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Determination of Phthalates, Including DEHP, by LC/MS/MS and GC/MS to Aid in Food Safety Analysis

Seyed Sadjadi, Matthew Trass, Jeff Layne and Sky Countryman

Phenomenex, Inc., 411 Madrid Ave.,Torrance, CA 90501 USA

The recent discovery by Taiwan’s Department of Health of the chemical Bis(2-ethylhexyl) phthalate (DEHP) in some food products resulted in many countries pulling juices, teas, and other products originating from Taiwan from grocery store shelves. Phthalates are carcinogenic and are banned by EU, Chinese, and Taiwanese legislation. This most recent scare has only served to emphasize the need for fast and accurate analytical techniques to ensure the safety of our food. In this work, we demonstrate the separation of 21 phthalates using both LC/MS/MS and GC/MS.

Analysis of phthalates by LC/MS/MS is challenging due to the low ionization potential for the analytes as well as background contamination in the system. Good system maintenance, source optimization and mobile phase selection are critical to achieving good sensitivity.

GC/MS has been widely used for phthalate analysis, but late elution of the heavier compounds can lead to low detection limits. In this study we utilized a high temperature stable column and fast ramping GC oven to maximize peak intensity.

Introduction

Representative Phthalate Structures

Bis(2-ethylhexyl) phthalate

Dimethyl phthalate

Diisobutyl phthalate

Dicyclohexyl phthalate

Di-n-octyl phthalate

Benzyl butyl phthalate

Synergi™ Polar-RP

Ether linkage increases aromaticity of the phenyl group and also provides pi-pi interactions with conjugated compounds

Polar endcapping increases selectiv-ity for phthalates by interaction

with ester functional groups.

Ultra-pure silica

LC/MS/MS Conditions

HPLC conditions:Column: Synergi Polar-RP 2.5 μm

Dimensions: 100 x 2.0 mmPart No.: OOD-4371-B0

Mobile Phase: A: 0.1 % Formic acid in Water B: 0.1 % Formic acid in Acetonitrile:Methanol 50:50

Gradient:

Flow Rate: 0.4 mL/minInjection Volume: 5 µL

Time (min) B (%)

0.00 40

1.00 50

6.00 50

11.00 85

14.00 85

14.10 40

19.00 40

MRM conditions:Q1 Q3 ID

195.1 163.1/77.0 Dimethyl phthalate (DMP)

223.1 149.1/177.1 Diethyl phthalate (DEP)

247.0 189.2 Diallyl phthalate (DAP)

251.0 149.1/191.2 Di-n-propyl phthalate (DPP)

279.1 205.2 Di-n-butyl phthalate (DBP)

279.1 223.2/205.2 Diisobutyl phthalate (DIBP)

283.1 59.0/207.2 Bis(2-methoxyethyl phthalate (DMEP)

307.2 149.2/219.2 Di-n-pentyl phthalate (DNPP)

311.2 211.1/73.0 Bis(2-ethoxyethyl phthalate (DEEP)

313.2 205.2/149.1 Benzyl butyl phthalate (BBzP)

331.2 167.1/149.1 Dicyclohexyl phthalate (DCHP)

335.2 149.1 Di-n-hexyl phthalate (DNHP)

335.2 167.2/149.1 Di-(4-methyl-2-pentyl) phthalate (DMPP)

363.1 247.0/149.2 Di-n-heptyl phthalate

367.2 101.1 Bis(2-n-butoxyethyl) phthalate (DBEP)

391.3 167.1/149.1 Bis(2-ethylhexyl phthalate (DEHP)

391.3 149.1 Di-n-octyl phthalate (DNOP)

419.1 275.3/149.2 Di-n-nonyl phthalate (DNP)

419.1 127.3/149.2 Diisononyl phthalate (DINP)

447.2 141.2/289.2 Diisodecyl phthalate (DIDP)

AB SCIEX™ API 4000™

Ion source conditions:IS: 5500 V

TEM: 600 °CGas1: 50Gas2: 50

DP: 30Scan Type: MRM

Results

2 4 6 8 10 12 14 16 min0.0

5.0e5

1.0e6

1.5e6

2.0e6

2.5e6

3.0e6

3.5e6

4.0e6

4.5e6

Inte

nsity

, cps

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

Analyte list: 1. Bis(2-methoxyethyl) phthalate2. Dimethyl phthalate3. Bis(2-ethoxyethyl) phthalate4. Diethyl phthalate5. Diallyl phthalate 6. Di-n-propyl phthalate 7. Diisobutyl phthalate 8. Di-n-butyl phthalate9. Bis(2-n-butoxyethyl) phthalate

10. Benzyl butyl phthalate11. Di-n-pentyl phthalate12. Dicyclohexyl phthalate13. Di-(4-methyl-2-pentyl) phthalate 14. Di-n-hexyl phthalate 15. Di-n-heptyl phthalate 16. Bis(2-ethylhexyl) phthalate 17. Di-n-octyl phthalate18. Diisononyl phthalate19. Di-n-nonyl phthalate20. Diisodecyl phthalate

Ap

p ID

199

32

Figure 1. Phthalates standard mix by LC/MS/MS on a Synergi Polar-RP 2.5 µm, 100 x 2.0mm column

Separation of Critical Isomers

Figure 3. Bis(2-ethylhexyl) phthalate & Di-n-octyl phthalate

Figure 4. Di-(4 methyl-2-pentyl) phthalate & Di-n-hexyl phthalate

Figure 2. Diisobutyl phthalate & Di-n-butyl phthalate

2 4 6 8 10 12 14 16 min

19935

14

13

Ap

p ID

199

35

1 2 3 4 5 6 7 8 9 10 11 12 13 14 min

19934

17

16A

pp

ID 1

9934

2 4 6 8 10 12 14 16 min

19933

8

7

Ap

p ID

199

33

Figure 5. Diisononyl phthalate & Di-n-nonyl phthalate

2 4 6 8 10 12 14 16 min

19936

19

18

Ap

p ID

199

36

Separation of Critical Isomers cont

Zebron ZB-50

High polarity column capable of high-temperature bake-out to remove contaminants

Temperature Limits: 40 to 320/340 ˚C (Isothermal/TPGC)

Si O

CH3

CH3

50 %

Si OPhenyl functionality improves selectivity

for phthalates

50 %

GC/MS

Figure 6. Phthalates standard mix by GC/MS on a ZB-50 30 m x 0.25 mm x 0.25 µm column

Column: Zebron ZB-50 Dimensions: 30 meter x 0.25 mm x 0.25 µm

Part No.: 7HG-G004-11Injection: Split 10:1@ 260ºC, 1 µL

Carrier Gas: Helium @ 1 mL/min

Oven: 135 ºC to 275 ºC @ 25 ºC/min for 3.5 min to 340 ºC @ 35 ºC/min

MSD: Scan mode 45-500 da.

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 10.51.5 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 min

19931

2

3

45

6,78

9

10 11

12

14

1516

17 18

19

13

1

20.21

Analyte list: 1. Dimethyl phthalate 2. Diethyl phthalate 3. Diallyl phthalate 4. Di-n-propyl phthalate 5. Di-n-butyl phthalate 6. Diisobutyl phthalate 7. Di-n-hexyl phthalate 8. Bis(2-methoxyethyl) phthalate9. Di-n-pentyl phthalate

10. Bis(2-ethoxyethyl) phthalate 11. Di-(4-methyl-2-pentyl) phthalate 12. Bis(2-ethylhexyl) phthalate 13. Benzyl butyl phthalate 14. Di-n-heptyl phthalate15. Bis(2-n-butoxyethyl) phthalate 16. Dicyclohexyl phthalate 17. Di-n-octyl phthalate 18. Diphenylhexyl phthalate19. Di-n-nonyl phthalate 20. Diisononyl phthalate21. Diisodecyl phthalate

Figure 7. Separation of Isobaric Compounds

Compound found in Taiwansese food

products (DEHP)

Ap

p ID

199

31

Figure 1 shows a representative chromatogram of the phthalates mixture. The analysis was run on a Synergi Polar-RP 2.5 µm, 100 x 2.0 mm column with a run time of under 13 minutes.

Despite their relatively inert functionality, phthalates are a difficult class of compound to analyze via LC/MS/MS for several reasons. Firstly, there are significant background levels of phthalates in the API (Atmospheric Pressure Ionization) source, which makes compound tuning problematic. Secondly, several of the analytes are isomers which makes it necessary to tune with individual standards, as well as separate the isomeric compounds chromatographically. Figures 2-5 show the chromatographic separation of each of the closely eluting isomeric phthalates.

In addition to LC/MS/MS, the same group of analytes, with the addition of diphenyl phthalate (diphenyl phthalate did not produce a useable precursor ion in the ESI source), were analyzed using GC/MS (Figure 6). The run-time on a ZB-50 30 m x 0.25 mm x 0.25 µm column was under 11 minutes. Similarly to the LC/MS/MS method, many of the

compounds required chromatographic separation due to virtually identical fragmentation patterns. (Figure 7).

Note that peaks 6 and 7 (Diisobutyl phthalate and Di-n-hexyl phthalate) could not be separated without compromising the resolution of DEHP (DEHP was given precedence for this method).

By the time peak 18 (Diphenylhexyl phthalate) elutes, the GC oven temperature is in excess of 300 ºC. In this instance, a high-temperature stable column is important for two reasons. Firstly, low bleed at high temperature means greater sensitivity since the signal to noise ratio remains high. Secondly, running at high temperature allows the analytes to elute more quickly from the column creating narrow peaks, again resulting in greater sensitivity.

The high temperature stability of the GC column is demonstrated by the virtually insignificant increase in the baseline signal during the final minutes of the run.

Results and Discussion

Conclusions

Phthalates, which are found in a very wide array of consumer products, can be successfully analyzed by both LC/MS/MS and GC/MS.

Because these compounds fragment similarly, chromatography is relied upon

to separate the analytes for quantitation. Therefore, co lumn se lec t ion and optimization of method conditions are of paramount importance.

TrademarksSynergi and Zebron are trademarks of Phenomenex, Inc. API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX is being used under license. © 2011 Phenomenex, Inc. All rights reserved.