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Journal of Pharmaceutical and Biomedical Analysis 120 (2016) 383–
Contents lists available at ScienceDirect
Journal of Pharmaceutical and Biomedical Analysis
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j p b a
A new method for rapid determination of indole-3-carbinol and its
condensation products in nutraceuticals using core–shell column
chromatography method
Jakub Fibigr, Dalibor ˇSatínsk ´y ∗, Lucie Havlíková, Petr Solich
Department of Analytical Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, Hradec Králové 500 05, Czech Republic
a r t i c l e i n f o
Article history: Received 27 October 2015 Received in revised form 17 December 2015 Accepted 20 December 2015 Available online 28 December 2015
Keywords: Indole-3-carbinol Nutraceuticals Condensation products Liquid chromatography Core–shell columns Quality control
a b s t r a c t
Indole-3-carbinol is a natural glucosinolate known for prevention of human breast, prostate and other types of cancer and it started to be used in commercial preparations, as food supplements. However no analytical method has been proposed for quality control of nutraceuticals with this substance yet. In this paper a new high-performance liquid chromatography (HPLC) method using core–shell col- umn for separation of indole-3-carbinol and its condensation/degradation products was developed and used for the quantitative determination of indole-3-carbinol in nutraceuticals. Separation of indole-3- carbinol, its condensation/degradation products and internal standard ethylparaben was performed on the core–shell column Kinetex 5 XB-C18 100A (100 × 4.6 mm), particle size 5.0 m, with mobile phase acetonitrile/water according to the gradient program at a flow rate of 1.25 mL min−^1 and at tempera- ture 50 ◦C. The detection wavelength was set at 270 nm. Under the optimal chromatographic conditions good linearity of determination was achieved. Available commercial samples of nutraceuticals were extracted with 100% methanol using ultrasound bath. A 5-L sample volume of the supernatant was directly injected into the HPLC system. The developed method provided rapid and accurate tool for qual- ity control of nutraceuticals based on cruciferous vegetable extracts with indole-3-carbinol content. The presented study showed that the declared content of indole-3-carbinol significantly varied in the different nutraceuticals available on the market. Two analyzed preparations showed the presence of condensa- tion/degradation products of indole-3-carbinol which were not officially declared by the manufacturer. Moreover, further two analyzed nutraceutical preparations showed absolutely no content of declared amount of indole-3-carbinol. © 2015 Elsevier B.V. All rights reserved.
- Introduction
Indole-3-carbinol (I3C) is the breakdown product of the nat- urally occurring glucosinolates, mainly in cruciferous vegetables such as broccoli, Brussels sprouts, cabbage, cauliflower and garden cress. I3C is released from its glucosinolate precursor glucobrassicin when brings into contact with myrosinase [1]. In an acidic solu- tion like the human stomach or under acidic conditions in vitro (0.05 M hydrochloric acid, pH 1.5 for 60 min), I3C is rapidly con- verted into an array of acid condensation products and modified derivatives [2]. In vivo assessment of I3C and its products suggest that 3, 3 ′-diindolylmethane (DIM), an I3C acid-condensation prod- uct, is one of the major bioactive compounds responsible for the
∗ (^) Corresponding author. Fax: +420 495067164. E-mail address: [email protected] (D. ˇSatínsk ´y).
benefits associated with I3C, because DIM has distinct targets and greater bioactivity [3,4]. I3C together with its metabolic products has indicated anti-tumor activity. These indole derivatives have been shown to suppress the proliferation of various cancer cell lines, including those of breast, colon, prostate, and endometrium, by targeting a wide spectrum of signaling pathways governing apo- ptosis, cell-cycle progression, hormonal homeostasis, DNA repair, angiogenesis, and multiple-drug resistance. Moreover, indole-3- carbinol proves to be an effective chemoprotective agent against estrogen responsive cancer such as breast and cervical cancer, in part, because it functions as a negative regulator of estrogen by inhibiting ER signaling and altering cytochrome P450-mediated estrogen metabolism [5]. The intrinsic instability of indole-3-carbinol in acidic milieu arises from the vinyl hemiaminal moiety of the indole ring. This unique structural feature underlies the high susceptibility of indole-3-carbinol to acid catalyzed dehydration and condensation
http://dx.doi.org/10.1016/j.jpba.2015.12. 0731-7085/© 2015 Elsevier B.V. All rights reserved.
to generate a complex series of oligomeric products in vivo, includ- ing DIM (3, 3 ′-diindoylmethane), ICZ (indolo[3,2b]-carbazole), LTr (a linear trimer), CTr (a cyclic trimer), and CTet (a cyclic tetramer) [1,4,5]. Prevention and protection against chemical carcinogens by phytochemicals presented in extensively consumed glucosinolate- containing cruciferous vegetables is of great interest. It provides a safe and cost effective means of cancer prevention [6]. Direct and indirect research evidences demonstrated the benefits of crucif- erous vegetable in prevention of metabolic disorders, asthma and Alzheimer’s disease, along with antimicrobial activity against the number of pathogens. A large number of plant derived compounds including indole-3-carbinol have been identified for prevention and treatment of cancer [7]. Cancer inhibition mechanisms by indole-3- carbinol were established for human breast cancer cells [8] and for human prostate cancer cells [9]. Other health protective effect were studied, e.g. long term treatment with 200 mg of indole-3carbinol twice a day has been demonstrated to be effective in chemopre- vention of respiratory papillomatosis caused by human papilloma virus [10,11]. The above mentioned beneficial biological properties create a wide scope for preparation of food supplements containing indole-3-carbinol. Therefore indole-3-carbinol started to be used in multicomponent commercial preparations, food supplements and nutraceuticals. Despite the benefits of indole-3-carbinol in the aux- iliary cancer treatment, no analytical method for quality control of this substance in nutraceutical products has been proposed.
Nutraceuticals and food supplements are known as dietary sup- plements that deliver a concentrated form of a presumed bioactive agent from food, presented in a non-food matrix, and used with the purpose of enhancing health in dosages that exceed those that could be obtained from normal foods [12]. Food supplements are taken alone or in combination and marketed in dose forms such as capsules, pastilles, tablets, pills and other similar forms, sachets of powder, ampoules of liquids, drop dispensing bottles and pow- ders designed to be taken in measured small unit quantities [13]. Nutraceutical products together with functional foods belong to the most rapidly growing sectors in the food and personal care product industry. The main problem associated with nutraceutical products is their legal classification. Being neither food nor pharmaceuti- cals, they often stay in a gray area between both, which makes the quality control and regulation very difficult [13,14]. No specific reg- ulation exists in Europe to control nutraceuticals, although they are considered under the same laws that regulate medicine and drug. The only aim is to harmonize the legislation and to ensure that these products are safe and appropriately labeled so that consumers can make informed choices of appropriate supplement [15]. In the USA, the Food and Drug Administration regulates dietary supplements under a different set of guidelines than those covering conventional foods and drug products [12]. Because of consumers trust in the safety, beneficial biological activity and efficiency of these products, there is a need of efficient quality control measures [16]. From this point of view, new modern analytical methods covering the quality control of nutraceuticals and food supplements must be developed.
Fig. 1. Chromatogram of indole-3-carbinol, ethylparaben (IS) and condensation products CP1, CP2 and CP3 separation in standard solution performed on the core–shell column Kinetex 5 XB-C18 100A (100 × 4.6 mm), particle size 5.0 m, with mobile phase acetonitrile/water according to the gradient program at temperature 50 ◦^ C.
(LOD) and the limit of quantification (LOQ) was defined as the low- est amount of analyzed compound that gives a reproducible peak with threefold (3) and tenfold (10) variations, respectively, of the baseline noise and signals analyte.
- Results and discussion
No fast method for determination of indole-3-carbinol in nutraceutical products with HPLC and core–shell particle columns has been reported in the literature so far. There were found only two papers in the literature dealing with the separation of the degra- dation/condensation products of indole-3-carbinol [2,27]. Both papers show the separation longer than 12 and 30 min, respec- tively. In our study, the method development and procedure of the sample preparation, conditions of mobile phases and station- ary phases were optimized before the validation and real samples analysis to shorten the separation time of a few minutes. The aim of the presented work was to show new and modern approach to fast separation of indole-3-carbinol and to show the reliability of a new method for the quality control of indole-3-carbinol content in commercial nutraceuticals.
3.1. Optimization of HPLC analysis and method development
The aim of this part was to develop a new chromatographic method which would allow fast and effective separation and quan- tification of indole-3-carbinol in nutraceuticals containing other biologically active compounds and impurities. Indole-3-carbinol is a relatively small middle-polar molecule without charge in almost whole spectrum of pH, but unstable in acidic conditions. Consider- ing this fact, columns with non-polar stationary phase were chosen for the method development. Mobile phase containing acetoni- trile was selected because acetonitrile provides lower viscosity and better peak symmetry of analysed compounds on reversed phase columns. Water without any additives was chosen as a second component of the mobile phase because of the stability of indole- 3-carbinol. For further decreasing mobile phase viscosity, column oven temperature at 50 ◦C was used. In the first step, retention study on different non-polar reversed phase columns with different particle sizes was performed: Ascen- tis Express C18 100 × 4.6 mm, 5 m; X-terra RP18 100 × 3.0 mm, 5 m; Discovery HS F5 100 × 4.6 mm, 3 m; Zorbax SB-C 50 × 4.6 mm, 1.8 m; Kinetex 5 Biphenyl 100 × 4.6 mm, 5 m; Kinetex 5 Phenyl–Hexyl 100 × 4.6 mm, 5 m; Kinetex 5 PFP 100 × 4.6 mm, 5 m; Kinetex 5 XB-C18 100 × 4.6 mm, 5 m. Dif- ferent compositions of mobile phase consisting of acetonitrile and water, and different flow rates were tested to obtain the data about retention of analysed compound. The tested ranges of mobile phase ratios from 10 to 60% (v/v) of acetonitrile with water were used for basic screening of the retention times on each used column. All columns showed similar retention considering the length of the columns and particle size, but columns with core–shell par- ticle technology showed better peak symmetry and more effective separation than the columns with fully porous particles. To achieve the request of short and effective separation, the core–shell parti- cle columns Kinetex and Ascentis-Express were used for further optimization of separation. In the second step a suitable gradient elution method was developed to get a faster elution of other compounds which were also present in multicomponent nutraceuticals and of con- densation/degradation products of indole-3-carbinol. The gradient elution led to shortening the time of analysis and to the separation of all peaks in the chromatograms. Columns with C18, phenyl- hexyl, biphenyl and pentafluorophenyl stationary phases showed similar chromatography separations of all compounds. This effect
could be explained that specific interactions of each stationary phase showed only of a minor influence on retention because of small middle-polar molecules were separated and gradient method was used. But only C18 stationary phase showed the best peak res- olution and no co-elutions with impurities. Kinetex C18 column with iso-butyl side chains and with TMS endcapping fulfilled the request of effective separation and was further used for the method validation. The choice of internal standard was made of the following compounds: indole-4-carboxyaldehyd, 5-hydroxymethylfurfural, nitrobenzoic acid, 2,3,3-trimethylindolenine, 3,5-dichlorphenole, methylparaben, and ethylparaben, owing to their availability and their similarity of retention times to the indole-3-carbinol. It was found that indole-4-carboxyaldehyd has absorption mini- mum at 261 nm, which was inappropriate to selected detection wavelength 270 nm. Other compounds showed unsatisfactory separation or inappropriate peak symmetry parameters. Under optimized conditions of the mobile phase and temperature, the peak of ethylparaben was well separated from the peaks of assayed compounds and also from other compounds present in samples of nutraceuticals. Moreover ethylparaben showed good response at 270 nm and the UV absorption maximum at 255 nm, which was close to the detection wavelength of indole-3-carbinol. The optimal chromatography conditions ofthe separation showed in Fig. 1, are suitable for separation and quantification of indole-3-carbinol concerning the resolution, retention factors, peak symmetry, and short time of analysis. Condensation products of I3C (CP1-CP3) were eluted after the peak of internal standard ethyl- paraben. The structures of the condensation products were not further defined, but they are in the introduction section mentioned structures such as DIM (3, 3 ′-diindoylmethane), ICZ (indolo[3,2b]- carbazole), LTr1 (a linear trimer), or CTr (a cyclic trimer).
3.2. Optimization of extraction conditions
Several procedures for extracting indole-3-carbinol based on the simplest methods currently found in the literature were tested. Due to no presence of indole-3-carbinol in two analysed nutraceu- ticals, several different solvents such as methanol, acetonitrile, ethanol, dimethylsulfoxide, propan-2-ol and others were tested to improve the extraction from homogenized capsule mixture, but direct extraction to methanol with the help of the ultrasound bath showed sufficient recoveries for the analysed compound. Method based on a simple extraction with methanol showed that no evap- oration and reconstitution steps are necessary before the sample injection. Results of the analysis showed that the use of about 0.08–0.15 g of each sample of nutraceuticals was sufficient for the extraction and precise quantification of indole-3-carbinol. Reached recovery values were in the range 97.2–104.5% for all nutraceu- tical samples. Optimal extraction procedure for indole-3-carbinol from various powdered samples was performed by 10-min sonica- tion with 50 mL of 100% methanol with internal standard addition, followed by centrifugation (5 min at 12,000 rpm).
3.3. Validation of the proposed method
Validation of the method includes the evaluation of following performance parameters such as linearity, repeatability, precision, accuracy, and selectivity in order to evaluate the reliability of the results provided by the method. Within the method valida- tion, the parameters of the HPLC system suitability test (Table 2) were measured and evaluated. The samples of standard solution were six times injected into the chromatographic system. Mean values and standard deviations of retention time, retention fac- tors, peak symmetry, resolution, and repeatability of analytical run were calculated according to the European Pharmacopoeia recom-
Table 2 HPLC system suitability parameters of the validated method.
Retention time (min) Retention time repeatability RSD (%)a^ Repeatability of peak areas RSD (%)a^ Retention factork Peak symmetry Indole-3-carbinol 2.35 0.1 0.2 1.62 1. Ethylparaben (IS) 3.73 0.5 0.2 3.16 1. a (^) Made in eight replicates at one concentration level 100 g mL- (^).
Table 3 Analytical characteristics of the validated HPLC method.
Parameter Indole-3-carbinol Standard calibration range (g mL -1^ )a^ 5– Regression coefficient 0. LOD (g mL -1^ ) 0. LOQ (g mL -1^ ) 0. Repeatability RSD (%)b^ 0.2–0. Method precision intraday RSD (%)c^ 0.75–3. Accuracy—spike recovery (%)±SDd^ 100.0–104.1 ± (0.2–2.9) a (^) Each concentration of calibration standard was measured in triplicate. b (^) Relative standard deviation (RSD) was calculated from six injections of standard solution at three concentration levels 5, 100, and 500 g mL -^. c (^) Relative standard deviation (RSD) for repeated injections of multiple prepara- tions of different dietary supplement samples (n = 6), three injections of each sample preparation. d (^) Accuracy was determined as a method recovery using fortified food supplement samples at one concentration level (added amount 10 % of indole-3-carbinol content for each food supplement) in six samples from one batch (± minimal and maximal standard deviation of recovery determination).
mendations [32]. Obtained validation results and chromatography system suitability parameters are summarized in Tables 2, 3.
3.4. Linearity, sensitivity and selectivity
Under the chromatographic conditions tested, linear relation- ships of standard solutions were verified. Ten standard calibration solutions ranging from 5 to 500 g mL−^1 of I3C were prepared by dilution of stock solution and tested for the linearity test. The detector response at 270 nm was found linear in the whole range of calibration curve. The linear regression curve was obtained by plotting peak area count of standards at each level against the con- centration of each standard solution in the mentioned range (500, 400, 300, 200, 100, 75, 50, 25, 10 and 5 g mL−^1 ). Sufficient linearity (regression coefficient r^2 = 0.9993) was achieved in the investigated range. Linear regression parameters were described by the follow- ing equations: A = (8,593 ± 113) c + (28,902 ± 26,740), where A is the absorbance, and c is the analyte concentration. All samples were measured in triplicates. The detection limit of indole-3-carbinol determination was 0.03 g mL−^1. The limit of quantification was estimated to be 0.10 g mL−^1. The values of LOD and LOQ are sum- marized in validation Table 3. The selectivity of the validated method was evaluated for different nutraceuticals samples by checking the peak purity of indole-3-carbinol and internal standard onto the chromatogram. Peak purity index of all compounds in food supplement sample chromatograms reached values greater than 0.9999. Ballast matrix components extracted with methanol were eluted in the front of the chromatogram and did not disturb the quantification of indole-3-carbinol as well as the other active biological compounds in multicomponent nutraceuticals and condensation products of indole-3-carbinol.
3.5. Repeatability, precision and accuracy
Repeatability of the proposed method was characterized by rel- ative standard deviation (RSD, (%)), which was calculated for eight consecutive measurements at three concentration levels 5, 100, and 500 g mL−^1 of standard solutions during the system suit-
ability test. The results in the form of RSD were determined for indole-3-carbinol 0.76, 0.23, and 0.27%; and for internal standard ethylparaben 0.65, 0.16, and 0.44%. To validate the precision of the method, a number of six deter- minations with the same food supplement were evaluated, which were prepared from the same batches and performed on the same day. This approach provides a means of covering the precision of the entire method, from sample extraction and analyte separation to data evaluation. The intraday precision values of indole-3-carbinol was calculated as RSD values which were 1.17% in Indonal Partner for Woman food supplement; 0.75% in GynMax food supplement; 2.77% in Indonal Man food supplement; and 3.25% in Prostain food supplement. In all tested nutraceuticals, the relative standard devi- ation values were in the range recommended in accordance with European Pharmacopoeia recommendations (less than 5%). The accuracy of the method was tested by analyzing nutraceuti- cals fortified with known quantities of analysed compounds. Spiked sample solutions (10% addition of compound content) and un- spiked sample solutions were compared for recovery evaluation. The method accuracy results were evaluated for all food supple- ment preparations individually. Average values of the recoveries and standard deviations (SD) were found as follows: 104.1 ± 0.2% in Indonal Partner for Woman food supplement; 100.0 ± 2.8% in GynMax food supplement; 100.1 ± 2.7% in Indonal Man food sup- plement; and 101.6 ± 2.9% in Prostain food supplement. Results of recoveries and standard deviations showed satisfactory and reproducible extraction procedure for determination of indole-3- carbinol in different commercial food supplement preparations in the presence of other components and complex matrix.
3.6. Robustness
The robustness of the method was determined by measuring the effect of small and deliberate changes in the analytical param- eters on retention time and peak area counts. The parameters that were taken into consideration were column oven tempera- ture and short-term stability of the samples. Differences in peak areas due to the changes in column temperature were + 0.08% for indole-3-carbinol and + 0.02% for internal standard ethylparaben (30 ◦C), and −0.16% for indole-3-carbinol and + 0.08% for internal standard ethylparaben (45 ◦C). Differences in peak areas of indole- 3-carbinol and internal standard ethylparaben between immediate injection of prepared samples and after 30 h in autosampler at 25 ◦C were −1.33% for indole-3-carbinol and −0.34% for internal stan- dard ethylparaben. The very low variations of peak areas are in agreement with the statement that the optimized and validated conditions of the method are robust and small changes of temper- ature in column oven and longer stocking of vials in autosampler will not significantly affect the results of analysis.
3.7. Determination of indole-3-carbinol in nutraceuticals
The newly developed method has been applied to the deter- mination of indole-3-carbinol in capsules of nutraceuticals. The samples were commercially available on the local market. High accuracy, precision, and repeatability of the analysis, and high peak purity index of analysed compound in samples showed that the method is suitable for the quantitative analysis of indole-3-carbinol
Fig. 3. Chromatogram of indole-3-carbinol and condensation products CP1, CP2 and CP3 determination in nutraceutical sample (Prostain capsules) with using validated conditions and internal standard ethylparaben.
confirmed that the quality of nutraceutical preparations differs widely according the type and producer. In general, the low qual- ity of nutraceuticals in the Czech region is an alarming problem for consumers. The solution of this problem will probably require specific regulations to control the whole nutraceutical’s market.
- Conclusion
This work has established a fast and simple method for the separation and quantitative analysis of indole-3-carbinol in the presence of the condensation products. It was proven that simple liquid extraction method using ultrasound bath, extraction with pure methanol and centrifugation to remove undissolved mate- rial was the efficient and fast sample pretreatment procedure for indole-3-carbinol extraction from capsules of the nutraceutical preparations. The core–shell chromatographic column with C stationary phase with iso-butyl side chains and with TMS endcap- ping and mobile phase consisting of acetonitrile/water according to the gradient program have shown optimal parameters of sepa- ration from the other tested columns. Due to easy handling, fast sample preparation procedure, and short analysis time 6.3 min, the developed method is reliable for quality control of indole- 3-carbinol content in presence of the condensation/degradation products in nutraceuticals. In conclusion, the present study showed that the declared content of indole-3-carbinol varied in different samples and was depend on different qualities of food supplement produc-
ers. Unsettling could be the fact, that two analyzed products showed absolutely no content of declared amount, which consid- ers the prizes of these nutraceuticals very alarming finding for the nutritionist and general public. Moreover, two of the analyzed samples showed the presence of huge amount of the condensa- tion/degradation products of indole-3-carbinol which were not officially declared by producer. Generally, the quality of the tested nutraceuticals was very low, because only one from the six tested commercial preparations showed the declared amount of the active substance.
Acknowledgment
The authors are grateful to the Charles University and they would like to acknowledge financial support of the project of spe- cific research, project no. SVV 260 184.
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