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Food Chemistry 117 (2009) 739–744

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Liquid chromatography–electrospray ionisation mass spectrometry method for the rapid identification of citrus limonoid glucosides in citrus juices and extracts
Andrew P. Breksa III *, Marlene B. Hidalgo, Michelle Lee Yuen
Department of Agriculture, Western Regional Research Center, Agricultural Research Service, 800 Buchanan St., Albany, CA 94710, United States

a r t i c l e

i n f o

a b s t r a c t
A rapid and selective liquid chromatography–electrospray ionisation mass spectrometry (LC–ESI-MS) method to screen citrus samples for limonoid glucosides and estimate their relative concentrations has been developed. This method utilises a phenyl stationary phase, whereas previous methods have relied on C-18. Samples may be analysed directly without treatment other than dilution. Peak areas from the extracted deprotonated molecular ion mass signals for individual limonoid glucosides were normalised against the sum of the areas to establish their relative concentrations. The method was successfully applied to the analysis of various juice, extracts, and liquid samples of partially purified limonoid glucosides. Published by Elsevier Ltd.

Article history: Received 2 October 2008 Received in revised form 10 March 2009 Accepted 12 April 2009

Keywords: Citrus Limonoids Juice analysis Limonin glucoside LC–MS

1. Introduction Research in our laboratory is focused on the isolation, identification, and quantification of citrus limonoids. Citrus limonoids are complex triterpenoid compounds found in significant quantities in a variety of citrus tissues as aglycones, glucosides, or A-ring lactones, the metabolic precursors of limonoid aglycones and glucosides (Fig. 1) (Zukas, Breksa III, & Manners, 2004). Citrus limonoids have been screened for a number of biological activities, and a summary of these activities including anti-tumour, anti-HIV, and cholesterol lowering properties are summarised in a recent review (Manners, 2007). The human bioavailability of limonin glucoside, the most abundant glucoside for most citrus species, has also been reported (Manners, Breksa, Schoch, Hasegawa, & Jacob, 2003). Recently we turned our attention to two areas: (1) isolating sub-kilogram quantities of limonin glucoside in preparation for a human study to examine the potential short- and medium-term health benefits obtained from consuming limonin glucoside and (2) cataloging the many samples which have been accumulated in our lab over the past 20 years. We concluded that both tasks would benefit from a streamlined method to rapidly evaluate limonoid glucoside content and character. Beside limonin glucoside (LG), the analysis must target the other most common glucosides (Fig. 1), namely nomilin (NG), deacetyl nomilin (DNG), nomilinic acid (NAG), deacetyl nomilinic acid (DNAG), and obacunone glucosides (OG) (Hasegawa, Bennett, Herman, Fong, & Ou, 1989).

* Corresponding author. Tel.: +1 510 559 5898; fax: +1 510 559 5849. E-mail address: andrew.breksa@ars.usda.gov (A.P. Breksa III). 0308-8146/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodchem.2009.04.050

Thin layer chromatography (TLC), high pressure liquid chromatography (HPLC), capillary electrophoresis (CE), and liquid chromatography coupled to mass spectrometry (LC–MS) methods have all been described for the identification of limonoid glucosides. None of these methods in their present form are suitable for our screening efforts. The TLC method is not rapid and is additionally encumbered with the need for trained judges to evaluate individual spot sizes. The HPLC (Fong, Hasegawa, Coggins, Atkin, & Miyake, 1992; Herman, Fong, Ou, & Hasegawa, 1990; Ohta, Fong, Berhow, & Hasegawa, 1993) and CE (Braddock & Bryan, 2001; Moodley, Mulholland, & Raynor, 1995) methods rely on UV detection and are plagued with long analysis times (12–60 min) and complex multi-step extraction protocols to remove interfering components because limonoid glucosides structurally lack a specific chromophore and exhibit only weak absorbance maxima in the range from 210 to 220 nm. In contrast, limonoid glucosides are readily detected and distinguished by mass spectrometry because each limonoid glucoside possess a readily ionizable, free carboxylic acid group and exhibits a unique mass ([MÀH]À m/z, 649 (LG), 693 (NG), 651, (DNG), 711 (NAG), 669 (DNAG), 633 (OG)). Of the three LC–MS-based methods available, two are limited to the detection of only one (Tian, Kent, Bomser, & Schwartz, 2004) or two (Tian & Ding, 2000) of the required compounds, and both are further hampered by either a long analysis time (Tian et al., 2004) or an extensive sample preparation step (Tian & Ding, 2000). The third method (Schoch, Manners, & Hasegawa, 2001), having utilised a C-18 guard column to achieve abbreviated analysis times and covering the required limonoid glucosides, appeared the closest to meeting our screening needs. However, we soon discovered in

H.5 bar N2. Injection volumes varied from 3 to 20 lL. Instrument control and data acquisition were accomplished using Masslynx (Version 4. FL and from fruit purchased from local grocery stores. with additional samples obtained from the USDA’s A. HPLC grade acetonitrile and methanol. R=H) Fig. NJ). Alfortville. Estimation of total limonoid glucoside content Before LC–MS analysis. First.0 V. the samples were analysed by LC–MS using the system and MS set- . 2.5. Billerica. Clifton. Pure crystalline limonin glucoside was available in our laboratory from previous studies.E. Desiring to exploit the advantages that LC–MS affords in reduced sample preparation and sample analysis time while avoiding the potential pitfalls simultaneously. Materials Solvents. practice that chromatographic results from this method were less than satisfactory and ultimately traced the root cause of the deficiency to the lot-to-lot variability found among guard columns. 2. Other limonoid glucosides used to establish chromatographic retention times were available as mixtures or obtained from extracts. 5 min. MA) and Sedex 55 ELS detector (50 °C. Based on these results. 2.3 generated by introduction of a limonin glucoside solution (5 ppm) into the mass spectrometer in the LC mobile phase at the flow rate used for analysis. 2. samples (200 lL) were simply diluted with water (500 lL) and acetonitrile (300 lL) and analysed directly without further treatment. 8 °C) and the resulting supernatant collected and filtered (0.0) with a SAT/IN module used to capture the signal from the ELS detector. Chemical structures of Citrus limonoids.45 lm. All method development experiments were conducted at 30 °C under isocratic conditions using a 50 Â 2. samples were diluted with water to concentrations between 75 and 125 mg LÀ1 limonin glucoside equivalents. we have succeeded in generating an alternative chromatographic method based on a standard micro-bore HPLC column. France) was used for initial method development experiments.2. Samples having concentrations in excess of the calibration range were further diluted using 30% acetonitrile (aq) and reassessed. the total limonoid glucoside content of each sample was estimated using the modified colorimetric method described above.P. the mass spectrometer was operated in the negative ion mode using a capillary temperature of 380 °C.. R=Ac) 4 Deacetyl Nomilin Glucoside (DNG.4) and coupled to a TSP UV 2000 detector (l = 210 nm) and Thermo Finnigan LCQ Advantage Mass Spectrometer (San Jose.E. spray voltage of 4. Screening method for evaluating limonoid glucoside content and character of samples The screening method consists of two steps. S. Syringe filtration is not required for colorimetric determination. Sample preparation Juice samples or samples containing limonoid glucosides were clarified by centrifugation (14.740 A.45 lm type HA membrane filter (Millipore. Described within this report is the development of this new chromatographic method and its application as a screening method to rapidly determine the content and character of limonoid glucosides found in a variety of citrus derived samples. Tuning of the mass spectrometer was accomplished in negative ion mode through optimisation on the limonin glucoside signal at m/z 649.5 mL/min) and were conducted using a Waters 2695 system controlled by Xcalibur (Version 1. Materials and methods 2.E. Whitmore Foundation Farm located in Groveland. / Food Chemistry 117 (2009) 739–744 O O O O O O 1 Limonin O O O O O O O O O O OH CO2H O O O O O OH O O H HO CO2H H H H H OH OH 2 Limonoate A-ring lactone O 3 Limonin glucoside (Limonin 17 β-D-glucopyranoside.3. Breksa III et al. ACS reagent grade) were purchased from Fisher Scientific Ltd. CA). but is done as a precaution to prevent fouling of the HPLC column. R=H) 5 Nomilinic Acid Glucoside (NAG. The mass spectrometer was set to acquire data over a mass range from 475–750 m/z. Subsequent experiments used a sample containing all six limonoid glucosides to examine the effects of solvent polarity (acetonitrile 10–30%) and flow rate (0. the limonoid glucoside content of each sample was estimated by the colorimetric method of Breksa and Ibarra (2007) with the following modification: In place of the solid phase extraction step. Next. 2.D. R=Ac) 7 Obacunone Glucoside (OG) 6 Deacetyl Nomilinic Acid Glucoside (DNAG. 1.2–0. LG) O OR O O O O OGlucose CO2H OR HO2C HO O O OGlucose CO2H O O OGlucose CO2H O O 3 Nomilin Glucoside (NG. Initial experiments determined the effects of mobile phase acidity (formic acid 1–50 mM) on the chromatographic properties of limonin glucoside.000g.1. and formic acid (88%. Whatman Inc. 2..1 MX/cm resistance using a Barnstead NANOpure Deionisation System (Dubuque. Water was deionized to P18. IA) and filtered through a 0. HPLC apparatus for method development A Waters HPLC system equipped with a Model 2695 Separations Module coupled to a Waters model 996 diode array detector (190– 250 nm) (Milford. MA).4. CA) equipped with a guard column of the same stationary phase.50 kV. MA) before use. Samples for this study were taken from our in-house limonin glucoside isolation efforts. 25 mm GD/X filter.R. Following tuning. and capillary voltage of 9. (Waltham.0 mm Phenomenex Phenosphere-Next-5l Phenyl column (Torrance.

Before starting these experiments.5 bar N2) were varied as 20 lL injections of LG (5 mg LÀ1) were introduced into an aqueous solvent stream flowing into the detector at a rate of 500 lL per min.2–0. Of the limonoid glucosides present in the standard solution. were evaluated in triplicate on three consecutive days to evaluate inter-run and inter-day variability of the method. The solvent composition was 15:85 acetonitrile: 4 mM formic acid. the LC–MS system was used to analyse a sample containing all six limonoid glucosides. Comparison of the ELS response to that of a UV (k = 210 nm) detector over a range of LG concentrations (1–50 mg LÀ1) revealed that ELS detection afforded at least a twofold increase in signal when the instrument’s gain was set to its maximum level. Results and discussion Initial experiments on the application of the phenyl stationary phase to the analysis of citrus limonoid glucosides were evaluated using an HPLC system coupled to an evaporative light scattering (ELS) detector. Thus. Ohta et al. / Food Chemistry 117 (2009) 739–744 741 tings described above and conducted as follows: Samples (10 lL) were injected into a 50 Â 2. and then spikes of limonin glucoside were added at two concentrations (10 and 25 mg LÀ1). Based upon the observed results. so that only 1/5 of the flow was introduced into the mass spectrometer. To reduce analysis time. R2 = 0.0 mm Phenosphere-Next-5l Phenyl column equipped with a guard column of the same stationary phase maintained at 30 °C and flowing isocratically at a rate of 1. raw peak areas of individual limonoid glucosides were normalised to the sum of the areas of the detected limonoid glucosides for each chromatogram and reported as a relative percent. Breksa III et al. For quantification of the limonoid glucosides. DNG. in contrast to the linear response of the UV detector (y = 8. To calculate the percent recovery. Concurrent with the increase in flow rate. a mobile phase composition of 85:15. DNG. 2 shows the effects of mobile phase acidity (formic acid 1–50 mM) on the retention of LG on the phenyl stationary phase. These subtle differences in selectivity between the two phases may be useful in the isolation of limonoid glucosides. and finally OG. NAG. Using the Xcalibur software package. Having established chromatographic conditions suitable to resolve limonoid glucosides we further optimised the method by reducing analysis time to maximize sample throughput. 2. whereas the phenyl phase in contrast is much shorter.. response of the ELS detector was parabolic and was fit using a polynomial equation (y = 1.987). are reversed. The differences observed in elution order between the two stationary phases are the result of the modes in which each phase can interact with the analytes in solution. The best response (data not shown) was observed using an operating temperature of 50 °C in conjunction with a nitrogen pressure of 2. R2 = 0. the analytes were not retained. NAG.P. Schoch et al. At the lowest acetonitrile concentration. In contrast. the elution order of two sets of compounds. col- .6.896x + 37..14 min with increasing acidity of the mobile phase. DNAG was the first of the analytes to be eluted. From these experiments the instrument limit of quantitation for LG by ELS was estimated to be approximately 5 ng. including juice. 1). DNAG was followed by LG. Subsequent data analysis including identification of the limonoid glucosides present and integration of individual peak areas was accomplished using Xcalibur. and finally OG (Herman et al. The long carbon chains of the C-18 phase are like long fingers that can interact with analytes with a significant degree of freedom in a number conformations. CA). The flow from the TSP UV 2000 detector was directed through an LC Packing’s Acurate flow splitter (Sunnyvale. A side-to-side comparison of chromatograms revealed that methanol runs were plagued with significant peak tailing (data not shown). Limonin glucoside (LG) was evaluated first because it is the predominant limonoid glucoside found in most Citrus and was available in our laboratory from previous studies. The choice of ELS detection during the method development stage was based upon recommendations found in the literature (Charlesworth. NG. mass traces for individual limonoid glucosides were extracted from the total ion chromatograms. 1993. 4 mM formic acid:acetonitrile was chosen as the optimal compromise between analysis time and resolution. and NG and NAG. and liquid samples of partially purified limonoid glucosides.. the ELS operating parameters of temperature and nebulizer pressure were optimised.09 min to 2.A. Evaluation of run-to-run and day-to-day variability A set of eleven randomly chosen samples. shifting from C-18 to a phenyl stationary phase. We thus focused our evaluation exclusively on acetonitrile. Whereas when 30% acetonitrile was used.0 min. A second reason for choosing ELS was to determine if ELS detection afforded any improvement over UV detection of limonoid glucosides since the ELS is mass dependent and not dependent on the presence of an appropriate chromophore. the analytes were eluted as broad peaks barely discernable over the baseline and required a run time in excess of 10 min to complete their elution. which is likely constrained to interact directly with the furan ring of limonoid glucosides and perhaps the double bond in the A-ring of obacunone. 2002) and made by instrument manufacturers suggesting that ELS would be a less expensive alternative to a mass spectrometer during the development of LC–MS methods. Webster & Diaz. we doubled the flow rate from 0. We wished to evaluate both methanol and acetonitrile as organic components of the mobile phase. On a per sample basis. 1978. Changes in flow rate under the conditions examined affected neither peak shape nor resolution of the analytes. Spike recovery experiments were conducted in triplicate. the reported order of elution for the same glucosides from a C-18 stationary phase was LG followed by DNAG. 2005). Spike recovery experiments were conducted on some samples. Operating temperature (30–55 °C) and nebulizer pressure (1.5 mL minÀ1). In those experiments. however. varying its concentration stepwise from 10% to 30%.5 to 1. a formic acid concentration of 4 mM was selected for subsequent experiments examining the effects of solvent polarity and flow rate (0.9–2. an approach commonly applied in metabolic profiling of arabidopsis plants was used (Nikiforova et al.5 bar. Fig. and the total run time was 4. For the next phase of the method development. Examining the structures of the six glucosides (Fig. The apparent contrast in the ability of individual phases to interact with analytes suggests that limonoid glucosides with open A-rings (NAG and DNAG) can have conformations in solution that allow additional interactions with a C-18 phase.318x + 57.9032x2 + 21. such that quantitation of each of the six glucosides in a mixture may be accomplished without worry of isotope contribution. Fig. Ultimately.0 mL minÀ1 and found chromatographic resolution of the analytes unaffected by increased flow.373. Retention time increased from 1.999). NG. Formic acid concentrations above 50 mM had no further effect on retention time. LG and DNAG. the observed concentration was divided by the expected concentration and resulting product multiplied by 100%..0 mL per min. samples were first diluted within the linear range of the LC–MS method. Limonoid glucosides contain one or two carboxylic acid groups and we anticipated that the pH of the mobile phase would affect retention time.437. extracts. but did result in an increase in baseline noise from the ELS detector. 2001). but were immediately co-eluted. ending with a planar phenyl ring. it is not surprising that DNAG was the first to be eluted considering that DNAG is the most polar of the six compounds. 3 illustrates by mass spectrometry data the resolution and order of elution. 1990. 3.

1990. Injection volume = 3. The compositional differences between these samples and those for the Hamlin. the total limonoid glucoside content of each sample was estimated by the modified colorimetric method described in Section 2.3 DNG m/z= 651. 2001). molasses or concentrated extracts) 50 to 1000-fold dilutions were required. Utilizing the LC–MS conditions described above. Herman et al. Therefore.0 mm. Considering that limonoid glucoside concentrations in orange juice and citrus molasses typically range from 250 to 396 mg LÀ1 (Fong. 1990) and 470–7960 mg LÀ1 (Schoch et al. After the concentration was determined. 1992). the sample was diluted with water to a total limonoid glucoside concentration between 50 and 100 mg LÀ1. TIC: 400-800 DNAG m/z= 669.4 NAG m/z= 711. We have observed that NG is slowly converted to DNAG under the acidic conditions found in juice it is probable that its conversion into DNAG prior to the analysis may have been the cause for the reduced NG composition reported for the orange juices. diluted 5:1 with water and analysed for their limonoid glucoside content. Washington Navel and Valencia juices were the most similar and exhibited LG compositions close to those previously reported for orange juices. Both results are consistent with a previous report on the detection of limonoid glucosides by LC–MS (Schoch et al. whereas for more concentrated samples (e. Additional details are found in Section 2. Total ion and selective ion chromatograms resulting from the LC–MS analysis of a standard solution containing the six limonin glucosides using a Phenosphere-Next-5l Phenyl column. isocratic mobile phase of ACN:4. Herman. A 5:1 dilution was sufficient for most juice samples. R2 > 0. Retention time of limonin glucoside on the phenyl stationary phase increases with increasing formic acid concentrations in the mobile phase. response was confirmed to be linear over three orders of magnitude (1– 150 mg LÀ1..g.. we evaluated the inter-run and inter-day variability associated with the .P. Experiments were conducted using a 25 mg LÀ1 solution of limonin glucoside and chromatograms monitored with an evaporating light scattering detector.0 mM formic acid in water (15:85).4 LG m/z= 649. Amongst the juices tested. Although the LCQ Advantage mass spectrometer could handle a flow rate of 0.3 OG m/z= 633. Washington Navel.5 mL minÀ1. & Ou. 2001). this concentration range was targeted to ensure that the concentrations of the predominate glucosides would be within the linear range of the MS detector.5 mL minÀ1. / Food Chemistry 117 (2009) 739–744 Fig.0 mL minÀ1 so that it was necessary to introduce a flow splitter (1:5) prior to the mass spectrometer to accommodate the increased flow rate.. respectively. flow rate = 0. Hamlin and Valencia sweet oranges purchased from a local grocery store were hand squeezed. it was overwhelmed by a flow of 1. we were concerned that analyte concentrations in neat samples would be out of the linear range of the detector unless diluted.3 NG m/z= 693..3 6 0 1 2 3 4 5 Time (min) Fig. before LC–MS analysis. 2.0 ll. Hasegawa. Since a single limonoid glucoside usually accounts for more than 50% of the total glucoside content of a given sample. column temperature of 30°. Results for the analysis of each of these samples along with the results previously reported for orange juices are listed in Table 1..742 A. umn backpressure doubled and reached a value around 2200 psi. the limonoid glucoside contents found in Citrus fruits are also greatly influenced by variety. Because we wished to apply this method on an ongoing basis and results were to be reported as relative percentages. Breksa III et al.98) and the limit of quantitation was estimated to be near 2 ng. 50 Â 2. 3. Pera Rio and Natal samples suggest that in addition to harvest time (Fong et al.

The more concentrated the analytes the smaller the variability. Results from the second two days of testing were similar (data not shown). Sinensis (Succari). Limonoid glucoside ([MÀH]À m/z) Variety Washington Navel Valencia Hamlin Previously reported Orange juice (Ave)a Orange juice (Ave)b Pera Rio orangec Natal orangec nd = not determined.1 DNAG (669) 8.0 0.e.9 NG (693) 26.3 0. such as this one and typical of many LC– MS methods. extracts and liquid samples of partially purified limonoid glucosides were evaluated in triplicate on three consecutive days. total ion and selective ion monitoring (SIM) chromatograms obtained for two representative samples are shown in Fig..5 ± 2.5 nd 0.6 743 OG (633) 0.7%). Detection of analytes by mass spectrometry can be hindered by matrix components. juice obtained from fruits from trees located within the collection were analysed.H. 5F) DNAG (19. 4. we found that the %CV increased by a factor of two or less for each of the samples tested and this result provided us with the confidence to move forward with our strategy to utilise relative concentrations as an alternative to reporting absolute concentrations and the need to run calibration curves on a daily basis. in particular by salts or other species in significant concentrations (e. Thus with abbreviated chromatographic methods.1 33. organic acids) that are often not bound by reverse phase stationary phases.6 57. Whitmore Foundation Farm. a set of eleven randomly chosen samples. sugars. For this evaluation. there is a need to resolve the majority of sample matrix components sufficiently from the analytes of interest.6 21.1 31. (1990).0%). the analytes are resolved from interfering matrix components. Comparison of the UV and SIM traces reveals that the limonoid glucosides are well-resolved from the unbound matrix components eluting in the void volume of the column. the %CV continued to increase. Results from the intrarun evaluation are shown in Fig.0 method in order to determine if there were any limitations with this strategy. including juice. LG (649) 53.4%). Each sample was diluted to within the linear range of the MS detector before analysis. considering the variability observed.4 9. Variability was more heavily influenced by concentration rather than by limonoid identity. 5A–F.2 82.1 2.0 NAG (711) 10.2 23.4 nd 14.4 ± 0.9 ± 1.05). LG (4. NAG (17. / Food Chemistry 117 (2009) 739–744 Table 1 Limonoid glucoside contents found in common commercial varieties and previously reported values as relative percentage.g.3 nd 25. The relative percentage concentration for each limonoid glucoside in these two representative samples was calculated as described above and in Section 2.7 nd nd 1.9 nd 6.3 53. For limonoid glucosides that accounted for 10% or more of the normalised total.2 56. NAG (74. (1990). Evaluation of intra-run variability: %CV and its relationship to percent total composition. For relative concentrations that were equal to or greater than 50%.A. 5C) LG (14.4 DNG (651) 0.2 9. A %CV of 10% was used for relative concentrations between 5 and 50% and for relative concentrations below 5% a %CV of 20% was applied. whereas for the second sample (Fig. . Evaluating the results obtained across the three days.6 ± 2. Breksa III et al.7 1. assigned uncertainty = relative concentration (%)  0.9 1.8 80. a %CV of 5% was utilised (i.7%).6 5.2 ± 1. for at least limonin glucoside.1 ± 1. Limonoid glucosides detected at greater than 1% for the first sample included (Fig. 10% and 20%) and recommend that these guidelines be used when evaluating results. DNG (54.0%). Spike recoveries observed after the addition of limonin glucoside (10 and 25 mg LÀ1) to juice and other samples were observed to range from 95% to 103% and provide further support that. We are currently continuing our analysis of this population in Fig.0%). 4.2 ± 1. we adopted specific uncertainty levels (%CV = 5%. Although.P. c Schoch et al.7%). However.2 3.7%) were detected.8 1.1 0.7 6. As the percent composition decreased further. the fruit samples came from two different trees that are members of a F1 population generated from a cross between C. Inset is a magnification of the same graph from 0% to 10%..0 ± 3. the relative concentrations of the limonoid glucosides found in the samples is distinctly different.8 0.2 5. and NG (3. the observed %CV was typically less than 5%.1 11.2 10. (2001). As part of an ongoing project to characterise chemical phenotypes of genetic resources found within the USDA’s A. a Fong et al. Grandis and Poncirus trifoliata (Nakon  Flying Dragon)  C. and NG (10. b Herman et al. The UV (k = 220 nm).

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