Food Chemistry 117 (2009) 739744

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Food Chemistry
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Analytical Methods

Liquid chromatographyelectrospray ionisation mass spectrometry method for the rapid identication 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 chromatographyelectrospray ionisation mass spectrometry (LCESI-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 puried 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 LCMS

1. Introduction Research in our laboratory is focused on the isolation, identication, and quantication of citrus limonoids. Citrus limonoids are complex triterpenoid compounds found in signicant 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 benets 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 benet 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 (LCMS) methods have all been described for the identication 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 (1260 min) and complex multi-step extraction protocols to remove interfering components because limonoid glucosides structurally lack a specic 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 ([MH] m/z, 649 (LG), 693 (NG), 651, (DNG), 711 (NAG), 669 (DNAG), 633 (OG)). Of the three LCMS-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

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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 -D-glucopyranoside, 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, R=Ac) 4 Deacetyl Nomilin Glucoside (DNG, R=H)

5 Nomilinic Acid Glucoside (NAG, R=Ac) 7 Obacunone Glucoside (OG) 6 Deacetyl Nomilinic Acid Glucoside (DNAG, R=H)

Fig. 1. Chemical structures of Citrus limonoids.

practice that chromatographic results from this method were less than satisfactory and ultimately traced the root cause of the deciency to the lot-to-lot variability found among guard columns. Desiring to exploit the advantages that LCMS affords in reduced sample preparation and sample analysis time while avoiding the potential pitfalls simultaneously, we have succeeded in generating an alternative chromatographic method based on a standard micro-bore HPLC column. 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. 2. Materials and methods 2.1. Materials Solvents, HPLC grade acetonitrile and methanol, and formic acid (88%, ACS reagent grade) were purchased from Fisher Scientic Ltd. (Waltham, MA). Water was deionized to P18.1 MX/cm resistance using a Barnstead NANOpure Deionisation System (Dubuque, IA) and ltered through a 0.45 lm type HA membrane lter (Millipore, Billerica, MA) before use. Pure crystalline limonin glucoside was available in our laboratory from previous studies. Other limonoid glucosides used to establish chromatographic retention times were available as mixtures or obtained from extracts. Samples for this study were taken from our in-house limonin glucoside isolation efforts, with additional samples obtained from the USDAs A.H. Whitmore Foundation Farm located in Groveland, FL and from fruit purchased from local grocery stores. 2.2. Sample preparation Juice samples or samples containing limonoid glucosides were claried by centrifugation (14,000g, 5 min, 8 C) and the resulting supernatant collected and ltered (0.45 lm, 25 mm GD/X lter, Whatman Inc., Clifton, NJ). Syringe ltration is not required for colorimetric determination, but is done as a precaution to prevent fouling of the HPLC column. 2.3. Estimation of total limonoid glucoside content Before LCMS analysis, the limonoid glucoside content of each sample was estimated by the colorimetric method of Breksa and Ibarra (2007) with the following modication: In place of the solid phase extraction step, samples (200 lL) were simply diluted with

water (500 lL) and acetonitrile (300 lL) and analysed directly without further treatment. Samples having concentrations in excess of the calibration range were further diluted using 30% acetonitrile (aq) and reassessed. 2.4. 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) and Sedex 55 ELS detector (50 C, 2.5 bar N2, S.E.D.E.R.E., Alfortville, France) was used for initial method development experiments. Instrument control and data acquisition were accomplished using Masslynx (Version 4.0) with a SAT/IN module used to capture the signal from the ELS detector. Initial experiments determined the effects of mobile phase acidity (formic acid 150 mM) on the chromatographic properties of limonin glucoside. Subsequent experiments used a sample containing all six limonoid glucosides to examine the effects of solvent polarity (acetonitrile 1030%) and ow rate (0.20.5 mL/min) and were conducted 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 negative ion mode through optimisation on the limonin glucoside signal at m/z 649.3 generated by introduction of a limonin glucoside solution (5 ppm) into the mass spectrometer in the LC mobile phase at the ow rate used for analysis. Following tuning, the mass spectrometer was operated in the negative ion mode using a capillary temperature of 380 C, spray voltage of 4.50 kV, and capillary voltage of 9.0 V. The mass spectrometer was set to acquire data over a mass range from 475750 m/z. All method development experiments were conducted at 30 C under isocratic conditions using a 50 2.0 mm Phenomenex Phenosphere-Next-5l Phenyl column (Torrance, CA) equipped with a guard column of the same stationary phase. Injection volumes varied from 3 to 20 lL. 2.5. Screening method for evaluating limonoid glucoside content and character of samples The screening method consists of two steps. First, the total limonoid glucoside content of each sample was estimated using the modied colorimetric method described above. Based on these results, samples were diluted with water to concentrations between 75 and 125 mg L1 limonin glucoside equivalents. Next, the samples were analysed by LCMS using the system and MS set-

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tings described above and conducted as follows: Samples (10 lL) were injected into a 50 2.0 mm Phenosphere-Next-5l Phenyl column equipped with a guard column of the same stationary phase maintained at 30 C and owing isocratically at a rate of 1.0 mL per min. The solvent composition was 15:85 acetonitrile: 4 mM formic acid, and the total run time was 4.0 min. The ow from the TSP UV 2000 detector was directed through an LC Packings Acurate ow splitter (Sunnyvale, CA), so that only 1/5 of the ow was introduced into the mass spectrometer. Using the Xcalibur software package, mass traces for individual limonoid glucosides were extracted from the total ion chromatograms. Subsequent data analysis including identication of the limonoid glucosides present and integration of individual peak areas was accomplished using Xcalibur. For quantication of the limonoid glucosides, an approach commonly applied in metabolic proling of arabidopsis plants was used (Nikiforova et al., 2005). On a per sample basis, 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. Spike recovery experiments were conducted on some samples. In those experiments, samples were rst diluted within the linear range of the LCMS method, and then spikes of limonin glucoside were added at two concentrations (10 and 25 mg L1). To calculate the percent recovery, the observed concentration was divided by the expected concentration and resulting product multiplied by 100%. Spike recovery experiments were conducted in triplicate. 2.6. Evaluation of run-to-run and day-to-day variability A set of eleven randomly chosen samples, including juice, extracts, and liquid samples of partially puried limonoid glucosides, were evaluated in triplicate on three consecutive days to evaluate inter-run and inter-day variability of the method.

3. 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. The choice of ELS detection during the method development stage was based upon recommendations found in the literature (Charlesworth, 1978; Webster & Diaz, 2002) and made by instrument manufacturers suggesting that ELS would be a less expensive alternative to a mass spectrometer during the development of LCMS methods. 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. Limonin glucoside (LG) was evaluated rst because it is the predominant limonoid glucoside found in most Citrus and was available in our laboratory from previous studies. Before starting these experiments, the ELS operating parameters of temperature and nebulizer pressure were optimised. Operating temperature (3055 C) and nebulizer pressure (1.92.5 bar N2) were varied as 20 lL injections of LG (5 mg L1) were introduced into an aqueous solvent stream owing into the detector at a rate of 500 lL per min. The best response (data not shown) was observed using an operating temperature of 50 C in conjunction with a nitrogen pressure of 2.5 bar. Comparison of the ELS response to that of a UV (k = 210 nm) detector over a range of LG concentrations (150 mg L1) revealed that ELS detection afforded at least a twofold increase in signal when the instruments gain was set to its maximum level; however, in contrast to the linear response of the UV detector (y = 8.896x + 37.373, R2 = 0.987), response of

the ELS detector was parabolic and was t using a polynomial equation (y = 1.9032x2 + 21.318x + 57.437, R2 = 0.999). From these experiments the instrument limit of quantitation for LG by ELS was estimated to be approximately 5 ng. Limonoid glucosides contain one or two carboxylic acid groups and we anticipated that the pH of the mobile phase would affect retention time. Fig. 2 shows the effects of mobile phase acidity (formic acid 150 mM) on the retention of LG on the phenyl stationary phase. Retention time increased from 1.09 min to 2.14 min with increasing acidity of the mobile phase. Formic acid concentrations above 50 mM had no further effect on retention time, but did result in an increase in baseline noise from the ELS detector. Based upon the observed results, a formic acid concentration of 4 mM was selected for subsequent experiments examining the effects of solvent polarity and ow rate (0.20.5 mL min1). For the next phase of the method development, the LCMS system was used to analyse a sample containing all six limonoid glucosides. We wished to evaluate both methanol and acetonitrile as organic components of the mobile phase. A side-to-side comparison of chromatograms revealed that methanol runs were plagued with signicant peak tailing (data not shown). We thus focused our evaluation exclusively on acetonitrile, varying its concentration stepwise from 10% to 30%. At the lowest acetonitrile concentration, 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. Whereas when 30% acetonitrile was used, the analytes were not retained, but were immediately co-eluted. Ultimately, a mobile phase composition of 85:15, 4 mM formic acid:acetonitrile was chosen as the optimal compromise between analysis time and resolution. Changes in ow rate under the conditions examined affected neither peak shape nor resolution of the analytes. Fig. 3 illustrates by mass spectrometry data the resolution and order of elution, such that quantitation of each of the six glucosides in a mixture may be accomplished without worry of isotope contribution. Of the limonoid glucosides present in the standard solution, DNAG was the rst of the analytes to be eluted. Examining the structures of the six glucosides (Fig. 1), it is not surprising that DNAG was the rst to be eluted considering that DNAG is the most polar of the six compounds. DNAG was followed by LG, DNG, NAG, NG, and nally OG. In contrast, the reported order of elution for the same glucosides from a C-18 stationary phase was LG followed by DNAG, DNG, NG, NAG, and nally OG (Herman et al., 1990; Ohta et al., 1993; Schoch et al., 2001). Thus, shifting from C-18 to a phenyl 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 stationary phases are the result of the modes in which each phase can interact with the analytes in solution. The long carbon chains of the C-18 phase are like long ngers that can interact with analytes with a signicant degree of freedom in a number conformations, whereas the phenyl phase in contrast is much shorter, ending with a planar phenyl ring, 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. 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. These subtle differences in selectivity between the two phases may be useful in the isolation of limonoid glucosides. Having established chromatographic conditions suitable to resolve limonoid glucosides we further optimised the method by reducing analysis time to maximize sample throughput. To reduce analysis time, we doubled the ow rate from 0.5 to 1.0 mL min1 and found chromatographic resolution of the analytes unaffected by increased ow. Concurrent with the increase in ow rate, col-

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Fig. 2. Retention time of limonin glucoside on the phenyl stationary phase increases with increasing formic acid concentrations in the mobile phase. Experiments were conducted using a 25 mg L1 solution of limonin glucoside and chromatograms monitored with an evaporating light scattering detector. Additional details are found in Section 2.

umn backpressure doubled and reached a value around 2200 psi. Although the LCQ Advantage mass spectrometer could handle a ow rate of 0.5 mL min1, it was overwhelmed by a ow of 1.0 mL min1 so that it was necessary to introduce a ow splitter (1:5) prior to the mass spectrometer to accommodate the increased ow rate. Utilizing the LCMS conditions described above, response was conrmed to be linear over three orders of magnitude (1 150 mg L1; R2 > 0.98) and the limit of quantitation was estimated to be near 2 ng. Both results are consistent with a previous report on the detection of limonoid glucosides by LCMS (Schoch et al.,

TIC: 400-800

DNAG

m/z= 669.4

LG

m/z= 649.3

DNG

m/z= 651.4

NAG

m/z= 711.3

NG

m/z= 693.3

OG

m/z= 633.3
6

Time (min)
Fig. 3. Total ion and selective ion chromatograms resulting from the LCMS analysis of a standard solution containing the six limonin glucosides using a Phenosphere-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 in water (15:85), ow rate = 0.5 mL min1.

2001). Considering that limonoid glucoside concentrations in orange juice and citrus molasses typically range from 250 to 396 mg L1 (Fong, Hasegawa, Herman, & Ou, 1990; Herman et al., 1990) and 4707960 mg L1 (Schoch et al., 2001), respectively, we were concerned that analyte concentrations in neat samples would be out of the linear range of the detector unless diluted. Therefore, before LCMS analysis, the total limonoid glucoside content of each sample was estimated by the modied colorimetric method described in Section 2. After the concentration was determined, the sample was diluted with water to a total limonoid glucoside concentration between 50 and 100 mg L1. Since a single limonoid glucoside usually accounts for more than 50% of the total glucoside content of a given sample, this concentration range was targeted to ensure that the concentrations of the predominate glucosides would be within the linear range of the MS detector. A 5:1 dilution was sufcient for most juice samples, whereas for more concentrated samples (e.g., molasses or concentrated extracts) 50 to 1000-fold dilutions were required. Washington Navel, Hamlin and Valencia sweet oranges purchased from a local grocery store were hand squeezed, diluted 5:1 with water and analysed for their limonoid glucoside content. Results for the analysis of each of these samples along with the results previously reported for orange juices are listed in Table 1. Amongst the juices tested, Washington Navel and Valencia juices were the most similar and exhibited LG compositions close to those previously reported for orange juices. 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. The compositional differences between these samples and those for the Hamlin, Pera Rio and Natal samples suggest that in addition to harvest time (Fong et al., 1992), the limonoid glucoside contents found in Citrus fruits are also greatly inuenced by variety. Because we wished to apply this method on an ongoing basis and results were to be reported as relative percentages, we evaluated the inter-run and inter-day variability associated with the

A.P. Breksa III et al. / Food Chemistry 117 (2009) 739744 Table 1 Limonoid glucoside contents found in common commercial varieties and previously reported values as relative percentage. Limonoid glucoside ([MH] 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. a Fong et al. (1990). b Herman et al. (1990). c Schoch et al. (2001). LG (649) 53.6 57.1 33.2 56.3 53.2 82.8 80.9 NG (693) 26.2 23.1 31.4 nd 14.2 5.7 6.4 DNG (651) 0.8 1.8 0.9 nd 6.3 0.0 0.0 NAG (711) 10.6 5.2 9.7 nd nd 1.1 2.1 DNAG (669) 8.1 11.6 21.3 nd 25.4 9.2 10.6

743

OG (633) 0.7 1.2 3.5 nd 0.9 1.1 0.0

method in order to determine if there were any limitations with this strategy. For this evaluation, a set of eleven randomly chosen samples, including juice, extracts and liquid samples of partially puried limonoid glucosides were evaluated in triplicate on three consecutive days. Each sample was diluted to within the linear range of the MS detector before analysis. Results from the intrarun evaluation are shown in Fig. 4. Variability was more heavily inuenced 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 normalised total, the observed %CV was typically less than 5%. As the percent composition decreased further, the %CV continued to increase. Results from the second two days of testing were similar (data not shown). Evaluating the results obtained across the three days, 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 condence 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. However, considering the variability observed, we adopted specic uncertainty levels (%CV = 5%, 10% and 20%) and recommend that these guidelines be used when evaluating results. For relative concentrations that were equal to or greater than 50%, a %CV of 5% was utilised (i.e., assigned uncertainty = relative concentration (%) 0.05). 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. As part of an ongoing project to characterise chemical phenotypes of genetic resources found within the USDAs A.H. Whitmore Foundation Farm, juice obtained from fruits from trees located

within the collection were analysed. The UV (k = 220 nm), total ion and selective ion monitoring (SIM) chromatograms obtained for two representative samples are shown in Fig. 5AF. Detection of analytes by mass spectrometry can be hindered by matrix components, in particular by salts or other species in signicant concentrations (e.g., sugars, organic acids) that are often not bound by reverse phase stationary phases. Thus with abbreviated chromatographic methods, such as this one and typical of many LC MS methods, there is a need to resolve the majority of sample matrix components sufciently from the analytes of interest. 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. Spike recoveries observed after the addition of limonin glucoside (10 and 25 mg L1) to juice and other samples were observed to range from 95% to 103% and provide further support that, for at least limonin glucoside, the analytes are resolved from interfering matrix components. The relative percentage concentration for each limonoid glucoside in these two representative samples was calculated as described above and in Section 2. Limonoid glucosides detected at greater than 1% for the rst sample included (Fig. 5C) LG (14.2 1.4%), NAG (74.0 3.7%), and NG (10.2 1.0%), whereas for 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 different trees that are members of a F1 population generated from a cross between C. Grandis and Poncirus trifoliata (Nakon Flying Dragon) C. Sinensis (Succari), the relative concentrations of the limonoid glucosides found in the samples is distinctly different. We are currently continuing our analysis of this population in

Fig. 4. Evaluation of intra-run variability: %CV and its relationship to percent total composition. Inset is a magnication of the same graph from 0% to 10%.

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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 two different trees that are members of a F1 population generated from a cross between C. grandis and Poncirus trifoliata (Nakon Flying Dragon) C. sinensis (Succari).

the hope that results from the analysis of their limonoid glucosides content and character will yield segregating populations that may be further examined by genomic tools in order to establish the genetic basis of the observed phenotypes. Our goal was to develop a rapid and robust LCMS method to characterise limonoid glucosides found in citrus juices, extracts, and fractions obtained from our isolation efforts. The majority of methods described thus far for the HPLC and LCMS analysis of limonoid glucosides have relied upon C-18 stationary phases. In this report, we describe our evaluation of a phenyl stationary phase as an alternative to C-18 and show that, in addition to resolving the analytes of interest, the phenyl stationary phase, when compared to C-18 phases, also exhibits differences in selectivity that might further aid in isolating limonoid glucosides. Additionally, we demonstrate that the chromatography is rapid and robust, and, when paired with mass spectral detection, provides a method that is applicable to the analysis of samples having complex matrices. Furthermore, we believe that this method and its use, in conjunction with the colorimetric estimation of total limonoid glucoside concentration, will be of value to those with experience in the analysis of citrus limonoid glucosides and equally valuable to other researchers who do not have the means or resources to obtain analytical standards, but wish to evaluate citrus samples for their limonoid glucoside content and character. References
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