Food Chemistry 117 (2009) 739–744

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Food Chemistry

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Analytical Methods

Liquid chromatography–electrospray ionisation mass spectrometry method forthe rapid identification of citrus limonoid glucosides in citrus juices and extracts

Andrew P. Breksa III *, Marlene B. Hidalgo, Michelle Lee YuenDepartment 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

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

Keywords:CitrusLimonoidsJuice analysisLimonin glucosideLC–MS

0308-8146/$ - see front matter Published by Elsevierdoi:10.1016/j.foodchem.2009.04.050

* Corresponding author. Tel.: +1 510 559 5898; faxE-mail address: andrew.breksa@ars.usda.gov (A.P.

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 hasbeen developed. This method utilises a phenyl stationary phase, whereas previous methods have reliedon C-18. Samples may be analysed directly without treatment other than dilution. Peak areas from theextracted deprotonated molecular ion mass signals for individual limonoid glucosides were normalisedagainst the sum of the areas to establish their relative concentrations. The method was successfullyapplied to the analysis of various juice, extracts, and liquid samples of partially purified limonoidglucosides.

Published by Elsevier Ltd.

1. Introduction Thin layer chromatography (TLC), high pressure liquid chroma-

Research in our laboratory is focused on the isolation, identifica-tion, and quantification of citrus limonoids. Citrus limonoids arecomplex triterpenoid compounds found in significant quantities ina variety of citrus tissues as aglycones, glucosides, or A-ring lac-tones, the metabolic precursors of limonoid aglycones and gluco-sides (Fig. 1) (Zukas, Breksa III, & Manners, 2004). Citrus limonoidshave been screened for a number of biological activities, and a sum-mary of these activities including anti-tumour, anti-HIV, and cho-lesterol 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 beenreported (Manners, Breksa, Schoch, Hasegawa, & Jacob, 2003).

Recently we turned our attention to two areas: (1) isolatingsub-kilogram quantities of limonin glucoside in preparation for ahuman study to examine the potential short- and medium-termhealth benefits obtained from consuming limonin glucoside and(2) cataloging the many samples which have been accumulatedin our lab over the past 20 years. We concluded that both taskswould benefit from a streamlined method to rapidly evaluate limo-noid 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), nomilinicacid (NAG), deacetyl nomilinic acid (DNAG), and obacunone gluco-sides (OG) (Hasegawa, Bennett, Herman, Fong, & Ou, 1989).

Ltd.

: +1 510 559 5849.Breksa III).

tography (HPLC), capillary electrophoresis (CE), and liquid chroma-tography coupled to mass spectrometry (LC–MS) methods have allbeen described for the identification of limonoid glucosides. Noneof these methods in their present form are suitable for our screen-ing efforts. The TLC method is not rapid and is additionally encum-bered with the need for trained judges to evaluate individual spotsizes. The HPLC (Fong, Hasegawa, Coggins, Atkin, & Miyake, 1992;Herman, Fong, Ou, & Hasegawa, 1990; Ohta, Fong, Berhow, & Hase-gawa, 1993) and CE (Braddock & Bryan, 2001; Moodley, Mulhol-land, & Raynor, 1995) methods rely on UV detection and areplagued with long analysis times (12–60 min) and complex mul-ti-step extraction protocols to remove interfering componentsbecause limonoid glucosides structurally lack a specific chromo-phore and exhibit only weak absorbance maxima in the range from210 to 220 nm. In contrast, limonoid glucosides are readily de-tected and distinguished by mass spectrometry because each limo-noid glucoside possess a readily ionizable, free carboxylic acidgroup and exhibits a unique mass ([M�H]� m/z, 649 (LG), 693(NG), 651, (DNG), 711 (NAG), 669 (DNAG), 633 (OG)). Of the threeLC–MS-based methods available, two are limited to the detectionof only one (Tian, Kent, Bomser, & Schwartz, 2004) or two (Tian& Ding, 2000) of the required compounds, and both are furtherhampered by either a long analysis time (Tian et al., 2004) or anextensive sample preparation step (Tian & Ding, 2000). The thirdmethod (Schoch, Manners, & Hasegawa, 2001), having utilised aC-18 guard column to achieve abbreviated analysis times and cov-ering the required limonoid glucosides, appeared the closest tomeeting our screening needs. However, we soon discovered in

H

2 Limonoate A-ring lactone

O

O

O

O

O

O

O

O

1 Limonin

OO

O

O

O

O

O

CO2HH

OH

OH

HOH

CO2H

OGlucose

O

O

OOO

3 Nomilin Glucoside (NG, R=Ac)4 Deacetyl Nomilin Glucoside (DNG, R=H)

OR

3 Limonin glucoside(Limonin 17 β-D-glucopyranoside, LG)

CO2H

OH

O

O

O

O

O

O

H

HO

OH

CO2H

OGlucose

O

O

O

HO2CHO

5 Nomilinic Acid Glucoside (NAG, R=Ac)6 Deacetyl Nomilinic Acid Glucoside (DNAG, R=H)

ORCO2H

OGlucose

O

O

OOO

7 Obacunone Glucoside (OG)

Fig. 1. Chemical structures of Citrus limonoids.

740 A.P. Breksa III et al. / Food Chemistry 117 (2009) 739–744

practice that chromatographic results from this method were lessthan satisfactory and ultimately traced the root cause of the defi-ciency to the lot-to-lot variability found among guard columns.Desiring to exploit the advantages that LC–MS affords in reducedsample preparation and sample analysis time while avoiding thepotential pitfalls simultaneously, we have succeeded in generatingan alternative chromatographic method based on a standard mi-cro-bore HPLC column. Described within this report is the develop-ment of this new chromatographic method and its application as ascreening method to rapidly determine the content and characterof limonoid glucosides found in a variety of citrus derived samples.

2. Materials and methods

2.1. Materials

Solvents, HPLC grade acetonitrile and methanol, and formic acid(88%, ACS reagent grade) were purchased from Fisher Scientific Ltd.(Waltham, MA). Water was deionized to P18.1 MX/cm resistanceusing a Barnstead NANOpure Deionisation System (Dubuque, IA)and filtered through a 0.45 lm type HA membrane filter (Millipore,Billerica, MA) before use. Pure crystalline limonin glucoside wasavailable in our laboratory from previous studies. Other limonoidglucosides used to establish chromatographic retention times wereavailable as mixtures or obtained from extracts. Samples for thisstudy were taken from our in-house limonin glucoside isolation ef-forts, with additional samples obtained from the USDA’s A.H. Whit-more Foundation Farm located in Groveland, FL and from fruitpurchased from local grocery stores.

2.2. Sample preparation

Juice samples or samples containing limonoid glucosides wereclarified by centrifugation (14,000g, 5 min, 8 �C) and the resultingsupernatant collected and filtered (0.45 lm, 25 mm GD/X filter,Whatman Inc., Clifton, NJ). Syringe filtration is not required for col-orimetric determination, but is done as a precaution to preventfouling of the HPLC column.

2.3. Estimation of total limonoid glucoside content

Before LC–MS analysis, the limonoid glucoside content of eachsample was estimated by the colorimetric method of Breksa andIbarra (2007) with the following modification: In place of the solidphase extraction step, samples (200 lL) were simply diluted with

water (500 lL) and acetonitrile (300 lL) and analysed directlywithout further treatment. Samples having concentrations in ex-cess of the calibration range were further diluted using 30% aceto-nitrile (aq) and reassessed.

2.4. HPLC apparatus for method development

A Waters HPLC system equipped with a Model 2695 SeparationsModule coupled to a Waters model 996 diode array detector (190–250 nm) (Milford, MA) and Sedex 55 ELS detector (50 �C, 2.5 bar N2,S.E.D.E.R.E., Alfortville, France) was used for initial method devel-opment experiments. Instrument control and data acquisitionwere accomplished using Masslynx (Version 4.0) with a SAT/INmodule used to capture the signal from the ELS detector. Initialexperiments determined the effects of mobile phase acidity (for-mic acid 1–50 mM) on the chromatographic properties of limoninglucoside. Subsequent experiments used a sample containing allsix limonoid glucosides to examine the effects of solvent polarity(acetonitrile 10–30%) and flow rate (0.2–0.5 mL/min) and wereconducted using a Waters 2695 system controlled by Xcalibur(Version 1.4) and coupled to a TSP UV 2000 detector (l = 210 nm)and Thermo Finnigan LCQ Advantage Mass Spectrometer (San Jose,CA). Tuning of the mass spectrometer was accomplished in nega-tive ion mode through optimisation on the limonin glucoside sig-nal at m/z 649.3 generated by introduction of a limonin glucosidesolution (5 ppm) into the mass spectrometer in the LC mobilephase at the flow rate used for analysis. Following tuning, the massspectrometer was operated in the negative ion mode using a cap-illary temperature of 380 �C, spray voltage of 4.50 kV, and capillaryvoltage of 9.0 V. The mass spectrometer was set to acquire dataover a mass range from 475–750 m/z. All method developmentexperiments were conducted at 30 �C under isocratic conditionsusing a 50 � 2.0 mm Phenomenex Phenosphere-Next-5l Phenylcolumn (Torrance, CA) equipped with a guard column of the samestationary phase. Injection volumes varied from 3 to 20 lL.

2.5. Screening method for evaluating limonoid glucoside content andcharacter of samples

The screening method consists of two steps. First, the totallimonoid glucoside content of each sample was estimated usingthe modified colorimetric method described above. Based on theseresults, samples were diluted with water to concentrations be-tween 75 and 125 mg L�1 limonin glucoside equivalents. Next,the samples were analysed by LC–MS using the system and MS set-

A.P. Breksa III et al. / Food Chemistry 117 (2009) 739–744 741

tings described above and conducted as follows: Samples (10 lL)were injected into a 50 � 2.0 mm Phenosphere-Next-5l Phenylcolumn equipped with a guard column of the same stationaryphase maintained at 30 �C and flowing isocratically at a rate of1.0 mL per min. The solvent composition was 15:85 acetonitrile:4 mM formic acid, and the total run time was 4.0 min. The flowfrom the TSP UV 2000 detector was directed through an LC Pack-ing’s Acurate flow splitter (Sunnyvale, CA), so that only 1/5 of theflow was introduced into the mass spectrometer. Using the Xcali-bur software package, mass traces for individual limonoid gluco-sides were extracted from the total ion chromatograms.Subsequent data analysis including identification of the limonoidglucosides present and integration of individual peak areas wasaccomplished using Xcalibur. For quantification of the limonoidglucosides, an approach commonly applied in metabolic profilingof arabidopsis plants was used (Nikiforova et al., 2005). On a persample basis, raw peak areas of individual limonoid glucosideswere normalised to the sum of the areas of the detected limonoidglucosides for each chromatogram and reported as a relative per-cent. Spike recovery experiments were conducted on some sam-ples. In those experiments, samples were first diluted within thelinear range of the LC–MS method, and then spikes of limonin glu-coside were added at two concentrations (10 and 25 mg L�1). Tocalculate the percent recovery, the observed concentration was di-vided by the expected concentration and resulting product multi-plied by 100%. Spike recovery experiments were conducted intriplicate.

2.6. Evaluation of run-to-run and day-to-day variability

A set of eleven randomly chosen samples, including juice, ex-tracts, and liquid samples of partially purified limonoid glucosides,were evaluated in triplicate on three consecutive days to evaluateinter-run and inter-day variability of the method.

3. Results and discussion

Initial experiments on the application of the phenyl stationaryphase to the analysis of citrus limonoid glucosides were evaluatedusing an HPLC system coupled to an evaporative light scattering(ELS) detector. The choice of ELS detection during the methoddevelopment stage was based upon recommendations found inthe literature (Charlesworth, 1978; Webster & Diaz, 2002) andmade by instrument manufacturers suggesting that ELS would bea less expensive alternative to a mass spectrometer during thedevelopment of LC–MS methods. A second reason for choosingELS was to determine if ELS detection afforded any improvementover UV detection of limonoid glucosides since the ELS is massdependent and not dependent on the presence of an appropriatechromophore. Limonin glucoside (LG) was evaluated first becauseit is the predominant limonoid glucoside found in most Citrusand was available in our laboratory from previous studies. Beforestarting these experiments, the ELS operating parameters of tem-perature and nebulizer pressure were optimised. Operating tem-perature (30–55 �C) and nebulizer pressure (1.9–2.5 bar N2) werevaried as 20 lL injections of LG (5 mg L�1) were introduced intoan aqueous solvent stream flowing into the detector at a rate of500 lL per min. The best response (data not shown) was observedusing an operating temperature of 50 �C in conjunction with anitrogen pressure of 2.5 bar. Comparison of the ELS response tothat of a UV (k = 210 nm) detector over a range of LG concentra-tions (1–50 mg L�1) revealed that ELS detection afforded at leasta twofold increase in signal when the instrument’s gain was setto its maximum level; however, in contrast to the linear responseof the UV detector (y = 8.896x + 37.373, R2 = 0.987), response of

the ELS detector was parabolic and was fit using a polynomialequation (y = 1.9032x2 + 21.318x + 57.437, R2 = 0.999). From theseexperiments the instrument limit of quantitation for LG by ELSwas estimated to be approximately 5 ng.

Limonoid glucosides contain one or two carboxylic acid groupsand we anticipated that the pH of the mobile phase would affectretention time. Fig. 2 shows the effects of mobile phase acidity (for-mic acid 1–50 mM) on the retention of LG on the phenyl stationaryphase. Retention time increased from 1.09 min to 2.14 min withincreasing acidity of the mobile phase. Formic acid concentrationsabove 50 mM had no further effect on retention time, but did resultin an increase in baseline noise from the ELS detector. Based uponthe observed results, a formic acid concentration of 4 mM was se-lected for subsequent experiments examining the effects of solventpolarity and flow rate (0.2–0.5 mL min�1).

For the next phase of the method development, the LC–MS sys-tem was used to analyse a sample containing all six limonoid glu-cosides. We wished to evaluate both methanol and acetonitrile asorganic components of the mobile phase. A side-to-side compari-son of chromatograms revealed that methanol runs were plaguedwith significant peak tailing (data not shown). We thus focusedour evaluation exclusively on acetonitrile, varying its concentra-tion stepwise from 10% to 30%. At the lowest acetonitrile concen-tration, the analytes were eluted as broad peaks barelydiscernable over the baseline and required a run time in excessof 10 min to complete their elution. Whereas when 30% acetoni-trile was used, the analytes were not retained, but were immedi-ately co-eluted. Ultimately, a mobile phase composition of 85:15,4 mM formic acid:acetonitrile was chosen as the optimal compro-mise between analysis time and resolution. Changes in flow rateunder the conditions examined affected neither peak shape norresolution of the analytes.

Fig. 3 illustrates by mass spectrometry data the resolution andorder of elution, such that quantitation of each of the six glucosidesin a mixture may be accomplished without worry of isotope contri-bution. Of the limonoid glucosides present in the standard solu-tion, DNAG was the first of the analytes to be eluted. Examiningthe structures of the six glucosides (Fig. 1), it is not surprising thatDNAG was the first to be eluted considering that DNAG is the mostpolar of the six compounds. DNAG was followed by LG, DNG, NAG,NG, and finally OG. In contrast, the reported order of elution for thesame glucosides from a C-18 stationary phase was LG followed byDNAG, DNG, NG, NAG, and finally OG (Herman et al., 1990; Ohtaet al., 1993; Schoch et al., 2001). Thus, shifting from C-18 to a phe-nyl stationary phase, the elution order of two sets of compounds,LG and DNAG, and NG and NAG, are reversed.

The differences observed in elution order between the two sta-tionary phases are the result of the modes in which each phase caninteract with the analytes in solution. The long carbon chains of theC-18 phase are like long fingers that can interact with analyteswith a significant degree of freedom in a number conformations,whereas the phenyl phase in contrast is much shorter, ending witha planar phenyl ring, which is likely constrained to interact directlywith the furan ring of limonoid glucosides and perhaps the doublebond in the A-ring of obacunone. The apparent contrast in the abil-ity of individual phases to interact with analytes suggests thatlimonoid glucosides with open A-rings (NAG and DNAG) can haveconformations in solution that allow additional interactions with aC-18 phase. These subtle differences in selectivity between the twophases may be useful in the isolation of limonoid glucosides.

Having established chromatographic conditions suitable to re-solve limonoid glucosides we further optimised the method byreducing analysis time to maximize sample throughput. To reduceanalysis time, we doubled the flow rate from 0.5 to 1.0 mL min�1

and found chromatographic resolution of the analytes unaffectedby increased flow. Concurrent with the increase in flow rate, col-

Fig. 2. Retention time of limonin glucoside on the phenyl stationary phase increases with increasing formic acid concentrations in the mobile phase. Experiments wereconducted using a 25 mg L�1 solution of limonin glucoside and chromatograms monitored with an evaporating light scattering detector. Additional details are found inSection 2.

742 A.P. Breksa III et al. / Food Chemistry 117 (2009) 739–744

umn backpressure doubled and reached a value around 2200 psi.Although the LCQ Advantage mass spectrometer could handle aflow rate of 0.5 mL min�1, it was overwhelmed by a flow of1.0 mL min�1 so that it was necessary to introduce a flow splitter(1:5) prior to the mass spectrometer to accommodate the in-creased flow rate.

Utilizing the LC–MS conditions described above, response wasconfirmed to be linear over three orders of magnitude (1–150 mg L�1; R2 > 0.98) and the limit of quantitation was estimatedto be near 2 ng. Both results are consistent with a previous reporton the detection of limonoid glucosides by LC–MS (Schoch et al.,

0 1 2 3 4 5 6

TIC: 400-800

m/z= 669.4

m/z= 649.3

m/z= 651.4

m/z= 711.3

m/z= 693.3

m/z= 633.3

DNAG

LG

DNG

NAG

NG

OG

Time (min)

Fig. 3. Total ion and selective ion chromatograms resulting from the LC–MSanalysis of a standard solution containing the six limonin glucosides using aPhenosphere-Next-5l Phenyl column, 50 � 2.0 mm. Injection volume = 3.0 ll,column temperature of 30�, isocratic mobile phase of ACN:4.0 mM formic acid inwater (15:85), flow rate = 0.5 mL min�1.

2001). Considering that limonoid glucoside concentrations in or-ange juice and citrus molasses typically range from 250 to396 mg L�1 (Fong, Hasegawa, Herman, & Ou, 1990; Herman et al.,1990) and 470–7960 mg L�1 (Schoch et al., 2001), respectively,we were concerned that analyte concentrations in neat sampleswould be out of the linear range of the detector unless diluted.Therefore, before LC–MS analysis, the total limonoid glucoside con-tent of each sample was estimated by the modified colorimetricmethod described in Section 2. After the concentration was deter-mined, the sample was diluted with water to a total limonoid glu-coside concentration between 50 and 100 mg L�1. Since a singlelimonoid glucoside usually accounts for more than 50% of the totalglucoside content of a given sample, this concentration range wastargeted to ensure that the concentrations of the predominate glu-cosides would be within the linear range of the MS detector. A 5:1dilution was sufficient for most juice samples, whereas for moreconcentrated samples (e.g., molasses or concentrated extracts) 50to 1000-fold dilutions were required.

Washington Navel, Hamlin and Valencia sweet oranges pur-chased from a local grocery store were hand squeezed, diluted5:1 with water and analysed for their limonoid glucoside content.Results for the analysis of each of these samples along with the re-sults previously reported for orange juices are listed in Table 1.Amongst the juices tested, Washington Navel and Valencia juiceswere the most similar and exhibited LG compositions close tothose previously reported for orange juices. We have observed thatNG is slowly converted to DNAG under the acidic conditions foundin juice it is probable that its conversion into DNAG prior to theanalysis may have been the cause for the reduced NG compositionreported for the orange juices. The compositional differences be-tween these samples and those for the Hamlin, Pera Rio and Natalsamples suggest that in addition to harvest time (Fong et al., 1992),the limonoid glucoside contents found in Citrus fruits are alsogreatly influenced by variety.

Because we wished to apply this method on an ongoing basisand results were to be reported as relative percentages, we evalu-ated the inter-run and inter-day variability associated with the

Table 1Limonoid glucoside contents found in common commercial varieties and previously reported values as relative percentage.

Limonoid glucoside ([M�H]� m/z) LG (649) NG (693) DNG (651) NAG (711) DNAG (669) OG (633)

VarietyWashington Navel 53.6 26.2 0.8 10.6 8.1 0.7Valencia 57.1 23.1 1.8 5.2 11.6 1.2Hamlin 33.2 31.4 0.9 9.7 21.3 3.5

Previously reportedOrange juice (Ave)a 56.3 nd nd nd nd ndOrange juice (Ave)b 53.2 14.2 6.3 nd 25.4 0.9Pera Rio orangec 82.8 5.7 0.0 1.1 9.2 1.1Natal orangec 80.9 6.4 0.0 2.1 10.6 0.0

nd = not determined.a Fong et al. (1990).b Herman et al. (1990).c Schoch et al. (2001).

A.P. Breksa III et al. / Food Chemistry 117 (2009) 739–744 743

method in order to determine if there were any limitations withthis strategy. For this evaluation, a set of eleven randomly chosensamples, including juice, extracts and liquid samples of partiallypurified limonoid glucosides were evaluated in triplicate on threeconsecutive days. Each sample was diluted to within the linearrange of the MS detector before analysis. Results from the intra-run evaluation are shown in Fig. 4. Variability was more heavilyinfluenced by concentration rather than by limonoid identity.The more concentrated the analytes the smaller the variability.For limonoid glucosides that accounted for 10% or more of the nor-malised total, the observed %CV was typically less than 5%. As thepercent composition decreased further, the %CV continued to in-crease. Results from the second two days of testing were similar(data not shown). Evaluating the results obtained across the threedays, we found that the %CV increased by a factor of two or less foreach of the samples tested and this result provided us with theconfidence to move forward with our strategy to utilise relativeconcentrations as an alternative to reporting absolute concentra-tions and the need to run calibration curves on a daily basis. How-ever, considering the variability observed, we adopted specificuncertainty levels (%CV = 5%, 10% and 20%) and recommend thatthese guidelines be used when evaluating results. For relative con-centrations that were equal to or greater than 50%, a %CV of 5% wasutilised (i.e., assigned uncertainty = relative concentration (%) �0.05). A %CV of 10% was used for relative concentrations between5 and 50% and for relative concentrations below 5% a %CV of 20%was applied.

As part of an ongoing project to characterise chemical pheno-types of genetic resources found within the USDA’s A.H. WhitmoreFoundation Farm, juice obtained from fruits from trees located

Fig. 4. Evaluation of intra-run variability: %CV and its relationship to percent to

within the collection were analysed. The UV (k = 220 nm), totalion and selective ion monitoring (SIM) chromatograms obtainedfor two representative samples are shown in Fig. 5A–F. Detectionof analytes by mass spectrometry can be hindered by matrix com-ponents, in particular by salts or other species in significant con-centrations (e.g., sugars, organic acids) that are often not boundby reverse phase stationary phases. Thus with abbreviated chro-matographic methods, such as this one and typical of many LC–MS methods, there is a need to resolve the majority of sample ma-trix components sufficiently from the analytes of interest. Compar-ison of the UV and SIM traces reveals that the limonoid glucosidesare well-resolved from the unbound matrix components eluting inthe void volume of the column. Spike recoveries observed after theaddition of limonin glucoside (10 and 25 mg L�1) to juice and othersamples were observed to range from 95% to 103% and provide fur-ther support that, for at least limonin glucoside, the analytes areresolved from interfering matrix components.

The relative percentage concentration for each limonoid gluco-side in these two representative samples was calculated as de-scribed above and in Section 2. Limonoid glucosides detected atgreater than 1% for the first sample included (Fig. 5C) LG(14.2 ± 1.4%), NAG (74.0 ± 3.7%), and NG (10.2 ± 1.0%), whereasfor the second sample (Fig. 5F) DNAG (19.6 ± 2.0%), LG (4.9 ±1.0%), DNG (54.5 ± 2.7%), NAG (17.1 ± 1.7%), and NG (3.4 ± 0.7%)were detected. Although, the fruit samples came from two differ-ent trees that are members of a F1 population generated from across between C. Grandis and Poncirus trifoliata (Nakon � FlyingDragon) � C. Sinensis (Succari), the relative concentrations of thelimonoid glucosides found in the samples is distinctly different.We are currently continuing our analysis of this population in

tal composition. Inset is a magnification of the same graph from 0% to 10%.

Fig. 5. UV (k = 220 nm), total ion and selective ion monitoring (SIM) chromatograms obtained for two representative juice samples. Juice samples were obtained from twodifferent trees that are members of a F1 population generated from a cross between C. grandis and Poncirus trifoliata (Nakon � Flying Dragon) � C. sinensis (Succari).

744 A.P. Breksa III et al. / Food Chemistry 117 (2009) 739–744

the hope that results from the analysis of their limonoid glucosidescontent and character will yield segregating populations that maybe further examined by genomic tools in order to establish the ge-netic basis of the observed phenotypes.

Our goal was to develop a rapid and robust LC–MS method tocharacterise limonoid glucosides found in citrus juices, extracts,and fractions obtained from our isolation efforts. The majority ofmethods described thus far for the HPLC and LC–MS analysis oflimonoid glucosides have relied upon C-18 stationary phases. Inthis report, we describe our evaluation of a phenyl stationary phaseas an alternative to C-18 and show that, in addition to resolving theanalytes of interest, the phenyl stationary phase, when comparedto C-18 phases, also exhibits differences in selectivity that mightfurther aid in isolating limonoid glucosides. Additionally, we dem-onstrate that the chromatography is rapid and robust, and, whenpaired with mass spectral detection, provides a method that isapplicable to the analysis of samples having complex matrices.Furthermore, we believe that this method and its use, in conjunc-tion with the colorimetric estimation of total limonoid glucosideconcentration, will be of value to those with experience in theanalysis of citrus limonoid glucosides and equally valuable to otherresearchers who do not have the means or resources to obtain ana-lytical standards, but wish to evaluate citrus samples for theirlimonoid glucoside content and character.

References

Braddock, R. J., & Bryan, C. R. (2001). Extraction parameters and capillaryelectrophoresis analysis of limonin glucoside and phlorin in citrusbyproducts. Journal of Agricultural and Food Chemistry, 49(12), 5982–5988.

Breksa, A. P., III, & Ibarra, P. Jr., (2007). Colorimetric method for the estimation oftotal limonoid aglycones and glucoside contents in citrus juices. Journal ofAgricultural and Food Chemistry, 55(13), 5013–5017.

Charlesworth, J. M. (1978). Evaporative analyzer as a mass detector for liquidchromatography. Analytical Chemistry, 50(11), 1414–1420.

Fong, C. H., Hasegawa, S., Coggins, C. W., Jr., Atkin, D. R., & Miyake, M. (1992).Contents of limonoids and limonin 17-beta-D-glucopyranoside in fruit tissue ofvalencia orange during fruit growth and maturation. Journal of Agricultural andFood Chemistry, 40(7), 1178–1181.

Fong, C. H., Hasegawa, S., Herman, Z., & Ou, P. (1990). Limonoid glucosides incommercial citrus juices. Journal of Food Science, 54(6), 1505–1506.

Hasegawa, S., Bennett, R. D., Herman, Z., Fong, C. H., & Ou, P. (1989). Limonoidglucosides in citrus. Phytochemistry, 28(6), 1717–1720.

Herman, Z., Fong, C. H., Ou, P., & Hasegawa, S. (1990). Limonoid glucosides in orangejuices by HPLC. Journal of Agricultural and Food Chemistry, 38(9), 1860–1861.

Manners, G. D. (2007). Citrus limonoids: Analysis, bioactivity, and biomedicalprospects. Journal of Agricultural and Food Chemistry, 55(21), 8285–8294.

Manners, G. D., Breksa, A. P., III, Schoch, T. K., Hasegawa, S., & Jacob, R. A. (2003).Bioavailability of citrus limonoids in humans. Journal of Agricultural and FoodChemistry, 51(14), 4156–4161.

Moodley, V. E., Mulholland, D. A., & Raynor, M. W. (1995). Micellar electrokineticcapillary chromatography of limonoid glucosides from citrus seeds. Journal ofChromatography A, 718(1), 187–193.

Nikiforova, V. J., Kopka, J., Tolstikov, V., Fiehn, O., Hopkins, L., Hawkesford, M. J., et al.(2005). Systems rebalancing of metabolism in response to sulfur deprivation, asrevealed by metabolome analysis of arabidopsis plants. Plant Physiology, 138(1),304–318.

Ohta, H., Fong, C. H., Berhow, M., & Hasegawa, S. (1993). Thin-layer and high-performance liquid chromatographic analyses of limonoids and limonoidglucosides in citrus seeds. Journal of Chromatography, 639(2), 295–302.

Schoch, T. K., Manners, G. D., & Hasegawa, S. (2001). Analysis of limonoid glucosidesfrom citrus by electrospray ionization liquid chromatography–massspectrometry. Journal of Agricultural and Food Chemistry, 49(3), 1102–1108.

Tian, Q., & Ding, X. (2000). Screening for limonoid glucosides in citrus tangerina(Tanaka) Tseng by high-performance liquid chromatography–electrosprayionization mass spectrometry. Journal of Chromatography A, 874(1), 13–19.

Tian, Q., Kent, K. D., Bomser, J. A., & Schwartz, S. J. (2004). Characterization oflimonin glucoside metabolites from human prostate cell culture medium usinghigh-performance liquid chromatography/electrospray ionization massspectrometry and tandem mass spectrometry. Rapid Communications in MassSpectrometry, 18(24), 3099–3104.

Webster, G. K., & Diaz, A. R. (2002). Evaluation of evaporative light scatteringdetection as a bridge to LC–MS for quality control. 2002 AAPS National Meeting.

Zukas, A. A., Breksa, A. P., III, & Manners, G. D. (2004). Isolation and characterizationof limonoate and nomilinoate A-ring lactones. Phytochemistry, 65(19),2705–2709.