Waters Corporation, Milford MA, USA; 2. Waters …...Hesperidin (@ λ 283 nm) (A) is not detected in...
1
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2013 Waters Corporation ADULTERATION IN FRUIT JUICES: A SOLUTION TO A COMMON PROBLEM WITH THE USE OF HIGH RESOLUTION LIQUID CHROMATOGRAPHY, UV DETECTION, QUADRUPOLE-TIME OF FLIGHT MS AND MULTIVARIATE DATA ANALYSIS Marian Twohig 1 , Antonietta Gledhill 2 , Jennifer A. Burgess 1 and Ramesh Rao 2 1. Waters Corporation, Milford MA, USA; 2. Waters Corporation, Manchester, United Kingdom INTRODUCTION Economically motivated adulteration of food has emerged as a growing problem in the food industry due to its extremely lucrative outcome. Economic adulteration has far reaching consequences in the food manufacturing chain, it impacts the profits of reliable food producers and can pose potential threats to the health of unsuspecting consumers. The most common forms of adulteration that occur within the fruit juice industry usually take the form of juice dilution, the addition of high fructose corn syrup (HFCS) or the addition of other fruit juices [1,2]. Analytical methods that have been used to identify adulteration have been reviewed [3-5]. There is a need for highly informative analytical testing methods to help to authenticate ingredients and finished products. In this study, pineapple juice samples were analysed by high resolution liquid chromatography coupled with photodiode array and accurate mass detection. Data interpretation involved multivariate analysis (MVA) and database searching in order to identify any key differences between authentic and adulterated pineapple juices. Several citrus compounds were identified in commercially available juice claiming to be pure pineapple. Figure 1. Juice profiling workflow. Process and extract peaks Use chemometrics to ID marker compounds (MarkerLynx XS) Structural elucidation of identified marker compounds (MS E , EleComp, Chemspider and MassFragment) Acquire standards using LC MS/MS and compare standard results with samples (UPLC Xevo G2QToF) Samples of unknown authenticity High resolution chromatographic separation High sensitivity and accurate mass (UPLC Xevo G2 QToF) METHODS Samples: Three pineapple juice concentrate samples were obtained from a collaborator. Additional pineapple juice samples were purchased from local grocery stores. All samples were centrifuged, filtered and diluted before analysis using UPLC/PDA/Xevo G2 QTof MS. A description of the pineapple samples is given in Table 1. Table 1. Description of the pineapple juice samples. Name Sample Description S10 Study sample - adulterated S11 Study sample - adulterated S12 Study sample - authentic LJ Bought – Unknown authenticity KJ Bought – Unknown authenticity RESULTS AND DISCUSSION The scores plot resulting from PCA shows that S12 and S11 group closely together (Figure 2). Sample S10 is separated indicating something about this sample is distinctly different. Figure 3 shows an S-Plot from the OPLS-DA model of S12 and S10. Four markers selected as examples are highlighted in the S-plot and a trend plot of these is shown in Figure 4. Figure 2. The PCA scores plot obtained from 6 replicate injections of all the samples. Figure 3. S-plot of S12 (authentic) versus S10 (adulterated): Each dot represents an EMRT pair and the dots on the top right are potential marker compounds found at higher levels in Figure 4. Trend plots of four potential markers. 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000 S12 S12 S12 S12 S12 S12 S12 S12 S11 S11 S11 S11 S11 S11 S11 S11 S10 S10 S10 S10 S10 S10 S10 S10 QC QC QC QC QC QC QC LWOPAJ LWOPAJ LWOPAJ LWOPAJ LWOPAJ LWOPAJ LWOPAJ LWOPAJ KnPAJ KnPAJ KnPAJ KnPAJ KnPAJ KnPAJ KnPAJ KnPAJ Sample Group Variables EZinf o 2 - Negativ e_Pineapple_w_QC (M6: PCA-X) - 2011-03-11 10:35:34 (UTC-5) QC S10 LJ KJ S11 S12 KJ LJ Purchased juice (LJ) S10 - Adulterated S12 - Authentic Purchased juice (KJ) S11 - Adulterated S12 S10 Rt 5.10 m/z 711.2862 Rt 5.55 m/z 579.1712 Rt 5.67 m/z 609.1616 Rt 5.55 m/z 579.1712 Rt 5.10 m/z 711.2862 Rt 5.03 m/z 649.2495 Figure 6. XIC of m/z 609.1820 from standard hesperidin (A) and adulterated pineapple juice, S10 (B) with their respective high energy MS E spectra in the insets. The trend plot shows that S10, LJ and the QC sample (a mix of all the samples) were the only samples to contain the marker with m/z 609.1816 (the turquoise trace in Figure 4). In addition, S10 also contained the three other example markers. The molecular formula of the potential marker with m/z 609.1816 was determined to be C 28 H 34 O 15 . A possible identification as hesperidin was given by Chemspider. The MS E fragment data matched the hesperidin fragmentation proposed by MassFragment (Figure 5). Figure 5. MS E high energy spectrum showing MassFragment assignment of hesperidin exact mass ions. Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 % 0 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 % 0 100 PA_24_2_098a 2: TOF MSMS ES- 609.182 0.0050Da 1.96e3 5.67 PA_24_2_093a 2: TOF MSMS ES- 609.182 0.0050Da 3.57e3 5.67 m/z 100 200 300 400 500 600 % 0 100 301.0707 609.1819 Hesperidin Standard S10 A B m/z 100 200 300 400 500 600 % 0 100 301.0706 609.1821 301.0707 609.1819 5.67 5.67 301.0706 609.1821 m/z 100 200 300 400 500 600 % 0 100 301.0706 609.1821 609.1820 (+0.1mDa) 28 H 33 O 16 (-H 2 ) 301.0706 609.1821 301.0712 (-0.6mDa) C 16 H 13 O 6 (-C 12 H 22 O 9 ) Figure 7. XIC for m/z 609.182 using a 5mDa window. A: Hesperidin standard, B: S10 (Adulterated juice) C: S12 (Authentic juice) D: LJ (purchased juice). Insets are at ~10X magnification. Figure 8. Hesperidin (@ λ 283 nm) (A) is not detected in the UV data due to lack of sensitivity. (B) XIC for m/z 609.1819 is clearly detected at 5.67 mins in the MS data. The trend plot (Figure 3) suggested that hesperidin was present in LJ (juice purchased locally). Using the same workflow the presence of hesperidin was confirmed. As can be seen in Figure 7 hesperidin was detected in the store purchased sample but not the authentic juice sample. The concentration, however, was too low for UV detection, as shown in the UV chromatogram at 283 nm (Figure 8). Time 2.50 5.00 7.50 % 0 100 PA_24_2_065a 1: TOF MS ES- 609.182 0.0050Da 1.08e4 5.67 Time 2.50 5.00 7.50 % 0 100 PA_24_2_062a 1: TOF MS ES- 609.182 0.0050Da 1.08e4 5.66 Time 2.50 5.00 7.50 % 0 100 PA_24_2_066a 1: TOF MS ES- 609.182 0.0050Da 1.08e4 5.66 Time 2.50 5.00 7.50 % 0 100 PA_24_2_061a 1: TOF MS ES- 609.182 0.0050Da 1.08e4 A C D B Time 5.00 5.50 6.00 6.50 % 0 100 PA_24_2_061a 1: TOF MS ES- 609.182 0.0050Da 1000 Time 5.00 5.50 6.00 6.50 % 0 100 PA_24_2_065a 1: TOF MS ES- 609.182 0.0050Da 1000 5.67 Purchased juice (LJ) S10 Adulterated S12 Authentic Hesperidin Standard Time 2.00 3.00 4.00 5.00 6.00 7.00 8.00 % 0 100 2.00 3.00 4.00 5.00 6.00 7.00 8.00 0.0 5.0e-2 1.0e-1 1.5e-1 2.0e-1 4_2_013 4: Diode Array 283 Range: 2.939e-1 4.88 4.28 1.29 2.45 4_2_013 1: TOF MS ES- 609.182 0.0030Da 274 5.67 A B PDA @ 283 nm Store purchased (LJ) ToF MS m/z 609.1819 Store purchased (LJ) The other example markers in the S-plot were also investigated. Three potential compounds narirutin, limonin 17-beta-D-glucopyranoside (LG) and nomilinic acid 17-beta-D-glucopyranoside (NAG) were identified. Hesperidin, narirutin, LG and NG are found in citrus fruits [7,8]. Hesperidin is the most abundant flavanoid found in sweet orange juices, the next most abundant is narirutin [7]. The commercial sample LJ also looks to contain a small quantity of orange juice, or possibly lemon juice. The adulteration of S10 with orange juice was later confirmed with the collaborator. CONCLUSIONS Without prior knowledge of the samples, four citrus compounds were identified in an adulterated pineapple juice sample. MS E enabled the simultaneous acquisition of exact mass precursor and fragment ions, which was used in the process of compound identification. The high sensitivity of the Xevo G2 QTof MS enabled the detection of hesperidin in a store-purchased product that claimed to be authentic pineapple juice. PDA detection was not able to detect this low level, demonstrating the requirement for ultra sensitive techniques in the search for potential adulterants. UPLC with QToF MS can be used to obtain highly detailed information to help in the process of food authentication. References 1. W. Simpkins and M. Harrison, Trends in Food Sci. and Technol., 6 (10), 321-328, 1995. 2. P.R. Ashurst, Chemistry and Technology of Soft Drinks and Fruit Juices, 2nd edition, Black- well Publishing, Oxford, 2005. 3. A. Yamamoto, M. Kawai, T. Miwa, T. Tsukamoto, S. Kodama and K. Hayakawa, J. Agric. Food Chem., 56 (16), 7302-7304, 2008. 4. D. Cautela, B. Laratta, F. Santelli, A. Trifirò, L. Servillo and D. Castaldo, J. Agric. Food Chem., 56 (13), 5407–5414, 2008. 5. Y. Zhang, D. Krueger, R. Durst, R. Lee, D. Wang, N. Seeram and D. Heber, J. Agric. Food Chem., 57 (6), 2550–2557, 2009. 6. L. Eriksson, E. Johansson, N. Kettaneh-Wold, J. Trygg, C. Wikström and S. Wold, “Multi- and Megavariate Data Analysis: Basic Principles and Applications”, 2nd edition, Umetrics Academy, 2006. 7. G. Gattuso, D. Barreca, C. Gargiulli, U. Leuzzi, C. Caristi., Molecules 12, 1641-1673, 2007. 8. S. Hasegawa, M. Miyake., Food Rev. Int. 12, 413-435, 1996. To confirm the identification, a standard of hesperidin was analysed and compared to the component in the sample, as shown in Figure 6. UV conditions: UV system: ACQUITY PDA Detector Range: 210-500nm Sampling rate: 20 pts /s QTof MS Conditions: MS System: Xevo™ G2 Quadrupole Time-of-flight (QToF) Ionization Mode: ESI Negative (ESI-) Analyser Mode: Resolution Capillary Voltage (kV): 2.0 Cone Voltage: 25 V Desolvation Temp: 450˚C Desolvation Gas: 900 L/Hr Source Temp: 130 ˚C MS E Low Energy Collision Energy: 4 eV High Energy Collision Energy: 15-45 eV Acquisition Range: 50—1200 m/z Scan time: 0.1 sec Lock Mass reference: Leucine Enkephalin Data Analysis: Data analysis and trending was performed using MarkerLynx XS. This software solution performs multivariate analysis (MVA) [6] on mass spectral data sets using the Exact Mass Retention Time (EMRT) pairs. Principal Component Analysis (PCA) was used for initial investigation followed by a more predictive multivariate model - orthogonal projection to latent structures-discriminate analysis (OPLS-DA). OPLS-DA allows a relationship to be drawn between the classes and the potential marker compounds in each sample group. Markers identified by MarkerLynx were then automatically transferred to EleComp, Chemspider and MassFragment, in order to obtain the elemental composition and potential structures for the key markers.
Transcript of Waters Corporation, Milford MA, USA; 2. Waters …...Hesperidin (@ λ 283 nm) (A) is not detected in...
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ADULTERATION IN FRUIT JUICES: A SOLUTION TO A COMMON PROBLEM WITH
THE USE OF HIGH RESOLUTION LIQUID CHROMATOGRAPHY, UV DETECTION,
QUADRUPOLE-TIME OF FLIGHT MS AND MULTIVARIATE DATA ANALYSIS
Marian Twohig1, Antonietta Gledhill2, Jennifer A. Burgess1 and Ramesh Rao2
1. Waters Corporation, Milford MA, USA; 2. Waters Corporation, Manchester, United Kingdom
INTRODUCTION
Economically motivated adulteration of food has emerged as a
growing problem in the food industry due to its extremely
lucrative outcome. Economic adulteration has far reaching
consequences in the food manufacturing chain, it impacts the
profits of reliable food producers and can pose potential
threats to the health of unsuspecting consumers.
The most common forms of adulteration that occur within the
fruit juice industry usually take the form of juice dilution, the
addition of high fructose corn syrup (HFCS) or the addition of
other fruit juices [1,2]. Analytical methods that have been
used to identify adulteration have been reviewed [3-5]. There
is a need for highly informative analytical testing methods to
help to authenticate ingredients and finished products.
In this study, pineapple juice samples were analysed by high
resolution liquid chromatography coupled with photodiode
array and accurate mass detection. Data interpretation
involved multivariate analysis (MVA) and database searching
in order to identify any key differences between authentic and
adulterated pineapple juices. Several citrus compounds were
identified in commercially available juice claiming to be pure
pineapple.
Figure 1. Juice profiling workflow.
Process and extract peaksUse chemometrics to ID marker compounds
(MarkerLynx XS)
Structural elucidation of identifiedmarker compounds
(MSE, EleComp, Chemspider and MassFragment)
Acquire standards using LC MS/MS and compare standard results with samples
(UPLC Xevo G2QToF)
Samples of
unknown authenticity
High resolution chromatographic separationHigh sensitivity and accurate mass
(UPLC Xevo G2 QToF)
METHODS
Samples:
Three pineapple juice concentrate samples were obtained from a collaborator. Additional pineapple juice samples were purchased from local grocery stores.
All samples were centrifuged, filtered and diluted before analysis using UPLC/PDA/Xevo G2 QTof MS. A description of the pineapple samples is given in Table 1.
Table 1. Description of the pineapple juice samples.
Name Sample Description
S10 Study sample - adulterated
S11 Study sample - adulterated
S12 Study sample - authentic
LJ Bought – Unknown authenticity
KJ Bought – Unknown authenticity
RESULTS AND DISCUSSION
The scores plot resulting from PCA shows that S12 and S11 group closely together (Figure 2). Sample S10 is separated indicating something about this sample is distinctly different.
Figure 3 shows an S-Plot from the OPLS-DA model of S12 and S10. Four markers selected as examples are highlighted in the S-plot and a trend plot of these is shown in Figure 4.
Figure 2. The PCA scores plot obtained from 6 replicate injections of all the samples.
Figure 3. S-plot of S12 (authentic) versus S10 (adulterated): Each dot represents an EMRT pair and the dots on the top right are potential marker compounds found at higher levels in
Figure 4. Trend plots of four potential markers.
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
S12
S12
S12
S12
S12
S12
S12
S12
S11
S11
S11
S11
S11
S11
S11
S11
S10
S10
S10
S10
S10
S10
S10
S10 QC
QC
QC
QC
QC
QC
QC
LW
OP
AJ
LW
OP
AJ
LW
OP
AJ
LW
OP
AJ
LW
OP
AJ
LW
OP
AJ
LW
OP
AJ
LW
OP
AJ
Kn
PA
J
Kn
PA
J
Kn
PA
J
Kn
PA
J
Kn
PA
J
Kn
PA
J
Kn
PA
J
Kn
PA
J
Sample Group
Variables
DS1: 5.55_579.1712DS1: 5.67_609.1816DS1: 5.03_649.2495DS1: 5.10_711.2862
EZinf o 2 - Negativ e_Pineapple_w_QC (M6: PCA-X) - 2011-03-11 10:35:34 (UTC-5)
QC
S10
LJ
KJS11S12
KJLJ
Purchased juice (LJ)
S10 - Adulterated
S12 - Authentic
Purchased juice (KJ)
S11 - Adulterated
S12 S10
Rt 5.10m/z 711.2862
Rt 5.55m/z 579.1712
Rt 5.67m/z 609.1616
Rt 5.55m/z 579.1712
Rt 5.10m/z 711.2862
Rt 5.03m/z 649.2495
Figure 6. XIC of m/z 609.1820 from standard hesperidin (A) and adulterated pineapple juice, S10 (B) with their respective high energy MSE spectra in the insets.
The trend plot shows that S10, LJ and the QC sample (a mix of all the samples) were the only samples to contain the marker with m/z 609.1816
(the turquoise trace in Figure 4). In addition, S10 also contained the three other example markers. The molecular formula of the potential marker with m/z 609.1816 was
determined to be C28H34O15. A possible identification as hesperidin was given by Chemspider. The MSE fragment data matched the hesperidin fragmentation proposed by MassFragment (Figure 5).
Figure 5. MSE high energy spectrum showing MassFragment assignment of hesperidin exact mass ions.
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00
%
0
100
PA_24_2_098a 2: TOF MSMS ES- 609.182 0.0050Da
1.96e3
5.67
PA_24_2_093a 2: TOF MSMS ES- 609.182 0.0050Da
3.57e3
5.67
m/z100 200 300 400 500 600
%
0
100
m/z100 200 300 400 500 600
%
0
100301.0707
609.1819
301.0706
609.1821
HesperidinStandard
S10
A
B
m/z100 200 300 400 500 600
%
0
100
m/z100 200 300 400 500 600
%
0
100301.0707
609.1819
301.0706
609.1821
301.0707
609.1819
5.67
5.67
301.0706
609.1821
m/z100 200 300 400 500 600
%
0
100
m/z100 200 300 400 500 600
%
0
100301.0707
609.1819
301.0706
609.1821
609.1820 (+0.1mDa)C28H33O16(-H2)
301.0706
609.1821
301.0712 (-0.6mDa)C16H13O6 (-C12H22O9)
Figure 7. XIC for m/z 609.182 using a 5mDa window. A: Hesperidin standard, B: S10 (Adulterated juice) C: S12 (Authentic juice) D: LJ (purchased juice). Insets are at ~10X
magnification.
Figure 8. Hesperidin (@ λ 283 nm) (A) is not detected in the UV data due to lack of sensitivity. (B) XIC for m/z 609.1819 is clearly detected at 5.67 mins in the MS data.
The trend plot (Figure 3) suggested that hesperidin was present in LJ (juice purchased locally). Using the same workflow the presence of hesperidin
was confirmed. As can be seen in Figure 7 hesperidin was detected in the store purchased sample but not the authentic juice sample. The concentration, however, was too low for UV detection, as shown in the UV chromatogram at 283 nm (Figure 8).
Time2.50 5.00 7.50
%
0
100
PA_24_2_065a 1: TOF MS ES- 609.182 0.0050Da
1.08e4
5.67
Time2.50 5.00 7.50
%
0
100
PA_24_2_062a 1: TOF MS ES- 609.182 0.0050Da
1.08e45.66
Time2.50 5.00 7.50
%
0
100
PA_24_2_066a 1: TOF MS ES- 609.182 0.0050Da
1.08e4
5.66
Time2.50 5.00 7.50
%
0
100
PA_24_2_061a 1: TOF MS ES- 609.182 0.0050Da
1.08e4
A
C D
B
Time5.00 5.50 6.00 6.50
%
0
100
PA_24_2_061a 1: TOF MS ES- 609.182 0.0050Da
1000
Time5.00 5.50 6.00 6.50
%
0
100
PA_24_2_065a 1: TOF MS ES- 609.182 0.0050Da
10005.67
Purchased juice (LJ)
S10 Adulterated
S12Authentic
HesperidinStandard
Time2.00 3.00 4.00 5.00 6.00 7.00 8.00
%
0
100
2.00 3.00 4.00 5.00 6.00 7.00 8.00
AU
0.0
5.0e-2
1.0e-1
1.5e-1
2.0e-1
PA_24_2_013 4: Diode Array 283
Range: 2.939e-1
4.88
4.28
1.29
2.45
PA_24_2_013 1: TOF MS ES- 609.182 0.0030Da
274
5.67
A
B
PDA @ 283 nmStore purchased
(LJ)
ToF MS m/z 609.1819Store purchased
(LJ)
The other example markers in the S-plot were also investigated. Three potential compounds narirutin, limonin 17-beta-D-glucopyranoside (LG) and
nomilinic acid 17-beta-D-glucopyranoside (NAG) were identified. Hesperidin, narirutin, LG and NG are found in citrus fruits [7,8]. Hesperidin is the most abundant flavanoid found in sweet orange juices, the next most
abundant is narirutin [7]. The commercial sample LJ also looks to contain a small quantity of orange juice, or possibly lemon juice.
The adulteration of S10 with orange juice was later confirmed with the collaborator.
CONCLUSIONS
Without prior knowledge of the samples, four citrus
compounds were identified in an adulterated pineapple juice sample.
MSE enabled the simultaneous acquisition of exact mass
precursor and fragment ions, which was used in the process of compound identification.
The high sensitivity of the Xevo G2 QTof MS enabled the
detection of hesperidin in a store-purchased product that
claimed to be authentic pineapple juice.
PDA detection was not able to detect this low level,
demonstrating the requirement for ultra sensitive
techniques in the search for potential adulterants.
UPLC with QToF MS can be used to obtain highly detailed
information to help in the process of food authentication.
References
1. W. Simpkins and M. Harrison, Trends in Food Sci. and Technol., 6 (10), 321-328, 1995.
2. P.R. Ashurst, Chemistry and Technology of Soft Drinks and Fruit Juices, 2nd edition, Black-well Publishing, Oxford, 2005.
3. A. Yamamoto, M. Kawai, T. Miwa, T. Tsukamoto, S. Kodama and K. Hayakawa, J. Agric. Food Chem., 56 (16), 7302-7304, 2008.
4. D. Cautela, B. Laratta, F. Santelli, A. Trifirò, L. Servillo and D. Castaldo, J. Agric. Food Chem., 56 (13), 5407–5414, 2008.
5. Y. Zhang, D. Krueger, R. Durst, R. Lee, D. Wang, N. Seeram and D. Heber, J. Agric. Food Chem., 57 (6), 2550–2557, 2009.
6. L. Eriksson, E. Johansson, N. Kettaneh-Wold, J. Trygg, C. Wikström and S. Wold, “Multi- and Megavariate Data Analysis: Basic Principles and Applications”, 2nd edition, Umetrics Academy, 2006.
7. G. Gattuso, D. Barreca, C. Gargiulli, U. Leuzzi, C. Caristi., Molecules 12, 1641-1673, 2007.
8. S. Hasegawa, M. Miyake., Food Rev. Int. 12, 413-435, 1996.
To confirm the identification, a standard of hesperidin was analysed and compared to the component in the sample, as shown in Figure 6.
UV conditions:
UV system: ACQUITY PDA Detector Range: 210-500nm
Sampling rate: 20 pts /s QTof MS Conditions:
MS System: Xevo™ G2 Quadrupole Time-of-flight (QToF) Ionization Mode: ESI Negative (ESI-)
Analyser Mode: Resolution Capillary Voltage (kV): 2.0 Cone Voltage: 25 V
Desolvation Temp: 450˚C Desolvation Gas: 900 L/Hr Source Temp: 130 ˚C
MSE Low Energy Collision Energy: 4 eV High Energy Collision Energy: 15-45 eV Acquisition Range: 50—1200 m/z
Scan time: 0.1 sec Lock Mass reference: Leucine Enkephalin
Data Analysis:
Data analysis and trending was performed using MarkerLynx XS. This software solution performs multivariate analysis (MVA) [6] on mass spectral data sets using the Exact Mass Retention Time (EMRT) pairs.
Principal Component Analysis (PCA) was used for initial investigation followed by a more predictive multivariate model - orthogonal projection to latent structures-discriminate analysis (OPLS-DA). OPLS-DA allows a
relationship to be drawn between the classes and the potential marker compounds in each sample group.
Markers identified by MarkerLynx were then automatically transferred to EleComp, Chemspider and MassFragment, in order to obtain the elemental composition and potential structures for the key markers.
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