application of accelerated solvent extraction (ase) and

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APPLICATION OF ACCELERATED SOLVENT EXTRACTION (ASE) AND THIN LAYER CHROMATOGRAPHY (TLC) TO DETERMINATION OF PIPERINE IN COMMERCIAL SAMPLES OF PEPPER (PIPER NIGRUM L.) a

Eveline De Mey , Hannelore De Maere a

a b

a

, Lore Dewulf , Hubert

c

a

Paelinck , Mieczysław Sajewicz , Ilse Fraeye & Teresa Kowalska c a

Research Group for Technology and Quality of Animal Products, KaHo Sint-Lieven, Ghent, Katholieke Universiteit Leuven , Leuven , Belgium b

ISA, Food Quality Laboratory , Lille , France

c

Institute of Chemistry, University of Silesia , Katowice , Poland Published online: 20 Jun 2014.

To cite this article: Eveline De Mey , Hannelore De Maere , Lore Dewulf , Hubert Paelinck , Mieczysław Sajewicz , Ilse Fraeye & Teresa Kowalska (2014) APPLICATION OF ACCELERATED SOLVENT EXTRACTION (ASE) AND THIN LAYER CHROMATOGRAPHY (TLC) TO DETERMINATION OF PIPERINE IN COMMERCIAL SAMPLES OF PEPPER (PIPER NIGRUM L.), Journal of Liquid Chromatography & Related Technologies, 37:20, 2980-2988, DOI: 10.1080/10739149.2014.907014 To link to this article: http://dx.doi.org/10.1080/10739149.2014.907014

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Journal of Liquid Chromatography & Related Technologies, 37:2980–2988, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6076 print/1520-572X online DOI: 10.1080/10739149.2014.907014


APPLICATION OF ACCELERATED SOLVENT EXTRACTION (ASE) AND THIN LAYER CHROMATOGRAPHY (TLC) TO DETERMINATION OF PIPERINE IN COMMERCIAL SAMPLES OF PEPPER (PIPER NIGRUM L.)

Eveline De Mey,1 Hannelore De Maere,1,2 Lore Dewulf,1 Hubert Paelinck,1 Mieczysław Sajewicz,3 Ilse Fraeye,1 and Teresa Kowalska3 1 Research Group for Technology and Quality of Animal Products, KaHo Sint-Lieven, Ghent, Katholieke Universiteit Leuven, Leuven, Belgium 2 ISA, Food Quality Laboratory, Lille, France 3 Institute of Chemistry, University of Silesia, Katowice, Poland

& In this preliminary study, an attempt was made to develop a simple yet reliable overall approach to quantification of piperine in pepper (Piper nigrum L.), and also in the other piperinecontaining spice compositions. Such an approach can prove useful for rapid screening of the commercially traded pepper (and other spices) in a pulverized form, with piperine as a single adulteration marker. The main features of this approach consist in a rapid (ca. 40 min) and exhaustive (as proved in a recovery experiment) extraction of plant material with use of Accelerated Solvent Extraction (ASE), followed by an easy thin-layer chromatographic quantification of piperine in the obtained extracts. Within the framework of this approach, dichloromethane (DCM) was selected as an extracting agent and the extraction temperature was fixed at 70 C. With use of the piperine standard, the calibration curve was developed and applicability of the proposed approach was tested upon the commercial samples of white and black pepper in a pulverized and peppercorn form. The piperine levels obtained with the commercial pepper samples remained in agreement with the data available from the literature. Moreover, the assumed quantification approach correctly differentiates between the lower piperine levels in an unripe (black) pepper and the higher ones in a ripe (white) pepper, and also between the lower piperine levels extractable from the whole peppercorn and the higher ones extractable from the ground pepper. Now on the basis of this preliminary study, a completely validated method of quantifying piperine in botanical matter with use of the proposed approach can be elaborated. Keywords accelerated solvent extraction (ASE), Piper nigrum L., piperine, spice adulteration marker, TLC-densitometry in fluorescence mode

Address correspondence to Teresa Kowalska, Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40-006 Katowice, Poland. E-mail: [email protected]

Piperine in Commercial Samples of Piper Nigrum L.

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INTRODUCTION The main pungent compound contained in black pepper (Piper nigrum L.) is piperine, and owing to its flavor, black pepper is the world’s most demanded and also the most traded spice. Black pepper is native to South India, and it is extensively cultivated there and also elsewhere in tropical regions. Owing to its recognized culinary and antimicrobial properties, black pepper has entered the Mediterranean region in remote antiquity and it is supposed to have been used in mummification rites of Egyptian pharaohs. In certain periods of European history, black pepper has been referred to as ‘‘black gold’’ and served as a kind of commodity money. It is not too exaggerated, if we state that black pepper has affected the world history and instigated the so-called Age of Discovery, commenced by the Portuguese under the lead of Vasco da Gama. The dried black pepper fruit is known as peppercorn and depending on the stage of its ripeness and a kind of pre-processing, it can appear as black, orange, pink, red, green, or white pepper. Colloquially, all varieties of Piper nigrum L. are referred to simply as pepper. Nowadays, quantification of piperine can serve several purposes. One important reason is relatively common adulteration of other pulverized spices (also known for their pungency, yet not due to the contents of piperine) with pepper powder, or adulteration of pepper powder with other botanical additives, which are known for an absence of piperine from their composition.[1–3] Even if it is not recommended to trade pulverized spices for the risk of adulteration, it still remains a widely accepted commercial practice, and in the pepper-related adulteration cases, piperine can serve as a single adulteration marker. Another important reason to quantify piperine is to investigate mechanisms of interaction between piperine contained in pepper and the nitrate- and nitrite-based meat curing brines, widely applied in meat industry as preservative agents. This mechanism is not yet fully understood, although a risk of piperine conversion to the health-endangering N-nitroso compounds cannot be excluded.[4] As thin layer chromatography (TLC) can be performed easily and inexpensively, many applications are already reported. Piperine was determined in the plant material, for example, in fruits of Piper nigrum[5,6] and roots of Piper longum,[7] as well as in the other matrices (such, as biological tissues), for example, in liver and serum.[8] Generally, in these methods, glass plates precoated with silica gel were used, whereas in the course of the separation of piperine isomers, application of the silver nitrate impregnated plates was recommended.[9] Prior to applying this separation technique, piperine has to be extracted from the plant matrix and for this purpose, the conventional Soxhlet extraction is still widely in use. However, certain efforts have already been made to increase extraction efficiency by using innovative techniques

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such, as microwave assisted extraction (MAE)[10] or supercritical fluid extraction (SFE) with carbon dioxide as an extracting agent.[11] It was an aim of this study to elaborate a simple overall approach to quantify piperine in the black and white varieties of pepper (Piper nigrum L.), based on a rapid and efficient Accelerated Solvent Extraction (ASE) followed by the thin-layer chromatographic analysis with use of densitometry in the fluorescence mode. EXPERIMENTAL


Materials and Reagents The molecular structure of piperine is given in Figure 1. The piperine (1-piperoylpiperidine) standard (97%) was purchased from Sigma-Aldrich (St Louis, MO, U.S.A.) and then it was dissolved in dichloromethane (DCM) at a concentration of 0.1 m g mL1. Black pepper samples (powder and peppercorns) originated from the local supermarket (Delhaize, Brussels, Belgium), and the two different varieties of the white pepper powder originated from the two suppliers, Raps (Kulmbach, Germany), and Rejo (Nazareth-Eke, Belgium). The commercial Si 60 F254 coated glassplates (20 cm 20 cm; Merck, Darmstadt, Germany; cat. no. 1.05715.0001) were used in the thin-layer chromatographic study. DCM used for dissolution of the piperine standard and the extraction of piperine from the pepper samples was of the HPLC purity grade (Merck), and acetone and n-hexane used as mobile phase components were of the analytical purity grade and purchased from PPH POCh (Gliwice, Poland). Accelerated Solvent Extraction The ASE 200 model extractor (manufactured by Dionex, Sunnyvale, CA, USA) was used to carry out the accelerated solvent extraction of the pepper samples. To this effect, the 0.5-g portions of the dry plant material

FIGURE 1 Chemical structure of piperine.



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were carefully weighed and placed in the stainless steel cell, and each portion underwent three consecutive extraction runs with use of DCM at 70 C, under the following working conditions: the pressure of 100 atm; the solvent volume, 24 mL; static time, 15 min; number of extraction cycles within one extraction run, 2. The extraction temperature of 70 C was selected upon the results of a separate optimization experiment carried out within the range from 50 C to 80 C, not described here.[12] Three 24-mL portions of an extract obtained from one and the same spice sample were merged in the 100-mL calibrated volumetric flask and filled up to 100 mL with DCM. This solution was used for the chromatographic quantification of piperine in the investigated spices. Contrary to the traditional extraction modes (e.g., by macerating spices under the reflux in a boiling extraction solvent, or using the Soxhlet apparatus) which can last up to dozen or more hours, a complete ASE extraction run described herewith lasts approximately 40 minutes only, which is a hard to overestimate time gain. Recovery Study In order to evaluate an efficiency of the ASE procedure, a recovery study was performed. Therefore, the ground black pepper fruit was used as a model spice for the preparation of the blank plant matrix sample. In order to exhaustively extract piperine from this sample, the aforementioned ASE procedure with DCM as an extracting solvent was repeated six consecutive times. In the final extract, piperine could not be detected any more, so the plant material after the sixth extraction run was dried with use of the vacuum evaporator and then used as a blank matrix. The 5-gram samples of the blank plant matrix were spiked with 5 mL of the standard solutions of piperine at the four different aliquots per one gram of this matrix. The four 1-gram portions of the dried spiked matrix were weighted out and each portion underwent a single ASE extraction run. Then the four extracts obtained in parallel from the four equally spiked matrix samples were condensed in the flow of nitrogen at 40 C to the volume of 1 mL each, with use of the TurboVap LV model evaporator (manufactured by Zymark, Hopkinton, MA, U.S.A.). These condensed solutions were then utilized for the chromatographic recovery studies and standard deviation was calculated from the recovery repetitions. Determination of Piperine by Means of TLC-Densitometry in Fluorescence Mode The commercial Si 60 F254 coated glassplates were used in the thin-layer chromatographic experiment. Before use, the plates were activated at 110 C for 15 min. The chromatograms were developed with use of acetone


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þn-hexane, 3:2 (v=v)[13] to the distance of 15 cm and each individual analysis was repeated six times. After the development, the plates were dried at ambient air for 3 hr and eventually evaluated by means of densitometry. Acquisition of the densitograms was carried out with a Desaga CD 60 model densitometer equipped with Windows compatible ProQuant software (Desaga). Concentration profiles of the development lanes were recorded in the fluorescence mode using a deuterium lamp at the irradiation wavelength k ¼ 360 nm. The dimensions of the rectangular light beam were 2.0 mm 0.1 mm. Application of the fluorescence mode (less common than the most frequently applied reflectance mode) was meant to enhance quantification sensitivity, owing to the presence of an aromatic moiety in the piperine molecule (see Figure 1). The retardation factor (RF) value for piperine was equal to 0.90 0.02. The calibration curve was obtained for the aliquots from 0.10 to 0.60 mg piperine spot1, in the 0.10 mg piperine spot1 intervals. A series of six different piperine aliquots needed for the calibration curve were spotted onto one and the same chromatographic plate, and six repetitions on the six different chromatographic plates were made (n ¼ 6), in order to obtain the calibration curve. The chromatographic peak heights were plotted against the nanogram amounts of piperine applied to the chromatographic plate. The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the formulas LOD ¼ 3.3 SD=a, and LOQ ¼ 10 SD=a, respectively, where SD is standard deviation of the peak height taken as a measure of noise, and a is the slope of the corresponding calibration curve (y ¼ ax þ b). Then the evaluation of the recovery performance and quantification of the piperine contents in the four commercial black and white pepper samples was performed. RESULTS AND DISCUSSION It was an aim of this study to elaborate a simple overall approach to quantify piperine in different varieties of pepper (Piper nigrum L.), based on an efficient Accelerated Solvent Extraction (ASE) followed by the thinlayer chromatographic analysis. Up to our best knowledge, this type of extraction combined with quantification of piperine by means of TLC has never before been applied to the Piper nigrum L. fruit (but to the roots of Piper longum only[6]). To this effect, the calibration curve was first developed, by plotting the chromatographic peak heights for the piperine standard against the sample aliquots spotted on to the chromatographic plate. In Figure 2, a typical densitogram of the black pepper extract is presented and in Table 1, the resulting calibration curve is provided. In the next step, an efficiency of ASE in extracting piperine from the spiked blank pepper matrix was evaluated. As it can be seen from the data


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FIGURE 2 Typical densitogram of the extract obtained from the black pepper powder (RF for piperine equals to 0.90 0.02).

provided in Table 2, extraction of piperine from this matrix was practically complete in a single extraction run, when 70 C as a working temperature and DCM as an extracting agent were applied. This result remains in conformity with our earlier observations regarding an excellent performance of ASE in phytochemical research, as an advantageous replacement of the traditional extraction methods.[14,15] Owing to this excellent performance of ASE, quantification of piperine in the four different (white and black) pepper samples could be carried out directly, that is, without a tedious spiking the blank plant matrix, in order to prepare the plant material based calibration curves. The obtained quantification results are provided in Table 3. Numerical results presented in Table 3 confirm reliability of the employed procedure. First, the contents of piperine in the white pepper powder (originating from a ripe pepper fruit without a pericarp) are higher than that in the black pepper powder (originating from an unripe fruit with pericarp). Then, the contents of piperine in the black peppercorns are considerably lower than in the ground black pepper powder, which is correct and obvious (as in the course of the extraction, peppercorns cannot be as deep penetrated by the extracting medium, as it is possible with the pulverized matter). In the literature on the applications of TLC[13,16] and HPTLC[11] for the determination of piperine in the fruits of Piper nigrum, somewhat higher piperine levels used to be reported than those provided in our study. However, it

TABLE 1 (n ¼ 6)

Calibration Curve Obtained for Piperine by TLC, and Respective LOD and LOQ Values

Calibration Curve y ¼ ax þ b y ¼ 8.43 x þ 2551.4

r2

SD

LOD (mg spot1)

LOQ (mg spot1)

0.9636

1616.24

0.59

1.80

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TABLE 2 Recovery and the Relative Standard Deviation of Piperine from 1 Gram Dry Blank Plant Matrix Derived from the Ground Black Pepper Fruit and Spiked with Four Different Concentrations of the Test Compounds (n ¼ 4) Spiked Amount (mg=g) 0.100 0.200 0.500 0.600

Recovery (1) (mg=g)

Recovery (2) (%)

RSD (%)

0.098 0.197 0.494 0.595

98.1 98.6 98.9 99.2

3.35 3.23 3.11 2.99

Recovery consists of (1) mg g1 dry blank plant matrix and (2) %; RSD, relative standard deviation.


TABLE 3 Contents (mg g1 pepper) of Piperine in the White and Black Pepper Samples (n ¼ 6), as Quantified by Means of TLC Pepper Sample White pepper (Piper nigrum, Piperaceae) powder (Raps) powder (Rejo) Black pepper (Piper nigrum, Piperaceae) powder (Delhaize) peppercorns (Delhaize)

mg g1 Pepper [RSD (%)]

15.50 (5.09) 20.78 (5.99) 12.50 (4.26) 2.10 (2.62)

has to be accepted that these concentrations (ranging from 20 to 90 mg g1) are valid for the samples directly obtained from the manufacturers. It is known that a prolonged storage,[17] thermal processing,[18] and exposure to light[19] easily lowers the piperine content in the commercial spice samples (as it was the case with the samples used in our study). Nevertheless, the developed method has proven to be precise and accurate. If required for any further purposes, the ASE-TLC method can be fully validated according to the guidelines of the International Conference on Harmonization[20] or, as recognized by the Codex Alimentarius, the IUPAC[21] guidelines can be followed for the food applications.

CONCLUSION . A novel approach that combines Accelerated Solvent Extraction (ASE) and thin-layer chromatography (TLC) was proposed for the determination of piperine in the different pepper (Piper nigrum L.) samples. . Main advantages of this approach are rapidity and effectiveness of the ASE extraction (as compared with the traditional extraction procedures); the effectiveness thoroughly confirmed in the recovery studies. . Another advantage is the use of TLC as a rapid and cost-friendly analytical technique for quantification of piperine in the investigated samples, with


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the determined piperine levels falling well within the magnitude order achieved with use of more sophisticated chromatographic techniques (as reported in the literature). . If necessary, this rapid ASE-TLC method can prove convenient for the fast screening of the internationally traded pulverized spices, with the piperine contents as a single adulteration marker.


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19. Kozukue, N.; Park, M.-S.; Choi, S.-H.; Lee, S.-U.; Ohnishi-Kameyama, M.; Levin, C. E.; Friedman, M. Kinetics of Light-Induced Cis-Trans Isomerization of Four Piperines and Their Levels in Ground Black Peppers as Determined by HPLC and LC=MS. J. Agr. Food Chem. 2007, 55 (17), 7131–7139. 20. ICH, Q2B Validation of Analytical Procedures: Methodology; International Conference on Harmonization: Geneva, Switzerland, November 1996. 21. Currie, L. A. IUPAC Commission on Analytical Nomenclature, Recommendations in Evaluation of Analytical Methods including Detection and Quantification Capabilities. Pure Appl. Chem. 1995, 67, 1699–1723.