Food Chemistry 120 (2010) 915920

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

Analytical Methods

Direct injection and determination of the active principles of spices using micellar liquid chromatography
Mei-Liang Chin-Chen a, Samuel Carda-Broch a, Devasish Bose b, Josep Esteve-Romero a,*
a b

Qumica Bioanaltica, Q.F.A., E.S.T.C.E., Universitat Jaume I, 12071 Castell, Spain Department of Criminology and Forensic Sciences, Dr. H.S. Gour University, Sagar, India

a r t i c l e

i n f o

a b s t r a c t
In this work, we develop a simple chromatographic method to identify and determine three active principles: curcumin, capsaicin and piperine. These compounds are present as spices in some plants: turmeric (Curcuma longa), red pepper (Capsicum annuum) and black pepper (Piper nigrum). The method includes a micellar mobile phase containing 0.15 M sodium dodecyl sulphate and 12.5% (v/v) propanol buffered at pH 7, a Kromasil C18 column (125 mm 4.6 mm, 5 lm particle size) and UV detection set at 210 nm. The micellar liquid chromatography (MLC) method herein reported is simple, sensitive, precise, robust, and samples can be directly injected into the column without any pre-treatment step. Under these conditions, analysis times were below 7 min for the complete resolution of the three compounds. Linearity (r > 0.9998), limits of detection of 0.7, 1.3 and 5 ng/mL and limits of quantication of 2, 4.4 and 15 ng/ mL, for capsaicin, curcumin and piperine, respectively, intra- and inter-day precision (RSD, %) less than 5.9, were studied in the method validation. Finally, the MLC method was applied to spiked samples, with recoveries of around 100%, and in nine real samples, and with amounts of spices in the 0.02 and 47.1 mg/g range. The simplicity of the method makes it a good candidate to be used in routine analyses in the area of food control and quality. 2009 Elsevier Ltd. All rights reserved.

Article history: Received 4 February 2009 Received in revised form 17 October 2009 Accepted 3 November 2009

Keywords: Curcumin, capsaicin, piperine Micellar mobile phase Direct injection Spices samples

1. Introduction For centuries, spices have been used in kitchens to add taste and avour to our dishes. Moreover in certain parts of the world, the use of turmeric, red pepper and pepper has therapeutic properties (Srinivasan, 2005). However, high prices and the possibility of spice adulteration, especially when sold in a powder form, encouraged the study of analytical methods to make spice consumption safe. Curcumin (1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6diene-3,5-dione), or turmeric extract, is a natural dye obtained from turmeric, catalogued with the European Union as E-100. Turmeric extract is raw, whilst curcumin is a puried compound. This substance gives a characteristic yellow colour to curry powder and its dye is used in mustard sauce. It is mainly cultivated in India and has been used since ancient times for various applications. Furthermore, it is known for its antitumour (Aggarwal & Shishodia, 2004), antioxidant, anti-arthritis, and anti-inammatory properties. Capsaicin (8-methyl-N-vanillyl-6-nonenamide), is the naturally active principle derived from chillies (Capsicum). It produces a
* Corresponding author. Tel.: +34 964728093; fax: +34 964728066. E-mail address: josep.esteve@qfa.uji.es (J. Esteve-Romero). 0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2009.11.003

strong burning sensation in the mouth. Pure capsaicin is a lipophilic compound that is odourless and an off-white solid. Because of the burning sensation that it produces, capsaicin is commonly used in food products to give them a hotter, more pungent taste. Capsaicin is the active component of chilli peppers, and it is used in hot sauces. Capsaicin is also employed as medicine (Hayman & Kam, 2008), or as tear gas. In large amounts, it can be very toxic. The symptoms of its poisoning are shortness of breath, blue skin and seizures. However, chilli poisoning is an extremely rare event. Piper nigrum is a plant of the piperaceae family, which is grown for its fruit and is used as a dry spice. The fruit is a berry which may be used whole or converted into powder to obtain different processed peppers such as black, white or green. Its active principle, piperine (1-[5-(1,2-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl] piperidine), is responsible for pungency (Parmar et al., 1997). It is used worldwide as a condiment and has also been used in some forms of traditional medicine and as an insecticide. Finally, Fig. 1 shows structures of curcumin, capsaicin and piperine. To date, there have not been many reports on the simultaneous determination of these three compounds, although several analytical methods have been developed for the individual analysis of these active principles. The most widely technique used is high performance liquid chromatography (HPLC) and thin layer chro-

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was 1.0 mL/min), a degasier for the mobile phase, an auto sampler (20 lL injection volume), which was nally coupled to an UVvisible detector (range 190700 nm). The column employed was a Kromasil C18 (125 mm 4.6 mm, 5 lm particle size) from Scharlab (Barcelona, Spain). Chromatographic signals were acquired and treated with the Agilent Chemstation (Revision B.03.01), which was used in the integration of peaks to obtain dead volumes, retention times, efciencies and asymmetry factors. These data were later treated with SPSS 17.0 (SPSS Inc., Chicago, Illinois, USA). The pH of the solutions was measured with a Crison GLP 22 (Barcelona) equipped with a combined Ag/AgCl/glass electrode. The analytical balance used was a Mettler-Toledo AX105 DeltaRange (Greifensee, Switzerland). The vortex shaker and sonication unit were purchased from Selecta (Barcelona). 2.2. Chemicals and reagents Curcumin and piperine were obtained from Acros Organics (New Jersey, USA), capsaicin was purchased from Fluka (Buchs, Germany). Sodium dodecyl sulphate was obtained from Merck (Darmstadt, Germany). Sodium dihydrogen phosphate and propanol were from Sharlau (Barcelona). Methanol was acquired from J.T. Baker (Deventer, Holland). Ultrapure water came from Millipore S.A.S. (Molsheim, France) and was used throughout. 2.3. Preparation of solutions, samples and mobile phases matography (TLC) with UVvisible detection (Paramasivam, Poi, Banerjee, & Bandyopadhyay, 2009; Suresh, Manjunatha, & Srinivasan, 2007), HPLC with UVvisible detection (Bajad, Singla, & Bedi, 2002; Heath, Pruitt, Brenner, & Rock, 2003), counter-current chromatography, and HPLCmass spectrometry for the purication of amides from piper longum (Wu, Sun, Pei, Lu, & Pan, 2004). Conventional methods prove tedious and usually require multiple chromatographic steps, such as gravity-eld column chromatography and TLC. Nonetheless, capillary zone electrophoresis with diode array detection (Yuan, Weng, Zhang, Xiong, & Xu, 2005) and solid phase micro-extraction coupled to gas chromatography and mass spectrometry (GCMS) (Pea-lvarez, Ramrez-Maya, & AlvaradoSurez, 2009) are also important techniques to determine these compounds. Micellar liquid chromatography (MLC) is an alternative to conventional HPLC. It uses mobile phases that contain a surfactant, usually sodium dodecyl sulphate, and an organic solvent, such as propanol, butanol or pentanol, which increases peak efciencies and reduces retention times, thus producing changes in selectivity (Berthod & Garcia-Alvarez-Coque, 2000). This behaviour can be easily predicted by using different equations, thus making the interpretative optimisation of separation possible. Our research group has acquired broad experience in the determination of different compounds, such as antiepileptic drugs, biogenic amines, barbiturates, benzodiazepines, and stimulants, amongst others (Esteve-Romero, Carda-Broch, Gil-Agust, Capella-Peir, & Bose, 2005). The purpose of this work is to develop an MLC method for the determination of spices, such as curcumin, capsaicin and piperine, in which food samples are injected directly into the chromatographic system without the need of any pre-treatment step. The micellar mobile phase was prepared by weighing amounts of SDS and selected buffer powder with 0.01 M sodium dihydrogen phosphate and then dissolving them in water, adjusting to the desired pH with NaOH or HCl. Finally, a suitable volume of alcohol, depending on the percentage, was added. Standard stock solutions of 100 lg/mL of the active principles were prepared by dissolving the compound in a few millilitres of methanol, with the aid of an ultrasonic bath, and they were lled up with 0.05 M SDS-pH 7. The stock solutions of the spices were also prepared with 0.05 M SDS-pH 7 to a ratio of 1:10. For the analyses, solutions were diluted to the desired concentration with 0.05 M SDS-pH 7 and spice (1:10) solutions, which were injected directly into the chromatographic system. All the solutions were ltered through lter paper of 0.45 lm nylon membranes (Micron Separations, Westboro, MA, USA) before analysis. 2.4. Recommended chromatographic conditions Separation was performed in a reversed phase Kromasil C18 column thermostatted at 25 C. The selected mobile phase composition was 0.15 M SDS, 12.5% (v/v) propanol, 0.01 M NaH2PO4 at pH 7. The ow rate, injection volume and UV wavelength were 1 mL/ min, 20 lL and 210 nm, respectively. Under these conditions, the retention times for curcumin, capsaicin and piperine were 3, 5 and 6 min, respectively. Chromatographic signals were acquired and processed with an Agilent ChemStation (Rev. A.10.01). Chromatographic parameters (efciencies, asymmetries and retention factors) were obtained using this software. 2.4.1. Method validation The Food and Drug Analysis (FDA) guidelines were followed to validate the method (Guidance for Industry Bioanalytical Method Validation, 2001). This study includes selectivity, linearity, limits of detection and quantication, determination of precision, and robustness. Recovery of spices was determined at different concentration levels for curcumin and piperine (2, 10 and 50 lg/mL) and for capsaicin (0.5, 5 and 25 lg/mL). Finally, the procedure was applied to analyse the different types of commercial spices.

Fig. 1. Structures of active principles of spices: curcumin (1), capsaicin (2) and piperine (3).

2. Experimental 2.1. Instrumentation The chromatograph used was an Agilent Technologies Series 1100 (Palo Alto, USA), equipped with a quaternary pump (ow-rate

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3. Results and discussion 3.1. Selection of chromatographic conditions First of all, SDS was selected as the surfactant because, apart from being the most widely used, it offers a high degree of sensitivity, and desorption and cleaning up processes are easy to perform. Using pure micellar mobile phases of SDS (M)-pH 7: 0.05, 0.10 and 0.15, the retention factors were: 20, 11 and 7 for curcumin, respectively; 60, 30 and 19 for piperine, respectively; 41, 22 and 14 for capsaicin, respectively. Although the retention factors were adequate to perform the analysis, efciencies were low (between 20 and 410 for all three spices) and the chromatographic peaks were very asymmetrical (asymmetry factors above two). For this reason, the introduction of propanol was considered to increase efciencies and to decrease asymmetry factors. In order to select the best analysis conditions, several mobiles phases containing SDS (M)-propanol (%, v/v): 0.052.5, 0.0512.5, 0.107.5, 0.109, 0.152.5, 0.157.5 and 0.1512.5, were tested. The retention factors, efciencies and asymmetry factors for the three spices were measured. The usual behaviour in MLC with SDS was observed in all mobile phases. Thus, the retention factors decreased for SDS and alcohol as the concentration of both increased, 19.5, 16.2, 11, 8.5, 7, 6 and 2 for curcumin, respectively; 37, 27, 15, 13.6, 12.7, 8.6 and 6.6 for piperine, respectively; 27.6, 15, 11, 10.5, 10, 8.6 and 5 for capsaicin, respectively. On the other hand, efciencies decreased when the surfactant concentration increased and, in contrast, efciencies increased at higher modier concentrations: 60, 500, 50, 130, 55, 100 and 500 for curcumin, respectively; 700, 2100, 1100, 1300, 640, 1200 and 2000 for piperine, respectively; 950, 1900, 900, 1000, 600, 1200 and 1700 for capsaicin, respectively. Chromatographic data were processed with the Michrom software, and the maximum resolution and minimum analysis times were taken into account. This software allows the changes in the chromatograms to be observed graphically when the user progressively varies the concentrations of the surfactant and the modier. It also makes it possible to predict the retention behaviour accurately, based on a veried model, thus speeding up the process of nding the optimal composition of the mobile phase for a given compound. The equation used to describe the retention of species was (Garca-lvarez-Coque, Torres-Lapasi, & Baeza-Baeza, 1997):

describes the association equilibria between the solute (A) in the stationary phase (S), micelle (M) or bulk water (D). This equation was non-linearly tted according to the Powell method using the retention data from injections of the drug solutions in several mobile phases. Using this equation, errors between the experimental and theoretical data were below 2%. After modelling, the optimum mobile phase selected was 0.15 M SDS12.5% (v/v) propanolNaH2PO4 at pH 7. The chromatographic parameters for compounds in this mobile phase (k, N and B/A) were: 2, 500 and 1.5 for curcumin, 5, 1700 and 1.1 for capsaicin, and 6.6, 2000 and 1.2 for piperine, respectively. 3.2. Method validation 3.2.1. Selectivity In order to study selectivity, spice samples were processed directly in the chromatographic system and analysed to determine the extent to which endogenous compounds may interfere with the retention time of the active principles. No interference by endogenous compounds was observed. 3.2.2. Linearity In order to study linearity, stock solutions of spices (100 lg/mL) were prepared in a micellar solution (1:10). For curcumin and piperine, eight dilutions (1100 lg/mL) and eight dilutions for capsaicin (0.5100 lg/mL) were prepared and made up from the stock solutions. Injections were performed in these ranges in triplicate. Finally, the calibration equations, y = a + bx (lg/mL), were constructed using the peak area vs. the compound concentration data. To study the variability of calibration, parameter curves were obtained for 10 days over a period of 2 months. The response of the tested compounds was linear (r = 0.999) and within the concentration range studied. 3.2.3. Detection and quantication limits The limit of detection (LOD) for spices was determined with the 3s criterion (three times the standard deviation of the lowest concentration solution included in the calibration divided by the slope of the calibration curve) using a series of 10 solutions containing a low concentration. The results were based not only on the standard deviation of the response, but also on the slope of a specic calibration curve containing the spice. LODs were 0.7, 1.3 and 5 ng/mL for capsaicin, curcumin and piperine, respectively. The limits of quantication (LOQ) for spices were determined using the 10s criterion. LOQs were 2, 4.4 and 15 ng/mL, for capsaicin, curcumin and piperine, respectively. The LOD and LOQ are good enough to control the active principles of the commercial spices under study.

1 K SD u 1 K AD u k 1 K MD u 1 K AM M 1 K AD u K AS
where k is the retention factor, [M] and u are the concentrations of the surfactant and modier, respectively; KAS, KAM, KMD, KSD and KAD

Table 1 Inter-day and intra-day precision and accuracy of active principles of spices in SDS solutions. Active principles Concentration added (lg/mL) Founda (mean SD) (lg/mL) 2.04 0.05 9.82 0.11 50.7 0.6 0.48 0.06 5.15 0.03 25.15 0.08 2.03 0.09 9.86 0.03 50.29 0.17 Accuracy (%) Intra-day C.V. (%) 2.6 1.2 1.1 2.5 0.7 0.3 3.6 0.3 0.3 Founda (mean SD) (lg/mL) 2.11 0.12 9.83 0.06 50.45 0.3 0.48 0.03 5.18 0.06 25.39 0.19 2.18 0.31 9.9 0.9 50.7 2.3 Accuracy (%) Inter-day C.V. (%) 5.8 0.6 0.6 8 1.1 0.7 13.8 9 4.5

Curcumin

2 10 50 0.5 5 25 2 10 50

2 1.8 1.3 4 3 0.6 1.5 1.4 0.6

5.5 1.7 0.9 4 3.6 1.6 9 0.8 1.3

Capsaicin

Piperine

n = 10.

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3.2.4. Precision and accuracy The intra- and inter-day precisions of spices were determined by analysing at three concentrations: 2, 10 and 50 lg/mL for curcumin and piperine and at 0.5, 5 and 25 lg/mL for capsaicin; solved in the micellar medium SDS 0.05 M at pH 7 (1:10). The intra-day analysis was determined by assaying these three test solutions ten times on the same day, and the inter-day analysis was the average of 10 measurements of intra-day values taken on 10 days over a two-month period, performed by a different analyst and with different equipment at the same three concentrations. Table 1 shows the results obtained for active principles of spices. 3.2.5. Robustness Robustness of the method was examined by replicate injections (n = 4) of standard solutions of the spices at a concentration of 2 lg/mL under slightly changed chromatographic parameters (ow rate, pH, percentage of propanol and SDS concentration). Insignicant differences (less than 7%) in peak areas and less variability in the retention times were observed. The results indicate that the selected factors were unaffected by the slight variations in these parameters (Table 2). 3.3. Stability Finally, stability studies were performed at room temperature. No degradation of curcumin, capsaicin and piperine was observed when inspecting the chromatograms after a three-day storage in

the micellar medium at room temperature. No other peaks were observed when solutions were kept at a low temperature (4 C) over a 30-day period.

3.4. Determination in a standard solution of spices Curcumin, capsaicin and piperine were determined by diluting with 2 mL of methanol at a ratio of 1:10 with 0.05 M SDS at pH 7 (10 replicates for each standard) with known amounts of the active principles at three different concentrations (1100 lg mL1 for curcumin and piperine) and for capsaicin (0.5100 lg mL1) within the calibration range. Standard solutions were processed and analysed with the above-described procedure. Relative (analytical) recovery was calculated by comparing the concentration obtained from the standard solution with actual added amounts. The data obtained showed satisfactory recoveries for the three active principles, which are shown in Table 3.

3.5. Determination of the active principles in commercial spice samples The applicability of the developed procedure to determine curcumin, capsaicin and piperine was checked by analysing them in spice samples. Samples of curry and white pepper (Taberner, Spain), curry and curcuma (Hacendado, Spain), Chinese condiment (Yuk Mein, China), pig sauce and vinegar sauce (Verdifresh, Spain), chilli pepper (Spain), black pepper (Lufthansa, Germany), black pepper (Ducros, Spain) and peppercorn multicolour (Cusina, Germany) were bought in a local supermarket and Chinese restaurant. Table 4 shows the results of the analysis of the three active principles determined in 0.1 g of spices and 1 g of sauce samples after grinding, homogenisation with methanol and the micellar solution 0.05 M at pH 7, centrifugation and ltration with a syringe-driven lter unit 0.45 lm and 13 mm nylon membranes. Peppers were dissolved in 20 mL or 30 mL of SDS for which the concentration was into of curve calibration. The other samples were dissolved in 10 mL. The results showed that the active principle concentration was different for the same commercial spice. The developed MLC procedure is good enough to determine curcumin, capsaicin and piperine concentrations in spices and sauce samples without heat processing for preparation purposes because these active principles diminished with cooking (Suresh et al., 2007), Finally, Fig. 2 shows the chromatograms of the standard solution (A) and of the analysed spice samples, curry (B), pepper (C), chilli (D) and curcuma (E) which were diluted in the 1:10 factor.

Table 2 Robustness evaluation of MLC method. Chromatographic changes Level Curcumine Area 77 71.4 74 74 3 4.0 75.5 71.4 77 75 3 3.9 77 71.4 78 76 4 4.7 78 71.4 71.3 74 4 5.2 Capsaicin Area 140 130 122 131 9 7.0 132 130 135 133 3 1.9 133 130 132 131.6 1.6 1.2 139.2 130 127.4 132 6 4.7 Piperine Area 79.5 79.2 74 78 3 4.0 78 79.2 78 78.4 0.7 0.9 74 79.2 78 77 3 3.5 74.4 79.2 72.1 75 4 4.8

A: Flow rate (mL/min) 0.9 0.1 1 0 1.1 +0.1 Mean SD RSD (%) B: SDS concentration (M) 0.1495 0.0005 0.15 0 0.1505 +0.0005 Mean SD RSD (%) C: Percentage of propanol (v/v) 12.4 0.1 12.5 0 12.6 +0.1 Mean SD RSD (%) D: pH of mobile phase 6.9 0.1 7 0 7.1 +0.1 Mean SD RSD (%)

Table 4 Determination of active principles in commercial spices samples. Active principle Curcumin Commercial spices Curry (Taberner) Curry (Hacendado) Chinese condiment (Yuk Mein) Pig sauce (Verdifresh) Curcuma (Hacendado) Chilli pepper (Guindilla) Curry (Taberner) Curry (Hacendado) Chinese condiment (Yuk Mein) Pig sauce (Verdifresh) Vinegar sauce (Verdifresh) White pepper (Taberner) Black pepper (Lufthansa) Black pepper (Ducros) Peppercorn multicolour (Cusina) Found SD mg/g 1.80 0.05 4.28 0.07 0.34 0.02 0.017 0.009 8.58 0.25 0.171 0.007 3.7 0.01 0.633 0.013 0.94 0.18 0.023 0.002 0.05 0.02 5.38 0.09 5.136 0.004 47.13 0.03 5.79 0.13

Table 3 Recovery of active principles. Recovery (%) c1 Curcumin Capsaicin Piperine 101.96 0.05 96.24 0.06 109.62 0.06
a

Capsaicin Piperine c2 98.3 0.1 99.04 0.03 99.45 0.02 c3 101.3 0.6 97.86 0.08 97.6 0.2

c1, c2 and c3 were 2, 10 and 50 lg/mL for curcumin and piperine, 0.5, 5 and 25 lg/ mL for capsaicin. a Average SD of 10 measurements.

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120

100

3
80 80

1 3
60

muA

muA
40

40

20

0 0 1 2 3 4 5 6 7

0 0 1 2 3 4 5 6 7

t, min

t, min

C
120

muA

60

0 0 1 2 3

t, min

D
20

300

200

muA

10 100

0 0 1 2 3

muA
0 5 6 7

t, min

t, min

Fig. 2. Chromatograms for: (A) a mixture of standards (25 lg/mL) of curcumin (1), capsaicin (2) and piperine (3), (B) curry, (C) pepper, (D) chilli and (E) curcuma.

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4. Conclusion The proposed procedure is useful in the area of food quality and control to determine the content of the active principles in commercial spice samples of different registered trademarks. The procedure could also be extended to the analysis of these compounds, used for adulteration purposes. Micellar liquid chromatography using mobile phases containing a micellised surfactant is a good technique to analyse these spices. An important property of MLC is the ability of micelles to solubilise untreated samples, thus allowing direct injection. Minimal sample handling reduces analyses costs and times, increases sample throughput, and decreases error sources owing to minimised risks of losses and chemical changes in the analytes with a reduced number of steps. Our results indicate that the MLC procedure developed can be used for the analysis of active principles in spice samples with analysis times below 7 min. Validation according to FDA guidelines was performed efciently with satisfactory results in terms of the selectivity, precision, accuracy and robustness studies. The correlation coefcients (r) were above 0.9998 for the dynamic range studied, up to 100 lg/mL. The recoveries in spike samples were very similar and varied between 96% and 110%. No interferences from the endogenous compounds of the matrices were noted. The limits of detection and quantication were good enough to monitor these spice samples. The method is sensitive enough for quality control routine analysis and food studies if we take into account that the spice samples were directly injected without any previous treatment to separate or concentrate the analytes.

References
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Acknowledgements This work was supported by the MEC CTQ 200764473/BQU and Fundaci Caixa Castell-Bancaixa P1-1B2006-12 projects. MeiLiang Chin-Chen also thanks the Foundation for her grant.