23

Focus on Food Analysis - Supplem

ent to AgroFOO

D industry hi-tech - Sep

temb

er/Octob

er 2011 - vol 22 n 5

Super fruit juice authenticityusing multivariate data analysis,

high resolution chromatography, UV and Time of Flight MS detection

MARIAN TWOHIG1, DANA A. KRUEGER2, ANTONIETTA GLEDHILL1, JINCHUAN YANG1, JENNIFER A. BURGESS1**Corresponding author

1. Waters Corporation , 34 Maple Street, Milford, MA 01757, USA2. Krueger Food Laboratories, 45 Manning Road, Suite 2, Billerica, MA 01821, USA

INTRODUCTION

The verifi cation of food sources and authenticity is an activity that has increased in importance over the last decade. Economic adulteration is defi ned as the intentional fraudulent modifi cation of a fi nished product or ingredient for economic gain. Economically motivated adulteration of food has emerged as a growing problem that can pose potential threats to the health of consumers. It also impacts the manufacturers of reliable food products economically and creates an enforcement challenge for food regulatory agencies (1). Economic adulteration and other types of counterfeiting in the food and consumer products industry is estimated to cost $10-$15 billion per year (2). The ways and reasons why food and beverage products are adulterated can be varied and whilst some can be harmful to health (melamine is a good example of this) others are misleading to the consumer. The process of adulteration includes unacceptable enhancement, dilution and/or substitution with less expensive ingredients, failure to declare contamination and inaccurate or misleading labelling of a product or ingredient (Figure 1). So

far the reported cases of adulterated fruit juices have been economically driven, for example, the inclusion of high fructose corn syrup (HFCS) or other fruit juices.In the case of pomegranate juice, recent epidemiological and clinical studies have identifi ed remarkable health benefi ts from the regular consumption of pomegranate juice, including prevention against cancer and cardiovascular disease(3-6). Pomegranate juice has gained a reputation for being a super food with demand often exceeding supply.As a result, the practice of adulteration with lower quality juices has become prevalent (7). Although many of the fruit juices available to consumers have been well documented and studied, consumer trend and buying preferences have changed in recent years with an increase in demand for more unusual fruit juices. For these types of products, there is a need for sophisticated testing methods to help authenticate ingredients and fi nished products. In this work, we illustrate how the combination of ultra-performance liquid chromatography (UPLC®) for high resolution separations, PDA along with accurate mass MS and MS/MS for detection and software tools applying chemometrics and database searching, allowed the facile identifi cation of differences between pomegranate juice samples.

EXPERIMENTAL

Pomegranate juice samplesThree pomegranate juice concentrate samples were obtained from a collaborator. One sample was known to be authentic and the two others were known to be adulterated. Further pomegranate juice samples and whole fruits were purchased from local grocery stores and via the internet. It is widely established that S4 is 100 percent authentic pomegranate juice. The juice concentrates were made up to single dose (16 Brix). One degree Brix is 1 gram of fruit soluble solids in 100 grams of solution and thus represents the strength of the solution as a percentage by weight (% w/w). A description of the pomegranate samples is given in Table 1.

ABSTRACT: Pomegranate juices from various sources and whole fruit pomegranate were analysed using Ultra Performance Liquid Chromatography (UPLC) coupled with Quadrupole Time-of-fl ight (QTof) mass spectrometry and multivariate data analysis tools. Chlorogenic acid was identifi ed in adulterated pomegranate juice samples and blends, but was absent from authentic pomegranate juice samples and pomegranate whole fruits grown in North America. In addition, an unknown compound with its molecular formula determined as C14H27O10, was found to exist in all pomegranate juices and pomegranate arils, but was not present in authentic cranberry, peach and blueberry juices. Without prior knowledge of the samples, the combination of UPLC, QTof-MS and multivariate data analysis tools enabled the detection of compounds that could be used as indicators of a pomegranate juice quality. MSE functionality allowed the acquisition of low energy precursor and high energy product ions in a single run. This information combined with exact mass measurement provided confi dence for structural elucidation.

Figure 1. Flow diagram showing various ways of food adulteration

Peer-reviewed industry perspective

M

onogra

phic supplement

24

Focu

s on

Food

Ana

lysis

- Su

pple

men

t to

Agro

FOO

D in

dustr

y hi

-tech

- Sep

tem

ber

/Oct

ober

201

1 - v

ol 2

2 n

5RESULTS AND DISCUSSION

Typical base peak intensity (BPI) chromatograms for the high and low collision energy (CE) MSE analysis are shown in Figure 3. The low collision energy chromatogram provides the exact mass precursor ion information, and the high collision energy gives the exact mass fragment ion data. These two pieces of data support structural identifi cation for

unknown analysis. In addition, data from the PDA provides useful chemical information that will also indicate the type of chemical structure of the compounds of interest. An initial overview of the data structure by principal component analysis (PCA-X) shows the six distinct pomegranate juice sample groups in the scores plot.

A scores plot explains the relationships between the observations in the data (Figure 4). Each point in the scores plot represents a single injection. To explain the patterns in the scores plot a loadings plot is used (Figure 5). Each symbol in the loadings plot represents a single EMRT pair. Interesting trends associated with two EMRT pairs with m/z 353.0868 at a retention time (Rt) of 3.18 min and 4.34 min were identifi ed using the loadings plot and variable trend plot. In Figure 6A and 6B, it can be seen that m/z 353.0868 at Rt 3.18 and Rt 4.34min is absent in S2 and S4, which are the samples known to be authentic pomegranate juice and also S5, which is a sample of unverifi ed authenticity.

Pomegranate fruitWhole fruit pomegranates were dissected into three components arils, pulp and skin. Each component was extracted with water, homogenised, centrifuged and fi ltered before analysis.

AnalysisAll samples were centrifuged, fi ltered and diluted prior to injection (3 µl) onto a Waters ACQUITY® HSS T3 2.1 x 100mm, 1.8mm UPLC column. UPLC separation was achieved using a gradient elution employing acetonitrile/10mM ammonium acetate. Detection involved simultaneously monitoring eluate using PDA detection over the range 210-500 nm at a rate of 20pts/sec and using an MSE enabled Waters Xevo® G2 Quadrupole Time-of-fl ight Mass Spectrometer operating in ESI mode. Both negative and positive ionization modes were investigated. The data from negative ionization mode (which was more information-rich) was selected for this investigation. Samples were analysed by UPLC ToF MS using MSE. MSE functionality allows the simultaneous acquisition of intact molecular ion and fragmentation information with high mass accuracy in a single run. Replicate pomegranate samples with a randomised injection order were analysed for reproducibility and stability assessment of the analytical method and instrumentation.

Data analysisChemometric data processing was carried out using the Waters MarkerLynx XS application manager and EZInfo using both the retention time and MS data. The software integrates and aligns the data converting them into exact mass retention time (EMRT) pairs. Those EMRT pairs can then be used for multivariate statistical analysis to visualize and interpret complex MS data sets (8). The workfl ow is shown in Figure 2.

Table 1. Description of the pomegranate samples.

Figure 2. Pomegranate juice profi ling workfl ow.

Figure 3. UPLC/PDA/Tof-MSE chromatograms from the analysis of a pomegranate juice sample.

Figure 4. The scores plot obtained on analysis of the six pomegranate juice samples using UPLC/Tof-MS data (Each color represents a sample and each sample was injected six times).

Focus on Food Analysis - Supplem

ent to AgroFOO

D industry hi-tech - Sep

temb

er/Octob

er 2011 - vol 22 n 5Focus on Food A

nalysis - Supplement to AgroFO

OD

industry hi-tech - Septem

ber/O

ctober 2011 - vol 22 n 5

25

Whole fruit experimentsSince the veracity of label claims regarding authenticity may always be in doubt, more information was needed to explain the presence/absence of chlorogenic acid in the juice samples. Whole fruit pomegranates all believed to be grown in North America were purchased and dissected into three components arils, pulp and skin. The pomegranate segments were extracted and analysed. Chlorogenic acid was not found in any part of the three pomegranates, as can be seen in the extracted ion chromatograms in Figure 8. Further LC/MS analysis on whole fruit pomegranates from different regions are required to fully

The presence of the high levels of these two EMRT pairs is clearly evident in the deliberately adulterated juice samples and the juice blend. The elemental composition for both components was determined to be C16H18O9. An online database search indicated chlorogenic acid as the top hit. Chlorogenic acid standard was analysed and found to have a retention time of 3.18 min (Figure 7A). The UV spectra of the standard chlorogenic acid and the peak in the sample were compared. UV spectra taken across the entire peak width indicated the presence of other compounds close to the retention time of chlorogenic acid (data not shown). The UV spectra did not provide suffi cient confi rmation that the component was indeed chlorogenic acid. The accuracy of the exact mass data for both the low energy precursor ions and high energy fragment ions increased the confi dence in the identifi cation of chlorogenic acid. Excellent agreement between the fragmentation pattern of the component at Rt 3.18 min in the samples and standard chlorogenic acid was observed in the MSE data, as shown in Figure 7C and D. The MSE data for the component at Rt 4.34 min revealed that it shared some common fragments with chlorogenic acid but also showed additional fragments. This suggests a possible isomer of chlorogenic acid (data not shown). A closer look at the chromatographic data suggested samples S3 and S1 (known to be adulterated) and a juice blend, S6, contained chlorogenic acid (m/z 353.0873) and possible isomers at 4.34 min and 3.09 min. Chlorogenic acid was not found in the authentic pomegranate juice samples S2 and S4. The authenticity of S5 was unverifi ed and it did not contain chlorogenic acid. Chlorogenic acid was also identifi ed in authentic peach, cranberry and blueberry juice (data not shown). Eleven different additional pomegranate juice samples, all claiming to be made from 100 percent authentic pomegranate juice or juice concentrate were purchased and analysed. Chlorogenic acid was found in 45 percent of these additional samples. Two samples were made from imported juice and juice concentrate from Turkey. Chlorogenic acid was found at low levels in both samples. This appears to be supported by the fi ndings of Poyrazoglu et al. who reported the presence of chlorogenic acid in 13 different pomegranate varieties from four growing regions in Turkey (9). However, these fi nding are based on LC/UV analysis alone. Further analysis using more specifi c techniques are required to confi rm these fi ndings.

Figure 5. Loadings plot obtained from the analysis of the six pomegranate juice samples (UPLC/Tof-MS data): where the 4.34 is the retention time and 353.0867 is the signifi cant exact mass at this retention time.

Figure 6. Variable trend plots for 2 possible markers with m/z 353.0868 at RT 3.18 min (A) and RT 4.34 min (B).

Figure 7. Extracted ion chromatograms for the chlorogenic acid [M-H]- m/z 353.0873 in a standard (A) and an adulterated pomegranate juice sample, S3 (B). High CE MSE spectra for standard (C) and sample (D) are also shown.

26

Focu

s on

Food

Ana

lysis

- Su

pple

men

t to

Agro

FOO

D in

dustr

y hi

-tech

- Sep

tem

ber

/Oct

ober

201

1 - v

ol 2

2 n

5understand the signifi cance of these results. We can conclude that in the North American pomegranates and the authentic pomegranate juice believed to be made from pomegranates harvested in North America, chlorogenic acid was not detected.

Marker of interestA marker of interest was observed from the loadings plot having an m/z of 353.1448 at a RT of 3.43 minutes. Whilst the nominal mass for this closely eluting substance is the same as for chlorogenic acid, the elemental composition for this component was determined to be C14H27O10. Extracted ion chromatograms of this m/z from the juice samples used in the study revealed it to be present in all pomegranate samples. However, whilst it was present at low levels in S6, the juice blend (Figure 9) it was present at highest levels in the authentic pomegranate juice (S2). Authentic cranberry, peach and blueberry juice were analysed and the compound was not found in any of these juice types (data not shown). Extracted ion chromatograms of m/z 353.1448 in the whole fruit extracts (arils, pulp and skin) showed that this marker was only present in the arils, but it was present in the arils of all three pomegranates (Figure 10B). Further studies may be warranted to complete identifi cation of this substance.

Summary of the resultsChlorogenic acid was absent in the authentic pomegranate juice analysed in this study. Similarly it was not found in three different whole fruit pomegranates all believed to be grown in North America. Chlorogenic acid and two possible isomers were found in deliberately adulterated pomegranate juice.

In other juice samples obtained in local shops and on the internet, chlorogenic acid was present in 45 percent of the samples.A component of interest found in all pomegranate juice samples and in the arils of the whole fruit pomegranates may provide a marker for authenticity of pomegranate juice measured at 16 Brix.

CONCLUSION

- Without prior knowledge of the samples, the combination of UPLC, QToF and data analysis tools enabled the detection of compounds that have the potential to be used as indicators of a product’s quality.

- MSE functionality allowed the acquisition of low energy precursor (MS) and high energy product ions in a single run. The fragment data together with exact mass measurement provided added confi dence and accuracy for structural elucidation.

- Chlorogenic acid was identifi ed and shown to be absent from authentic pomegranate juice and pomegranate fruit believed to be grown in North America. For these varieties it may have potential to provide an indication of adulteration.

- Whilst chlorogenic acid has been previously reported to be present in pomegranates of Turkish origin, based on identifi cation using HPLC RT alone, a more defi nitive mass spectrometric study is necessary to confi rm this identifi cation.

- Intelligent analytical methods, such as the workfl ow presented here, provide reliable and highly detailed information that can help in the process of food authentication.

REFERENCES AND NOTES

1. M.T. Roberts, Cheaters Shouldn’t Prosper and Consumers Shouldn’t Suffer: The Need for Government Enforcement Against Economic Adulteration of 100% Pomegranate Juice and Other Imported Food Products, Michael T. Roberts is Special Counsel for Roll Law Group P.C (2010) http://works.bepress.com/michael_roberts/1/

2. A.T. Kearney, Grocery Manufacturers Association, Consumer Product Fraud: Deterrence and Detection (2010).

3. N.P. Seeram et al., J Nutr Biochem., 16(6), pp. 360-367 (2005). 4. R.E. Mehta, E.P. Lansky, Eur J. Cancer Prev., 13(4), pp. 345-348 (2004).5. E.P. Lansky el al., Invest New Drugs., 23(2), pp. 121-122 (2005).6. M. Aviram et al., Clin Nutr., 23(3), pp. 423-433 (2004).7. Y. Zhang, D. Heber et al., J. Agric. Food Chem., 57, pp. 2550-2557

(2009).8. L. Eriksson, Multi- and Megavariate Data Analysis: Basic Principles and

Applications, 2nd edition (2008).9. E. Poyrazoglu, N. Artuk et al., J. Food. Comp. Anal., 15, pp. 567-575

(2002).

Figure 8. Extracted ion chromatograms for chlorogenic acid, m/z 353.0873 in a standard and the skin, pulp and arils, of a whole pomegranate.

Figure 9. Variation of the levels of component 3.43_353.1448 in the pomegranate juice samples.

Figure 10. (A) Extracted ion chromatograms for marker 3.43_353.1448 in the arils, pulp and skin of a whole pomegranate. (B) The marker was found in the arils of all pomegranates used in the study.