Drastically Increase Laboratory Productivity in Food Authentication

Fruit juices and concentrates are natural products with a very limited range of allowed additives. Unfortunately, some juice products may be adulterated easily either by dilution or by adding specific components such as acids and sugars. Diversity in adulteration techniques, as well as other factors such as natural variation and processing techniques, make the detection and prevention of juice adulteration an analytical challenge.

In this App Note, you'll learn how to drastically increase laboratory productivity in food authentication through multiple factors, including:

Simplicity of sample preparation
A unique column and single mobile phase composition
Easy instrument method setup
Robustness and stability of the overall configuration
And more!

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Goal

To develop an analytical method that merges three conventional LC-UV methods into one LC-MS/MS method that incorporates polarity switching, improving laboratory productivity while maintaining high data confidence.

Introduction

According to the European Association of the Industry of Juices and Nectars (AIJN), 9.1 billion liters of fruit juices and nectars were consumed across the EU in 2018. Fruit juices and concentrates are natural products with a very limited range of allowed additives. Unfortunately, some juice products or fruit purees may be adulterated easily either by dilution with water and other juices or by adding specific components, such as acids and sugars.1

Diversity in adulteration techniques, natural variation due to geographical origins and species, as well as storage conditions and processing techniques make the detection and prevention of juice adulteration an analytical challenge. In that scope, this study describes a single LC-MS/MS approach for monitoring specific nutraceutical markers like polymethoxylated flavones (PMF), limonin, and phenols

in various juices and fruit purees. The objective is to simultaneously measure the following compounds: arbutin (phenols), phloridzin (phenols), 7-methoxycoumarin (PMF), limettin (PMF), bergapten (PMF), isopimpinellin (PMF), sinensetin (PMF), tangeritin (PMF), nobiletin (PMF), and limonin.

Currently, three separate methods are used to analyze all of the listed nutraceuticals. Each method is used to analyze subsets of compounds due to ionization potential (cation or anion formation) and the chromatographic conditions. In some instances, different columns and mobile phases may be needed to differentially retain all target compounds.

Experimental

Sample preparation

The calibration range was different for each compound. As some molecules are very hydrophobic (practically insoluble in water), the dilutions were made in a 50/50 (V/V) mix of methanol and water. Nine standards were made by serial dilutions as shown in Table 1.

These markers are the fingerprints of the juice. The presence and concentration of these compounds in juice depend on the nature of fruit as well as the geographical and varietal origin. Different sample preparations were made as a function of the type of matrices as not all the compounds were present in every matrix (Table 2).

PMF		Limonin	Arbutin	Phloridzin
Standard 1	1.25	–	–	–
Standard 2	2.5	–	–	–
Standard 3	5	–	–	–
Standard 4	12.5	–	–	–
Standard 5	25	20	125	62.5
Standard 6	50	40	250	125
Standard 7	125	100	625	312.5
Standard 8	250	200	1250	625
Standard 9	500	400	2500	1250

Table 1. Standards concentrations in ng/mL

Table 2. Research compounds in each of the matrices

Type of matrices	Compounds to quantify
Orange juice	PMF and limonin
Lime juice	PMF and limonin
Apple puree	Phenols
Pear puree	Phenols

For the PMF and limonin analysis, the sample was homogenized then an aliquot of 2 mL was transferred and centrifuged at 4,500 rpm for 5 minutes. The supernatant was diluted ten-fold (for samples for PMF analysis) and fifty-fold (for samples for limonin analysis) with a 50/50 (V/V) mix of methanol/water.

For the phenols analysis, the sample was homogenized and diluted ten-fold with a 50/50 (V/V) mix of methanol/ water. It was then filtered with a 0.2 µm syringe filter.

LC conditions

LC analysis was performed on a Thermo Scientific™ Vanquish™ Flex Binary UHPLC system. The column used was a Thermo Scientific™ Hypersil GOLD™ VANQUISH™ column (100 × 2.1 mm, 1.9 µm particle size) maintained at 30 °C. Mobile phases A and B consisted of 2 mM ammonium formate with 0.05% formic acid in Fisher Chemical™ Optima™ grade water and methanol, respectively. The total UHPLC gradient was 13 minutes (Table 3), with 0.5 µL injection volume.

Table 3. UHPLC gradient information

Time (min)	Flow rate (mL/min)	B (%)
0.00	0.400	5.0
3.00	0.400	80.0
6.00	0.400	100.0
8.00	0.400	100.0
8.10	0.400	5.0
13.00	0.400	5.0

MS conditions

The Thermo Scientific™ TSQ Fortis™ Plus triple-stage quadrupole mass spectrometer was used for this analysis. All compounds for this study were analyzed in positive and negative heated electrospray mode (HESI). The experimental conditions were optimized with a static spray voltage, and a cycle time of 0.5 s, while Q1 and Q3 resolutions were maintained at 0.7 and 1.2 Da FWHM, respectively. The source parameters and SRM table along with other critical MS features for all the target analytes are listed in Tables 4 and 5, respectively. Individual standards were infused into the mass spectrometer to determine the optimum tube lens settings and collision energies and source fragmentation voltage for the product ions. Timed-SRM scan mode and automated dwell time calculation (based on the expected peak width and desired number of data points across the peak) help users to easily set up instrument parameters and at the same time get maximum performance (sensitivity and chromatographic resolution).

Software

Data acquisition and processing were conducted using Thermo Scientific™ TraceFinderTM software version 5.1. Additionally, Thermo Scientific™ Chromeleon™ 7.3 Chromatography Data System (CDS) can be used for data processing and provides efficient and compliant software to manage all analytical processes, from instrument control to final report generation.

Table 4. Source parameters for analysis of nutraceuticals on the TSQ Fortis Plus triple quadrupole mass spectrometer

Ion source parameter	Value
Spray voltage	Static
Positive ion	1,500 V
Negative ion	2,000 V
Sheath gas	50 Arb
Aux gas	10 Arb
Sweep gas	1 Arb
Ion transfer tube temperature	300 °C
Vaporizer temperature	350 °C
CID gas	1.5 mTorr

Table 5. Optimized mass spectrometer transitions for the nutraceuticals with retention time of 6.5 min and retention time window of 13 min

Compound	Polarity	Precursor (m/z)	Product (m/z)	Collision energy (V)	Tube lens (V)	Source fragmentation (V)
Arbutin	Negative	317.02	108.16	25	52	5
Arbutin	Negative	317.02	271.14	5	52	5
Phloridzin	Negative	435.128	167.11	25	71	19.6
Phloridzin	Negative	435.128	273.16	15	71	19.6
7?methoxycoumarin	Positive	177.05	121.08	20	62	21.2
7?methoxycoumarin	Positive	177.05	133.18	15	62	21.2
Limonin	Positive	471.201	161.02	35	94	13.1
Limonin	Positive	471.201	425.15	20	94	13.1
Ispimpinellin	Positive	247.06	216.99	25	72	14.7
Ispimpinellin	Positive	247.06	231.96	20	72	14.7
Limettin	Positive	207.065	164.05	25	73	18
Limettin	Positive	207.065	192.04	20	73	18
Bergapten	Positive	217.049	174.03	30	76	18
Bergapten	Positive	217.049	201.967	20	76	18
Sinensetin	Positive	373.128	312.05	25	80	11.4
Sinensetin	Positive	373.128	343	30	80	11.4
Nobiletin	Positive	403.138	373.05	30	78	9.8
Nobiletin	Positive	403.138	388.05	20	78	9.8
Tangeritin	Positive	373.128	183.03	45	81	4.9
Tangeritin	Positive	373.128	343	25	81	4.9

Results and discussion

Due to the presence of isobaric compounds like sinensetin and tangeritin (similar parent and daughter ions) in the mix, a good chromatographic separation is critical for quantitation purposes. Different columns and mobile phase compositions were thoroughly evaluated to obtain the most appropriate separation. The use of a Hypersil GOLD VANQUISH column resulted in robust and consistent separation of sinensetin and tangeritin as shown in Figure 1.

Figure 1. Separation of two isobaric compounds, sinensetin and tangeritin

With the increasing demand for greater sample throughput, advancement in triple quadrupole technology is required. One of the ways of increasing sample throughput is by decreasing the time spent on polarity switching. With the new TSQ Fortis Plus MS, a polarity switching speed of 5 ms is possible for better cycle time management. The current method for this study allows the analysis of phenols (negative mode) as well as limonin and PMFs (positive mode) in a single run. Figure 2 shows the detection of all markers in a standard solution (1.25 ng/mL for PMF, 40 ng/mL for limonin, 62.5 ng/mL for phloridzin, and 125 ng/mL for arbutin) using this method. Table 6 and Figure 3 show excellent reproducibility when experiments were carried out both with and without polarity switching at dwell times less than 5 ms. This will allow the user to run methods with both polarities at a shorter LC gradient or addition of more transitions to the method without compromising confidence in quantitation.

Figure 2. Positive and negative extracted ion chromatograms at LLOQ level (a) arbutin, (b) phloridizin, (c) 3-methoxycoumarin, (d) limonin, (e) ispimpinellin, (f) limettin, (g) bergapten, (h) sinensetin, (i) nobiletin, and (j) tangeritin

Figure 2 (continued). Positive and negative extracted ion chromatograms at LLOQ level (a) arbutin, (b) phloridizin, (c) 3-methoxycoumarin, (d) limonin, (e) ispimpinellin, (f) limettin, (g) bergapten, (h) sinensetin, (i) nobiletin, and (j) tangeritin

Table 6. Area count comparison for polarity switching experiment against Cal I and 5 levels for positive and negative mode only experiments

Compound	PN switching		Positive only		Negative only
Compound	Area	%RSD	Area	%RSD	Area	%RSD
7?methoxycoumarin	3148.2	10.0	3151.6	8.5	NA	NA
Bergapten	6071.8	3.2	6063	5.9	NA	NA
Ispimpinellin	4917	6.5	5181.8	6.9	NA	NA
Limettin	1215.8	8.4	1207	6.9	NA	NA
Nobiletin	6948.8	2.0	6858.4	1.9	NA	NA
Sinensetin	1828.8	8.4	1928.4	6.2	NA	NA
Tangeritin	7250.2	4.8	6788.2	8.9	NA	NA
Arbutin	1078.4	3.1	NA	NA	1048.2	2.7
Phloridzin	4984	3.5	NA	NA	4521.4	5.2

Figure 3. Area count comparison for polarity switching experiment against Cal I and 5 levels for positive and negative mode only experiments, respectively

MS calibration curves fit with a linear model for all PMF compounds, limonin, and phenol compounds were used. The calibration curve plotted with the data satisfied the following criteria: R2 value of ≥ 0.98 and with variability (%CV) of < 15%. The percent accuracy of the ion ratio for each sample was averaged at ± 20%, and the required signal-to-noise ratio had a minimum threshold of 3:1.

All calibration details and lower limit of quantification (LLOQ) are displayed in Table 7 and Figure 4.

Arbutin is a glycosylated hydroquinone, which is not very sensitive and most of the time not retained with a reversed phase column. Appropriate chromatographic conditions provide sufficient retention for this compound. The TSQ Fortis Plus MS sensitivity in negative mode helps to detect arbutin at the targeted concentration level without any compromise regarding analysis of other markers. Figure 5 displays a chromatogram of arbutin at 1,250 ng/mL (at least four times less than the amount detected in purees).

Table 7. External calibration curve details

Compound	Polarity	Transition	Calibration range (ng/mL)	R2	LLOQ (ng/mL)
Arbutin	Negative	317 ? 271	125 to 2500	0.9985	125
Phloridizin	Negative	435 ? 273	62.5 to 1250	0.9984	62.5
7?methoxycoumarin	Positive	177 ? 121	1.25 to 500	0.9970	1.25
Limettin	Positive	207 ? 192	1.25 to 500	0.9989	1.25
Bergapten	Positive	217 ? 201	1.25 to 500	0.9988	1.25
Isopimpinellin	Positive	247 ? 216.9	1.25 to 500	0.9995	1.25
Sinensetin	Positive	373 ? 312	1.25 to 500	0.9972	1.25
Tangeritin	Positive	373 ? 343	1.25 to 500	0.9989	1.25
Nobiletin	Positive	403 ? 373	1.25 to 500	0.9995	1.25
Limonin	Positive	471 ? 425	40 to 400	0.9946	40

Figure 4. Calibration curves for the 10 compounds (a) arbutin, (b) phloridizin, (c) 3-methoxycoumarin, (d) limonin, (e) ispimpinellin, (f) limettin, (g) bergapten, (h) sinensetin, (i) nobiletin, and (j) tangeritin

Figure 4 (continued). Calibration curves for the 10 compounds (a) arbutin, (b) phloridizin, (c) 3-methoxycoumarin, (d) limonin, (e) ispimpinellin, (f) limettin, (g) bergapten, (h) sinensetin, (i) nobiletin, and (j) tangeritin

Figure 5. Chromatogram of arbutin’s solution standard at a concentration of 1,250 ng/mL

Different sample matrices (orange juice, lime juice, apple puree, and apple pear puree) have been analyzed to confirm the performance of this method and its compliance with conventional method validation criteria. Analytical results have been cross-confirmed by an external reference partner laboratory using a conventional three run approach (data not shown).

Table 8 shows that the pattern of PMF, phenols, and limonin in several juices is very different from one sample to the other and allows accurate differentiation of them. The adulteration by adding water or other cheaper juices would easily be identified.

Table 8. Comparison of amounts obtained for different type of juices and purees (all results are given in ng/mL)

As only a simple dilution is performed for sample preparation, a reproducibility test has been performed. Peak areas of the four nutraceuticals detected in orange juice showed remarkable reproducibility over twenty consecutive injections. Calculated precisions for limonin, sinensetin, tangeritin, and nobiletin were 4.6%, 2.5%, 2.2%, and 2.0%, respectively. Figure 6 shows the response stability, sample after sample.

The combining of three separate methods for PMF, phenols, and limonin into one method using a shorter LC gradient and faster polarity switching speed leads to an impressive productivity improvement.

Compound	Orange juice	Lime juice	Apple puree	Pear puree
Arbutin	0	0	0	5714.3
Phloridizin	0	0	2535.8	0
7?methoxycoumarin	0	0	0	0
Limettin	0	40.7	0	0
Bergapten	0	27.6	0	0
Isopimpinellin	0	155.6	0	0
Sinensetin	195.8	0	0	0
Tangeritin	56.8	0	0	0
Nobiletin	271.2	0	0	0
Limonin	444.6	906.3	0	0

Figure 6. Twenty consecutive runs of orange juice showing reproducible area counts. %RSD of 4.6%, 2.5%, 2.2%, and 2.0% for limonin, sinensetin, tangeritin and nobiletin, respectively.

Conclusion

This method provides a very robust analytical solution to authenticate fruit juices and purees in only 13 minutes by combining three Thermo Scientific premium products: the Hypersil GOLD VANQUISH 100 × 2.1 mm 1.9 µm LC column, the Vanquish Flex Binary UHPLC system, and the TSQ Fortis Plus mass spectrometer.

It also demonstrates how to drastically increase laboratory productivity in food authentication by merging three conventional LC methods into a single LC-MS/MS method. Several factors have contributed to this:

The simplicity of sample preparation involving a simple dilution
A unique column and a single mobile phase composition for retaining all compounds and appropriately separating the two isobaric ones
The polarity switching efficiency of TSQ Fortis Plus mass spectrometer for monitoring all compounds in positive and negative mode in the same run
The sensitivity of the TSQ Fortis Plus MS system in SRM mode for reaching target LOQs
Easy instrument method setup using timed-SRM and automated dwell time adjustment
Robustness and stability of the overall configuration after multiple injections of juices or purees

Reference

1. FI Handbook on Food Authenticity Issues and Related Analytical Techniques https://secure.fera.defra.gov.uk/foodintegrity/index.cfm?sectionid=83

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