the difficult sample matrix, peanut butter, while maintaining absolute recoveries above 80 %. Improved separation of aflatoxins is achieved with Kinetex™ 2.6 µm PFP core-shell columns, compared to traditional C18 columns, allowing for rapid run times under 2 minutes with great precision, and accuracy.

IntroductionMycotoxins are secondary metabolites generated by several kinds of molds. Aflatoxins are a subclass of mycotoxins mainly produced by Aspergillus flavus and Aspergillus parasiticus which can contam-inate a wide spectrum of foodstuffs. Due to their high toxicity and carcinogenicity, aflatoxins are of major concern for food producers, the food processing industry, and consumers. Most countries have legislation setting maximum permissible limits for aflatoxins, which are in the low micrograms per kilogram for food matrices.1,2 Two types of limits exist, one for aflatoxin B1 and the other for the sum of the four aflatoxins (B1, B2, G1 and G2).

The most frequently used analytical protocol for aflatoxins is HPLC coupled with either fluorescence or mass spectrometric detection.3,4 The toxins are solvent extracted from food matrices and the extracts cleaned either by immunoaffinity (IAC) or solid-phase extraction (SPE) columns prior to analysis. Sample cleanup with IAC suffers from several disadvantages, the major ones being inactivation of the affinity sites or masking of the toxin structure by matrix contaminants from food samples resulting in lower recovery yields.5

Several sorbents have been used for the SPE cleanup of solvent extracts from food matrices including C18, silica, florisil, phenyl-silica and functionalized polymers. It has been reported that SPE provides cleanliness comparable to IAC. In this technical note, we demonstrate a new SPE protocol for aflatoxins analysis from pea-nut butter which gives ultra-clean extracts. Samples are analyzed by LC/MS/MS using the new Kinetex™ core-shell particle technol-ogy achieving full resolution of all compounds in less than 2 min-utes.

Experimental ConditionsAflatoxin standards were obtained from Sigma, St. Louis, MO. Peanut butter with no aflatoxins was purchased from a local gro-cery store. The Strata Florisil and Silica cartridges as well as the Kinetex™ PFP columns were obtained from Phenomenex, Torrance, CA. All solvents and buffers are reagent grade from Sigma. Two

LC/MS/MS systems were used: Applied Biosystems® API 3000™ with TurboIonSpray® Source and 3200 Q TRAP® with Turbo V™ Ion Source.

To 5 g of peanut butter, 40 mL of methanol/water (80:20) containing 0.2 g of sodium chloride was added and the mixture was mechani-cally stirred for 2 hours. The residual solids were filtered off on a Whatman filter paper and rinsed three times with 5 mL methanol. The combined extracts were dried over anhydrous magnesium sul-fate, the solvent removed under nitrogen at 45 °C and the residue reconstituted in 500 µL of methanol/water (80:20).

Since this process was quite time consuming, peanut butter matrix extracts were prepared by extracting larger quantities of un-spiked peanut butter (50-100 g). These extracts were refrigerated and then could be spiked with a known amount of aflatoxin as needed. Ex-tracts were spiked with aflatoxin standards at 50 ppb for LC/MS/MS for use with SPE.

Data generated on the API 3000 LC/MS/MS was done at Phenom-enex, Torrance, CA. Work on the API 3200 Q TRAP was done at AB/MDS Analytical Technologies in Toronto, Canada.

Results and DiscussionPeanut butter is extremely complex consisting of carbohydrates, proteins, vitamins, phytosterols, poly/mono-unsaturated fatty acids and several inorganic ingredients. Solvent extraction will co-extract many of these constituents with the aflatoxins. Therefore, a thor-ough sample cleanup protocol is required to eliminate these matrix components, especially when parts per billion level aflatoxins are to be quantitated. In addition, the lactone ring present in aflatoxins can be cleaved under basic conditions. The G1 and G2 isomers are especially prone to this because they contain two such rings (Fig-ure 1). Moreover, aflatoxins possess high melting points and are stable to heat in the dry condition, but when moisture is present, heating may lead to cleavage of the ring as well as decarboxyla-tion. On the other hand, strong acids convert aflatoxins to adducts, as for example, trifluoroacetic acid forms the addition compound across the double bond of the furan ring. Thus, care must be ex-

ecised in isolating the aflatoxins before the analytical step

APPLICATIONSTN-0022

Page 2 of 8 For additional technical notes, visit www.phenomenex.com

Food safety testing often requires extremely fast turn around time between samples to allow proper action to be taken. When looking to achieve fast chromatography, there has been a lot of discussion in the past several years on the use of sub-2 µm or other high reso-lution columns. Since backpressure generated by the sub-2 µm materials is higher than the pressure limits of the standard HPLC system, many labs may have trouble implementing these fast LC solutions.

To overcome this challenge, Phenomenex has developed the new Kinetex™ HPLC columns that utilize core-shell technology to pro-vide sub-2 µm performance at backpressures compatible with standard HPLC instruments. Kinetex™ columns have a solid 1.9 µm core with a 0.35 µm porous shell, giving a total particle diameter of 2.6 µm. The 2.6 µm particle gives backpressures similar to a 3 µm particle, but the improved mass transfer kinetics of the 0.35 µm porous shell significantly improve resolving power giving sub-2 µm performance on any HPLC system.

Most of the published work on aflatoxins reference the use of C18 type phases. When using C18 phases, resolution problems be-tween B2 and G1 are common. The conjugated aromatic struc-ture of aflatoxins suggests that they would be well retained through π-π type interactions using phenyl-based phases. The Kinetex™ PFP is a pentafluorophenyl phase that is highly prone to elec-trophilic interactions due to the delocalized electrons in π orbit-als above and below the planar ring. Solutes containing aromatic rings participate in a stacking interaction with the benzene ring of the PFP ligand resulting in increased interaction and resolution.

When we combine the high efficiency Kinetex™ particle with the PFP chemistry, we were able to separate all four aflatoxins in

APPLICATIONSTN-0022


The two-stage SPE protocol presented here was designed to re-move the maximum amount of matrix contamination. The peanut butter extract from methanol/ water (80:20) was directly loaded on the Strata Florisil and thoroughly washed with a four-step wash se-quence. This protocol was expected to eliminate most hydrophilic impurities. However, since peanut butter contains a high amount of fatty acids, an optional second cleanup step was added to remove additional impurities. The eluate from the Strata Florisil was recon-stituted in 1:1 chloroform/hexane and loaded onto a Strata Silica cartridge. The afl atoxins were recovered by non-retentive SPE, while the impurities were presumably retained on the cartridge.

Post-column infusion studies were conducted to determine the effectiveness of the two-stage SPE cleanup. Figure 3 shows the total ion current when a blank (30 % acetonitrile) is injected while post-column infusing all four afl atoxins. As expected, no suppres-sion was observed. When a blank peanut butter matrix, which has undergone no cleanup procedure, was injected, there was a signifi -cant suppression region from about 0.2-1.0 min (Figure 4). Sup-pression in this region interferes with the internal standard M1 and results in poor sensitivity and quantitation. In Figure 5 the same peanut butter extract is re-analyzed after cleanup using the two-stage SPE procedure. The suppression region is signifi cantly re-duced, now only affecting peaks eluting in the 0.30 – 0.60 minute window. Since none of our target analytes or internal standard elute in this region, no suppression was observed.

It should be noted that the ion suppression work was performed on an API 3000™ system with a TurboIonSpray® source. The Tur-boIonSpray source will show a greater degree of ion suppression and take longer for signals to recover making good sample cleanup even more critical. Although suppression studies have not been performed with the Turbo V™ source on the 3200 Q TRAP®, it is presumed that suppression would be further reduced allowing for shorter run times.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 min020

40

60

80

100

120

140

160

180

200

240

220

260

280

300

320

340

360

380

400

420

440

460

475

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 min020

40

60

80

100

120

140

160

180

200

240

220

260

280

300

320

340

360

380

400

420

440

460

475

Figure 4. Infusion of Afl atoxin Standard (G2, B2, G1, B1) and Injection of Extracted Peanut Butter Matrix before SPE Cleanup

Figure 5. Infusion of Afl atoxin Standard (G2, B2, G1, B1) and Injection of Peanut Butter Matrix after Two-Stage SPE Cleanup

Strata® Silica (Si-1) 200 mg/3 mL

Part No.: 8B-S012-FBJ

Condition: 2 x 3 mL of hexane

Load: 2 mL of reconstituted sample from the Strata Florisil®

Wash: 1. 2 x 2 mL of methanol/chloroform (1:1)

2. 1 x 1 mL of methanol/chloroform (1:1)

Load and wash solutions from the silica SPE were pooled together and dried down under nitrogen and reconstituted in 500 µL of the mobile phase used for LC/UV or LC/MS analysis

Strata® Florisil® 500 mg/3 mL

Part No.: 8B-S013-HBJ

Load: A 1.5 mL aliquot of peanut butter extract was spiked with afl atoxin standards and loaded

Condition: No conditioning was performed as this led to reduced recoveries of afl atoxins

Wash: 1. 2 x 3 mL of methanol/water (80:20)

2. 2 x 3 mL of 100 % methanol

Elute: 2 x 3 mL of acetone/water/0.5 % formic acid (96:3.5:0.5)

The combined eluate was dried under nitrogen and the residue reconstituted in 2 mL of 1:1 hexane/chloroform and loaded onto the Strata Silica cartridge for further cleanup

SPE Procedure

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 min0

2040

60

80100

120

140160

180

200

220240

260

280300

320

340360

380

400

420440

460

480500

520535

Figure 3. Infusion of Afl atoxin Standard (G2, B2, G1, B1) and Injection of Solvent Blank (30 % ACN/Water)

Ap

p ID

184

76A

pp

ID 1

8476

Ap

p ID

184

76

APPLICATIONSTN-0022


Calibration curves for aflatoxins using the ABI 3200 Q TRAP® were generated from 5 to 500 ppb. When the concentration range is re-duced to 5 – 100 ppb, the data fits well to a linear equation with R2 values > 0.993 for all analytes. When the concentration ranges are extended to 500 ppb, a quadratic fit now yields the best quantita-tive results (Figure 6). At 500 ppb the response (height in cps) is well within the linear dynamic range of the detector suggesting that the suppression is originating in the ion source. This phenomenon has been observed in the past for other compounds and is not well understood. Further work must be done to better characterize and explain this observation.

Four replicate QC spiked samples were prepared at the 50 ppb level and processed using the Florisil® SPE procedure only. Two calibration curves were prepared, one in mobile phase and another in a blank peanut butter matrix that had been processed through the Florisil SPE cartridges.

Accuracy for the QC spiked samples was between 132.8 – 144.3 % with % CVs

APPLICATIONSTN-0022


20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500Analyte Conc. / IS Conc.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

20.0

21.0

22.0

B1R = 0.9880


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

B2R = 0.9972


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

G1R = 0.9970


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.25.4

G2R = 0.9961

Figure 6. Non-extracted Standard Calibration Curves

Table 3Performance of QC spiked samples at 50 ng/ml quantitatedagainst the non-extracted calibration curve

Analyte S/N * %CV** Recovery ***

Aflatoxin B1 275.9 5.0 141.7

Aflatoxin B2 302.2 4.7 144.3

Aflatoxin G1 224.5 3.2 132.8

Aflatoxin G2 180.0 6.5 136.4

* Signal to Noise (S/N) being the peak height divided by the noise measured at three standard deviations of the noise. ** % CV estimates from 4 replicate samples, with 2 injections for each sample (n=8) *** Recovery was obtained using non-extracted standard calibration curve.

APPLICATIONSTN-0022


Figure 7. Spiked Standard Calibration Curves

Table 4Performance of QC spiked samples at 50 ng/ml quantitatedagainst the extracted calibration curve

* Signal to Noise (S/N) being the peak height divided by the noise measured at three standard deviations of the noise. ** % CV estimates from 4 replicate samples, with 2 injections for each sample (n=8) *** Recovery was obtained using spiked standard calibration curve


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

17.0

18.0

19.0

20.0

21.0

22.0

B1R = 0.9980


0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

12.0

13.0

14.0

15.0

B2R = 0.9987


0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

G1R = 0.9990


0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

4.6

4.8

5.0

5.25.4

G2R = 0.9984


Aflatoxin B1 275.9 4.8 96.7

Aflatoxin B2 302.2 4.5 94.3

Aflatoxin G1 224.5 3.1 97.6

Aflatoxin G2 180.0 6.3 94.9

APPLICATIONSTN-0022


ConclusionsThe current work describes a quick and easy method for cleanup and analysis of aflatoxins from peanut butter samples. The two-stage SPE procedure removes a majority of matrix interferences observed in LC/MS/MS chromatography. The SPE phases also provide a good alternative to immunoaffinity products.

Combining the high efficiency Kinetex™ core-shell particle with the highly selective PFP chemistry enabled ultra fast separation of all four aflatoxins on any LC/MS/MS system configuration. Precision and accuracy of this method were excellent when us-ing a matrix matched calibration curve. The estimated detection limits for each analyte are well below 5 ppb, which are more than sufficient to allow the method to be used for either confirmation or quantitation.

Although this work focused only on the analysis of peanut butter, future work will be done to apply this method to other food products including peanuts and maize. To facilitate sample sharing between labs, sample stability will also be evalu-ated to determine the best way to preserve and ship samples.

Acknowledgements We would like to specially thank David Lavorato, Yun Yun Zou, Adam Latawiec, and Andre Schreiber from Applied Biosystems in Toronto, Canada for running samples, interpreting data, and providing advice regarding this project.

References1. H.P. van Egmond, Analytical Bioanalytical Chemistry 2004, 378, 1152-1160.

2. Food and Agriculture Organization. Worldwide Regulations for Mycotoxins in Food and Feed, 2003, FAO Food and Nutr. Paper, 2004, 81.

3. M.C. Spanjer, P.M. Rensen and J.M. Scholten, Food Additives and Contami-nants, 2008, 25, 472-489.

4. V.A. Vega, J. AOAC International 2005, 88, 1383-1386.

5. M. Castegnaro, M. Tozlovanu, C. Wild, An Molinie, A. Sylla and A. Pfohl-Leszkowicz, Mol. Nutr. Food Res. 2006, 50, 480-487.

6. V. Sobolev, J. Agric. Food Chem. 2007, 55, 2136-2141.

Table 5Representative Signal to Noise Ratios at 5 ng/ml spiked Standard level

* Signal to Noise (S/N) being the peak height divided by the noise measured at three standard deviations of the noise. ** % CV estimates from 2 replicate samples, with 2 injections for each sample (n=4).


Aflatoxin B1 185.5 5.6 97.8

Aflatoxin B2 63.5 3.4 99.2

Aflatoxin G1 120.5 5.4 96.2

Aflatoxin G2 49.5 11.0 97.5

APPLICATIONSTN-0022

Page 8 of 8

t:f:

Australia02-9428-6444 02-9428-6445 [email protected]

t:f:

Austria 01-319-1301 01-319-1300 [email protected]

t:f:

Belgium+31 (0)30-2418700 +31 (0)30-2383749 [email protected]

t:f:

Canada(800) 543-3681(310) 328-7768 [email protected]

t:f:

Denmark4824 80484810 6265 [email protected]

t:f:

France01 30 09 21 10 01 30 09 21 11 [email protected]

t:f:

Germany 06021-58830-0 06021-58830-11 [email protected]

t:f:

Ireland 01 247 5405 +44 1625-501796 [email protected]

t:f:

Italy 051 6327511 051 6327555 [email protected]

t:f:

Luxembourg+31 (0)30-2418700 +31 (0)30-2383749 [email protected]

t:f:

Netherlands030-2418700 030-2383749 [email protected]

t:f:

New Zealand09-478095109-4780952 [email protected]

t:f:

Puerto Rico (800) 541-HPLC (310) 328-7768 [email protected]

t:f:

United Kingdom 01625-501367 [email protected]

t:f:

All other countries:Corporate Offi ce USA (310) 212-0555(310) 328-7768 [email protected]

www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at [email protected].

TrademarksStrata is a registered trademark of Phenomenex, Inc. Kinetex and SecurityGuard are trademarks of Phenomenex, Inc. TurbolonSpray and Q TRAP are registered trademarks, and API 3000, API 3200, and TURBO V are trademarks of Life Technologies Corporation and its affi liated company Applied Biosystems. Florisil is a registered trademark U.S. Silica Co.

DisclaimerPhenomenex is not affi liated with Applied Biosystems or U.S. Silica Co. Subject to Phenomenex Standard Terms & Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions.

© 2009 Phenomenex, Inc. All rights reserved.

TN68

8304

09_L

If Strata® SPE products and Kinetex™ core-shell HPLC columns do not provide at least equivalent results and separations as com-pared to products of similiar dimensions, phase, and particle size, return the product with your comparative data within 45 days for a FULL REFUND.

Strata® Ordering Information Sample PreparationPart No. Description Unit8B-S013-HBJ Strata Florisil® (500 mg/3 mL) 50/box8B-S012-FBJ Strata Silica (200 mg/3 mL) 100/box

KrudKatcher™ Ultra In-Line Filter Ordering Information

Disposable in-line fi lter fi ts virtually all UHPLC/HPLC columns 1.0 to 4.6 mm ID. Extremely low dead volume minimizes sample peak dispersion. Pressure rated to 20,000 psi(1,375 bar).

KrudKatcher™ Ultra In-Line FilterPart No. Description UnitAF0-8497 KrudKatcher Ultra In-Line Filter,

0.5 µm Porosity x 0.004 in. ID 3/pk

Installation wrench not provided. KrudKatcher Ultra requires 5/16 in. wrench.

Kinetex™ Ordering Information

2.6 μm Minibore Columns (mm)

50 x 2.1 100 x 2.1 150 x 2.1

PFP 00B-4477-AN 00D-4477-AN 00F-4477-AN

2.6 μm Solvent Saver Midbore Columns (mm)50 x 3.0 100 x 3.0 150 x 3.0

PFP 00B-4477-Y0 00D-4477-Y0 00F-4477-Y0

2.6 μm Analytical Columns (mm)

50 x 4.6 100 x 4.6 150 x 4.6

PFP 00B-4477-E0 00D-4477-E0 00F-4477-E0

tn-0022 applications · 2016. 12. 16. · applications tn-0022 page 2 of 8 for additional technical...

Documents