Development of an integrated approach for the stability testing of flavonoids and ascorbic acid in powders

Food Chemistry 129 (2011) 51–58

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Development of an integrated approach for the stability testing of flavonoids and ascorbic acid in powders Megan E. West, Lisa J. Mauer ⇑ Purdue University, Department of Food Science, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907, USA

a r t i c l e

i n f o

Article history: Received 2 December 2010 Received in revised form 27 January 2011 Accepted 29 March 2011 Available online 12 April 2011 Keywords: Flavonoids Ascorbic acid Stability Powders NMR HPLC GLC

a b s t r a c t An integrated approach for determining the stability of flavonoids and ascorbic acid in powders was developed. A rapid analyte solubility procedure was used to decide whether HPLC, GLC, or NMR would best serve to measure the stability of each flavonoid. Both the flavonoid under study and ascorbic acid were measured in one analysis with any of the determinative methods. Seventeen flavonoids of differing types, as well as ascorbic acid, were evaluated to establish the approach. The effects of storage relative humidity (RH) on the stability of several flavonoid powders with and without ascorbic acid were then determined. Quercetin, luteolin, taxifolin, naringenin, and naringin were stable for up to 8 weeks in RHs up to 98%, and ascorbic acid was significantly destroyed in the presence of quercetin, luteolin, and taxifolin at 98% RH. Both grape anthocyanins and ascorbic acid were unstable in the presence of each other at 98% RH. The integrated approach could be used for shelf-life testing of powdered flavonoids under a variety of storage conditions. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Flavonoids are a very large family of natural products with thousands of structural variants described to date (BenaventeGarcía & Castillo, 2008; Higdon & Drake, 2008). The family is divided into numerous classes, including anthocyanidins and their glycosides, the anthocyanins, flavonols, flavanonols, flavanones, flavones, isoflavones, and flavonols. Research increasingly supports an important protective role for flavonoids against the risk of chronic diseases, such as cancer, cardiovascular, and neurodegenerative diseases (Benavente-García & Castillo, 2008; Higdon & Drake, 2008). While tea, citrus fruit juices, wine, and citrus fruit lead the list of foods for delivering the greatest mean daily intake of flavonoids (Chun, Chung, & Song, 2007), consumer product manufacturers have responded by marketing a plethora of products containing flavonoids as functional ingredients. To illustrate this point, the term ‘‘flavonoids’’ when entered into a popular consumer product Internet search engine resulted in a listing of almost 35,000 consumer choices (Google Product Search, 2010). In these products, flavonoids are universally formulated with additional materials, often substances with other reported health benefits. Vitamin C (ascorbic acid, sodium ascorbate) is one of the most commonly added nutrients to foods. Further to this point, mixtures of flavonoids and vitamin C are popular (Porjes, 2002). While liquid ⇑ Corresponding author. Tel.: +17 654949111. E-mail address: [email protected] (L.J. Mauer). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.131

products are available, the majority of flavonoids and vitamin C combinations are in powdered form. Considering the increasing use of powdered systems to deliver bioactive components, either in finished form (as powdered food products) or as premixes (for liquid or other food products), it is essential to understand the stability of the added ingredients throughout the variety of formulations, processes, packaging, storage, and distribution systems encountered. Additionally, the food industry often relies on accelerated shelf-life and stress testing to determine product shelf-life, using elevated temperature, relative humidity (RH), light exposure, or other treatment. It is therefore interesting to find limited information published on the stability of flavonoid powders with and without ascorbic acid exposed to these treatments. Previous research has demonstrated that the major flavonoids in tea powders, ( ) – epicatechin, ( ) – epicatechin gallate, ( ) – epigallocatechin, ( ) – epigallocatechin gallate, were degraded in the presence of moisture and this degradation was accelerated by coformulation with ascorbic acid (Oritz, Ferruzzi, Taylor, & Mauer, 2008). However, ascorbic acid enhanced the stability of tea catechins in buffered (pH 7.4) solutions (Chen, Zhu, Wong, Zhang, & Chung, 1998). The discrepancy between these results indicates that stability trends likely differ between powders and solutions. Although not previously demonstrated in powders, it is also well known that anthocyanins and ascorbic acid are mutually destructive in solution (Brenes, Pozo-Insfran, & Talcott, 2005; Clegg & Morton, 1968). Interestingly, other flavonoids have been reported to give opposite

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results; quercetin and taxifolin (dihydroquercetin); for example, were not only stable in dilute solution, but also protected ascorbic acid from decomposition (Clegg & Morton, 1968). Studies of vitamin C powders reported that the temperature and RH affect degradation, with vitamin degradation increasing significantly once the RH exceeded the deliquescence RH of the ingredient (Hiatt, Ferruzzi, Taylor, & Mauer, 2008). Powdered vitamin C stability is dependent upon the form used, with ascorbic acid possessing better stability than sodium ascorbate under similar conditions (Oritz et al., 2008; Hiatt et al., 2008). Owing to this rather limited number of studies with perhaps conflicting results and the importance of the subject, the need to better understand the stability of a broader range of flavonoid types with and without added ascorbic acid is clearly indicated. A limitation for determining the effect of environmental moisture or other stress conditions on the stability of a broad range of flavonoid types with or without added ascorbic acid in powders is the lack of a suitable testing methodology. Methods for measuring flavonoids (Marston & Hostettmann, 2006; Valls, Millán, Martí, Borràs, & Arola, 2009) and ascorbic acid (Nováková, Solich, & Solichová, 2008) have been extensively reviewed and summarised; these procedures were overwhelming designed for use with biological, food, and/or cosmetic samples. Unfortunately the techniques were not tailored to the special requirements of experimental moisture stability, stress testing of powders over time, or designed for measuring both flavonoids and ascorbic acid in a single chromatographic determination. The objective of this study was to develop an integrated approach for the analysis of numerous powdered flavonoids with and without ascorbic acid that could facilitate further study of the time-course effects of a variety of stressing factors (e.g. environmental moisture, temperature, headspace gas composition, and light) on powdered products. The approach was applied to monitor the effects of environmental relative humidity on the stability of flavonoid and/or ascorbic acid powders. 2. Materials and methods 2.1. Chemicals Methanol (HPLC grade), water (HPLC grade), dimethyl sulfoxide (DMSO, ACS grade), potassium carbonate 1.5 hydrate, (ACS grade), potassium sulfate (ACS grade), sodium chloride (ACS grade), sodium hydroxide (ACS grade), hydrochloric acid (ACS grade), and hesperedin 97% were obtained from VWR Scientific (West Chester, PA, USA). Ethyl alcohol, absolute (ACS/USP grade) was purchased from Pharmco-Aaper (Brookfield, CT, USA). Formic acid (p.a. grade), pyridine (HPLC grade), quercetin dihydrate P 98%, naringin > 90%, naringenin 95%, rutin hydrate 95%, (+) - catechin hydrate 98%, ( ) – epigallocatechin gallate 95%, chrysin 97%, apigenin 95%, genistein P 98%, hesperetin P 95%, luteolin P 98%, taxifolin P 85%, quercetin 3-b-D-glucoside P 90% (isoquercetrin), cyanidin chloride P 95%, cyanidin-3-glucoside chloride P 95% (kuromanin Cl), L-ascorbic acid (#A7506), methyl-a-D-glucopyranoside, calcium sulfate (DrieriteÒ – 4 mesh), and dimethyl sulfoxide-d6 containing 0.1% tetramethylsilane (TMS) were all purchased from Sigma–Aldrich (Milwaukee, WI, USA). Tri-SilÒ Z (1 part trimethylsilylimidazole: 4 parts pyridine) and N-methyltrimethylsilyltrifluoroacetamide (MSTFA) + 1% trimethylchlorosilane (TMCS) were obtained from Pierce-Thermo Fisher Scientific (Rockford, IL, USA). A proprietary water soluble grape skin extract (containing anthocyanins) was obtained from Roha Dyechem Pvt Ltd (Mumbai, India). 2.2. Solubility testing procedure for selecting the determinative method For solubility testing, 5 mg of the test flavonoid were weighed into each of four scintillation vials. Into two of the vials, 5 mg of

ascorbic acid were added. To two vials, one with and one without ascorbic acid, 5 ml of 2% V/V formic acid were added (prepared by adding 24.4 g formic acid to 980 g water). To the remaining vials, 5 ml of a freshly prepared 1:1 V/V absolute ethanol and 2% V/V formic acid solution were added. Samples were placed in an ultrasonic bath for up to 30 s to test the complete dissolution of all components. If a clear solution was not obtained with either of the two solvents, four more samples were prepared in an identical manner as described above except that 25 mg of the analytes were weighed into the vials. To two vials, one with and one without ascorbic acid, had 5 ml of pyridine added, while the remaining vials received 5 ml of DMSO. Samples were again observed for complete dissolution of analytes. 2.3. HPLC procedures Duplicate 10 mg single and 20 mg two-component samples were accurately weighed into scintillation vials and 10 ml of a freshly prepared 1:1 V/V solution of absolute ethanol and 2% V/V formic acid were added to each vial. Each sample was placed in a sonic bath (Thermo Fisher Scientific sonicator model FS30) for a few seconds to ensure complete dissolution of all of the components. A portion of the sample was filtered using Millipore Corp. (Billerica, MA, USA) MillexÒ – LCR HPLC filters and analysed. Samples were not batched, but were individually solubilised and immediately analysed. The HPLC was a Waters (Milford, MA, USA) 2695 HPLC equipped with a 2998 PDA detector and Empower 2 software. All chromatography was performed on a Grace (Deerfield, IL, USA) Prevail C18 5 l analytical column, 250  4.6 mm and Prevail C18 5 l guard column 7.5  4.6 mm at 30 °C. For the analysis of flavonoids with or without ascorbic acid, except anthocyanin-containing extracts, the mobile phase was a linear gradient of 2% V/V formic acid in water and methanol starting at 10% methanol for 5 min increasing to 65% methanol over 20 min. For the analysis of anthocyanin-containing extracts with or without ascorbic acid, the mobile phase was a linear gradient of 2% V/V formic acid in water and methanol starting at 0% methanol increasing to 25% methanol in 10 min and then to 50% methanol in 30 min. For ascorbic acid without the presence of flavonoids, an isocratic mobile phase of 2% V/V formic acid in water and 10% methanol was used. The flow rate was 1.0 ml/min and the injection size was 10 ll for all samples. UV detection was 290 nm for flavonoids, except anthocyanins and anthocyanidins where 520 nm was also used. Ascorbic acid was quantified with a detector setting of 250 nm. The linearity of the detector response was determined in a single experiment by weighing a range of sample weights directly into scintillation vials followed by dissolution and analysis. The limit of detection was also determined in a single experiment using the lowest sample weight from the linearity study and serially diluting 1:10 with solvent followed by analysis until the analyte signal was reduced to twice that of the baseline noise. 2.4. GLC procedures Duplicate 5 mg single and 10 mg two-component samples were accurately weighed into culture tubes and 20 ll of the internal standard solution were pipetted into each tube before placing on a SP Industries Inc. (Warminster, PA, USA) Virtis model 273466 freeze-dryer to remove all traces of water (from the internal standard and any moisture absorbed over storage) overnight at a pressure of 40 Pa and at room temperature. After drying, 1 ml of Tri-SilÒ Z or MSTFA + 1% TMCS was added and heated at 70 °C for 20 min in a Thermo Fisher Scientific (Rockford, IL, USA) MultiBlokÒ model 2050 heater with initial vortex mixing (Thermo Fisher Scientific Vortex Genie 2 model G-560 mixer) and then again at

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10 min. Samples were analysed immediately after derivatisation and prepared singly to ensure against any decomposition if allowed to stand before analysis. The GLC was an Agilent Technologies (Santa Clara, CA, USA) HP 5890 Series equipped with a FID detector and 3396C integrator. Chromatography was performed with 3% OV-17 on 100/120 Supelcoport™ contained in a 2ft (0.61 m)  2 mm I.D.  1/4 in. (6.4 mm) O.D. glass column purchased from Sigma–Aldrich. The column temperature was programmed from 100 to 300 °C at 10 °C/min. The carrier gas was helium at 20 ml/min. The injector and detector temperatures were 325 and 350 °C, respectively. The injection size was 1 ll. The internal standard for GLC analyses was prepared by dissolving 25 g of methyl-a-D-glucopyranoside in 100 ml of water. Aliquots of approximately 10 ml were kept frozen and thawed immediately before use. The linearity of detector response was determined in a single experiment by weighing a range of sample weights directly into culture tubes followed by derivatisation and analysis. The limit of detection was also determined in a single experiment using the lowest sample weight from the linearity study and serially diluting 1:10 with derivatisation reagent followed by analysis until the analyte signal was reduced to twice that of the baseline noise.

Scientific Model 818 (Rockford, IL, USA) temperature controlled incubator. RH was monitored in each chamber by inclusion of a traceable hygrometer purchased from Control Co. (Friendswood, TX, USA). Controls were stored tightly capped in a Revco (Rockford, IL, USA) ULT 1786–3-D-U-A freezer set at 80 °C. All frozen materials were placed in a desiccator containing DrieriteÒ and allowed to reach room temperature before opening.

2.5. 1H NMR procedures

The solubility of each analyte with and without ascorbic acid in 2% V/V formic acid and the absolute ethanol/2% V/V formic acid solution at a concentration of 1 mg/ml per compound was determined and used as the first step in the integrated stability testing approach (a schematic of the entire solubility and stability testing protocol is presented in Fig. 1). Samples that readily dissolved in either solution at room temperature were deemed good candidates for analysis by HPLC. Those analytes that were poorly soluble in both of these solutions were evaluated in pyridine at a concentration of 5 mg/ml. Compounds that were readily soluble at room temperature and whose TMS (trimethylsilyl) derivatives were thought to be volatile at GLC injector temperatures were considered good candidates for analysis by GLC. Flavonoids were also evaluated in DMSO at 5 mg/ml at room temperature. Readily soluble compounds were deemed good candidates for analysis by NMR. The solubility test results, analysis method choices, and selected flavonoid information for seventeen representatives from seven classes of flavonoids are reported in Table 1. With the exceptions that higher molecular weight flavonoid glycosides (MW > 600) and flavonoids with less than three hydroxyls (e.g. chrysin) may suggest analysis by NMR and GLC, respectively, no other obvious factors, based upon chemical structure, are apparent to predict a preferred analysis choice when following this solubility screening approach. The lack of simple rules for predicting the solubility of flavonoids has been observed by others (Chebil et al., 2007). It is also likely that the higher molecular weight observation is not valid with anthocyanins where even higher molecular weight species would be expected to have good solubility in 2% V/V formic acid (West, 2010, personal communication). As the database generated by this research is expanded, predictive trends may become apparent, but for now the solubility testing protocol remains a key component of this integrated approach.

Duplicate 10 mg single and 20 mg two-component samples were accurately weighed into culture tubes and placed on the freeze-dryer to remove any traces of water followed by the addition of 1 ml of DMSO-d6 to each tube. After complete dissolution of the contents, 750 ll of the solution were transferred to an NMR tube and analysed. The 1H NMR was a Varian (Santa Clara, CA, USA) Inova-300 with a 5 mm 4-nucleus probe. Data were processed using ACD/Labs (Toronto, Ontario, Canada) 1D NMR Processor software (version 12.0). NMR tubes were purchased from Kimble Chase LLC (Vineland, NJ, USA). The linearity of the detector response was determined in a single experiment by weighing a range of sample weights directly into culture tubes followed by dissolution and analysis. The limit of detection was also determined in a single experiment using the lowest sample weight from the linearity study and serially diluting 1:10 with DMSO-d6 followed by analysis until the analyte signal was reduced to twice that of the baseline noise. 2.6. Environmental relative humidity (RH) stability testing procedures Following the solubility testing for choosing the analytical method to be used for monitoring stability, samples were prepared as follows for storage followed by HPLC, GLC, or NMR analysis. When HPLC was selected for the determinative method, multiple sets of duplicate 10 mg single and 20 mg two-component samples were accurately weighed into scintillation vials. When analysing samples by GLC, 5 mg single and 10 mg two-component samples were accurately weighed into culture tubes – for NMR, the sample size was doubled. One set of duplicate samples for each analysis time period were placed in each of the RH chambers (43–98% RH) while freezing a matching set of controls. For a typical study, samples at time 0 and weeks 2, 4, and 8 were routinely analysed. Glass desiccators, 325  250 mm purchased from Corning Inc. (Corning, NY, USA), were used as RH chambers and contained the following saturated salt solutions: potassium carbonate (43% RH), sodium chloride (75% RH), and potassium sulfate (98% RH). Samples were placed into the RH chambers uncapped and on their sides to avoid any condensed water entering the containers from the chamber lids during removal. Condensation was especially prevalent on the surfaces of the 98% RH chamber. All storage experiments were conducted in the dark at 24.9 ± 0.1 °C in a Precision

2.7. Statistical analysis Data were analysed using SAS (Cary, NC, USA) version 9.2 software. Bonferroni’s, Tukey’s, and Scheffé’s multiple comparison methods along with ANOVA were used to determine the statistical significance of the data. All tests were conducted at a = 0.05. The percentage relative standard deviation (%RSD) of repeat analyses was calculated by dividing standard deviations by their respective averages and multiplying by 100. 3. Results and discussion 3.1. Solubility screening for analysis selection

3.2. HPLC analyses Of the 17 flavonoids in the test series, 12 were soluble in 2% V/V formic acid or the absolute ethanol/2% V/V formic acid solution with and without added ascorbic acid at 1 mg/ml (Table 1) and therefore subjected to analysis by HPLC. Both the flavonoid and ascorbic acid can be quantified with one analysis – an improvement over the methodology employed by others when measuring

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Fig. 1. Solubility testing procedure for selecting the analysis method.

Table 1 Solubility test results, analysis method choices, and selected flavonoid information. Compounds

a b c d e f g h

Solubility Resultsa

Number of OH Groups

C-ring double bond b,c

Mol. Wt.

Primary Analysis Methodd

Retention Time (min)

Y Y Y

8 (glycoside) 5 10 (glycoside)

Y Y (with OH) Y

464 302 611

HPLC GLC NMR

13.8 17.4 N/A

Y

Y

5

Y (with OH)

323

HPLC

13.8

Y N

Y N

Y Y

8 (glycoside) N/A (mixture)

Y Y

485 Variable

HPLC HPLC

11.8 or 23.7g 5 peaksh

N

Y

Y

Y

5

N

304

HPLC

12.7

N N N N

N Y Y Y

Y Y Y Y

Y Y Y Y

8 + 1 OCH3 (glycoside) 3 + 1 OCH3 3 8 (glycoside)

N N N N

611 302 272 581

NMR HPLC HPLC HPLC

N/A 16.3 15.9 12.9

Flavones Luteolin Apigenin Chrysin

N N N

Y N N

Y Y Y

Y Y Y

4 3 2

Y Y Y

286 270 254

HPLC GLC GLC

17.5 17.1 14.8

Flavanols (+) – Catechin ( ) – Epigallocatechin gallate

Y Y

Y Y

Y Y

Y Y

5 7 (ester)

N N

290 458

HPLC HPLC

10.8 11.2

Isoflavones Genistein Ascorbic acid

N Y

Y Y

Y Y

Y Y

3 4

Y N/A

270 N/A

HPLC HPLC, GLC, NMR

17.3 3.1 or 4.1g, 6.5, N/A

2% FAe

E/2% FAf

Pyridine

DMSO

Flavonols Isoquercetrin Quercetin Rutin

N N N

Y N N

Y Y Y

Anthocyanidins Cyanidin

Y

Y

Anthocyanins Cyanidin-3-glucoside Grape skin extract

Y Y

Flavanonols Taxifolin Flavanones Hesperedin Hesperetin Naringenin Naringin

N means not soluble and Y means soluble for solubility results. For location of C-ring, refer to rutin (Fig. 4). Y means yes for C-ring double bond and N means no for C-ring double bond. For details of HPLC, GLC, and NMR conditions refer to the materials and methods section. 2%FA means 2% formic acid. E/2% FA means 1:1 V/V absolute ethanol/2% formic acid. Longer retention time when using anthocyanin-containing extract mobile phase. Delphinidin-, cyanidin-, petunidin-, peonidin- and malvidin-3-glucosides (retention times 21.5, 23.7, 25.3, 27.3 and 28.2 min. respectively).

both types of analytes (Oritz et al., 2008). This method was based upon the use of a single column type and mobile phase (although the composition, not the solvents themselves, was varied to analyse anthocyanin-containing extracts, as well as ascorbic acid, when not combined with a flavonoid). This greatly facilitates the analysis of the different flavonoid types and ascorbic acid since no system change-over is required. The column selected is especially useful for mobile phases containing high water content and

the chromatography of very polar analytes such as ascorbic acid (Oey, Verlinde, Hendrickx, & Loey, 2006). Formic acid (added to HPLC water only) was selected as a mobile phase modifier to improve the chromatography of anthocyanins, anthocyanidins, and ascorbic acid, while not negatively impacting the chromatography of other flavonoid types. In addition, this modifier is compatible with further analyses using HPLC/MS (Rijke, Zappey, Ariese, Gooijer, & Brinkman, 2003).

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Fig. 2. Typical HPLC chromatogram of a flavonoid (naringin) with added ascorbic acid – both analytes at 1 mg/ml.

Samples for HPLC may be dissolved in 2% V/V formic acid or the absolute ethanol/2% V/V formic acid solution. In those cases where a sample was soluble in either solvent, the absolute ethanol/2% V/V formic acid solution was selected. Absolute ethanol was chosen over other alcohols for incorporation in the dissolution solvent owing to its safety and ease of disposal. The choice of HPLC sample filtration units was based upon studies demonstrating the low retention of flavonoids, especially anthocyanins, compared to filters manufactured with other membrane materials (Lunn, Schumacher & West, 2009, unpublished results). Linearity of the HPLC standard curves for triplicate samples of ascorbic acid (0.15–1.5 mg/ml 2% V/V formic acid), and representative flavonoids (naringin [0.43–1.5 mg/ml 1:1 V/V absolute ethanol/2% V/V formic acid solution] and naringenin [0.45–1.5 mg/ml 1:1 V/V absolute ethanol/2% V/V formic acid solution] resulted in correlation coefficients of 0.99. Repeated analyses routinely yielded percentage relative standard deviations of less than 1.5% for the triplicate samples of any analyte. The practical limit of detection was 1 nmol/injection for ascorbic acid and 2 nmol/ injection for a flavonoid (naringenin). Typical HPLC chromatograms of ascorbic acid and a single flavonoid (naringin) as well as an anthocyanin mixture with ascorbic acid are presented in Figs. 2 and 3, respectively. 3.3. GLC analyses All of the test flavonoids and ascorbic acid were soluble in pyridine at 5 mg/ml making them potential candidates for analysis by GLC (while the grape skin extract did not completely dissolve, it should not be considered typical since it is a crude mixture containing many non-flavonoid moieties). The only exceptions would be the glycosides, which are suspected of being insufficiently volatile for GLC except perhaps if using very high temperatures or columns with unusually low loadings of stationary phase. For example, naringin (the rhamnoglucoside of naringenin) failed to elute with our method, whereas Katagi et al. (1973) were successful using OV-17 at the very low loading of 0.5%. However, this low loading is not likely to retain ascorbic acid sufficiently as to be useful for our purposes. With the test samples, three substances (quercetin, apigenin, and chrysin) were chosen for GLC since of the five not soluble in the absolute ethanol/2% V/V formic acid solution or 2% V/V formic acid, two were glycosides deemed not amenable to the GLC method (Table 1). GLC of flavonoid TMS derivatives is a well established procedure

(Creaser, Koupai-Abyazani, & Stephenson, 1991a; Koupai-Abyazani, Creaser, & Stephenson, 1992). Similar to HPLC, additional structural information can be obtained when the GLC is coupled to mass spectrometry (Creaser, Koupai-Abyazani, & Stephenson, 1991b). The GLC procedure used a short packed column to minimise the time for analysis while maintaining sufficient resolving power to separate likely decomposition products. In addition, an internal standard was used to verify the formation of TMS derivatives and facilitate quantitation. As with the HPLC technique, both the ascorbic acid and the flavonoid can be analysed on a single chromatogram. A disadvantage of the GLC method is that only trace amounts of water can be tolerated. This necessitates the need for a drying step prior to analysis for samples exposed to RH treatments in order to ensure the reduction of water to a minimum. Tri-SilÒ Z can tolerate more water than MSTFA + 1% TMCS and is therefore preferred except in those cases where multiple peaks may be present due to chalcone formation (Creaser et al., 1991a). Multiple peak formation is not evident with MSTFA + 1% TMCS. Fortunately, should a sample with excessive water be inadvertently derivitised, it is easily observed and rejected during chromatography since the internal standard area is inconsistent with the other samples. GLC was not chosen over HPLC as the method of choice due to the additional steps of drying and derivitisation and analyst exposure to pyridine when using Tri-SilÒ Z. However, this procedure was especially useful for the study of quercetin with or without added ascorbic acid. Using the GLC method, the linearity of standard curves for triplicate samples of ascorbic acid (4.2–5.7 mg/ml Tri-SilÒ Z) and a representative flavonoid (quercetin [4.4–7.3 mg/ml Tri-SilÒ Z]) resulted in correlation coefficients of 0.99. Repeat analyses routinely yielded percent relative standard deviations of 3.0% for triplicate samples for any analyte. The practical limit of detection was 0.1 nmol/injection for both ascorbic acid and quercetin. A typical GLC chromatogram of ascorbic acid and a flavonoid (quercetin) is presented in Fig. 4. 3.4. 1H NMR analyses For samples not suitable for either HPLC or GLC based upon the solubility experiments (rutin and hesperedin in Table 1) and as a method for corroborating chromatographic results, an NMR procedure using 1H NMR was developed. NMR spectral data of many flavonoids are widely available (Davis, Cai, Davies, & Lewis, 1996; Moco, Tseng, Spraul, Chen, & Vervoort, 2006) and the utility of

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Fig. 3. Typical HPLC chromatogram of an anthocyanin mixture (grape skin extract) with ascorbic acid – both analytes at 1 mg/ml.

NMR for quantification of flavonoids has been recently demonstrated (Veit, 2010). Every test flavonoid and ascorbic acid were readily soluble in DMSO at the desired concentration, therefore all of the samples had the potential to be evaluated by NMR. However, NMR was not selected as the analysis method of choice over HPLC mainly due to the high expense of the equipment required. For flavonoids, the signal of the proton on carbon atom 6 (C6) of ring A was chosen for quantitative measurements. Likewise, for ascorbic acid the proton on carbon atom 4 was selected (Spectral Database for Organic Compounds, 2010). A spectrum of a standard mixture of a representative flavonoid (rutin) and ascorbic acid, identifying the positions of the protons selected for making quantitative measurements is presented in Fig. 5. Any chance of interference and signal suppression from water was minimised by drying each sample. A weakness of the procedure is possibly envisioned for those cases where the structures of the flavonoid decomposition products have not changed sufficiently to alter the intensity or position of the proton on C6. To guard against this possibility, careful inspection of the entire spectrum should be undertaken. The linearity of NMR standard curves for duplicate samples of ascorbic acid (0.9–8.2 mg/ml DMSO-d6) and representative flavonoids (quercetin [1.5–14.2 mg/ml DMSO-d6], naringin [3.6– 31.3 mg/ml DMSO-d6], and naringenin [3.0–23.4 mg/ml DMSOd6]) resulted in correlation coefficients of P 0.98. The practical limit of detection was 2 mmol for each analyte. Repeat analyses routinely yielded percent relative standard deviations of 1.4%, 1.2% and 2.5% for duplicate samples of naringin, naringenin, and ascorbic acid respectively. 3.5. Application of the integrated approach in RH stability studies The integrated approach was evaluated for usefulness in longer term studies by determining the effects of co-formulation with

ascorbic acid and the environmental moisture stability (43–98% RH) of several typical flavonoids. Naringenin, and its corresponding glycoside, naringin, as well as quercetin and two related flavonoids, luteolin and taxifolin, were chosen as representative structural types. Quercetin was analysed using GLC and all others by HPLC. While all these flavonoids were stable with and without ascorbic acid in powdered form in air at 43–98% RH and at 25 °C over 8 weeks in the dark, co-formulation of ascorbic acid with quercetin, luteolin, and taxifolin significantly (P < 0.05) accelerated the destruction of ascorbic acid at 98% RH while naringenin and naringin did not. Ascorbic acid alone did not significantly degrade under the study conditions. There are important similarities and differences between results obtained with these new studies and those reported by others. For example, catechins in green tea powders behaved differently from the test flavonoids of this study; the tea catechins significantly degraded either alone or when co-formulated with ascorbic acid at RHs of P69% and P75%, respectively (Oritz et al., 2008). While the key structural differences between tea catechins and the test flavonoids may well explain the results reported here, e.g. the degree of hydroxyl substitution of the B-ring (Chen et al., 1998), Ortiz et al. used green tea powders and not purified catechins. It cannot be ruled out that the degradation was caused, at least partly, by other factors present in the tea. The stability of powdered quercetin over differing RHs is also consistent with a previous solution study, but the instability of ascorbic acid (80% destroyed) in the presence of quercetin at 98% RH is very different (Clegg & Morton, 1968). Flavonoids related to quercetin, specifically luteolin (quercetin without the 3C hydroxyl) and taxifolin (dihydroquercetin) with added ascorbic acid yielded similar results during 98% RH storage. Both flavonoids appeared completely stable, but >94% of the ascorbic acid was destroyed. Naringenin and naringin (a glycoside containing

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Fig. 4. Typical GLC chromatogram of a flavonoid (quercetin) with added ascorbic acid – ascorbic acid at 5 mg/ml and quercetin at 7 mg/ml.

Fig. 5. Typical 1H NMR spectrum of a) flavonoid (rutin) with added b) ascorbic acid – ascorbic acid at 5 mg/ml and rutin at 15 mg/ml.

naringenin) did not destabilise ascorbic acid at 98% RH. These compounds differ chemically from quercetin and its analogs by not possessing a hydroxyl group or a double bond on the C-ring and have only one hydroxyl on the B-ring. The degree of hydroxyl

substitution on the B-ring has been suggested to influence the stability of certain flavonoids (Chen et al., 1998), but changes in the B- or C-ring on the stability of co-formulated ingredients is not well understood.

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Results of a separate 4 week study at 98% RH examining the stability of grape skin extract anthocyanins in ascorbic acid revealed a loss of almost 98% of the anthocyanins (five different glycosides including cyanidin-3-glucoside) and a 15% loss of ascorbic acid. This mutual degradation of both materials in powdered form parallels that reported when both are together in solution (Brenes et al., 2005; Clegg & Morton, 1968), although the stability of vitamin C was found to be far greater in powders. Unfortunately, it is difficult to make satisfactory comparisons between these studies as the types and purity of the anthocyanin-containing extracts used were certainly very different. It is well established that anthocyanins of varying chemical structure possess differing degrees of stability in a variety of environments and that the presence or absence of other substances, e.g. co-pigments, can significantly impact anthocyanin stability (Torskangerpoll & Andersen, 2005). 4. Conclusion A systematic and integrated approach for determining the stability of flavonoids with and without ascorbic acid in powdered formulations was developed. Analytes were weighed directly into the containers used for storage and subsequent dissolution, thereby not requiring any sub-sampling or reweighing of the samples before analysis. The procedure provides a logical framework for selecting a measurement technique based upon simple solubility determinations. When HPLC was indicated, a single column and mobile phase may be used for a broad variety of flavonoids and ascorbic acid with only changes to the mobile phase gradient required. For GLC, only a simple packed column system is required. With the exception of certain anthocyanin mixtures, the chromatography described here was designed for the analysis of individual flavonoids. If the stability of flavonoid mixtures is to be determined, the chromatography may require some modifications to ensure the adequate resolution of all analytes. NMR may also be used as the primary method for analysis, but is especially useful for verifying results obtained by chromatography. The developed approach will enable long term stability studies evaluating powdered flavonoid and ascorbic acid combinations under differing conditions of RH, temperature, headspace gas composition, light, and time. Acknowledgment The authors wish to thank Dr. Leslie West of Kraft Foods Global for his assistance and helpful advice. This project was supported in part by USDA project INDE-2007-04381. References Benavente-García, O., & Castillo, J. (2008). Update on uses and properties of Citrus flavonoids: New findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry, 56, 6185–6205.

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