Assessment of the N-nitrosopiperidine formation risk from piperine and

piperidine contained in spices used as meat product additivesEveline De Mey1*, Hannelore De Maere1,2, Lore Dewulf1, Hubert Paelinck1, Mieczysław Sajewicz3, Ilse Fraeye1,

Teresa Kowalska3

1Research Group for Technology and Quality of Animal Products, KaHo Sint-Lieven, , Gebr. Desmetstraat 1,

9000 Ghent, Belgium, member of Leuven Food Science and Nutrition Research Centre (LFoRCe), Department

M2S, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium

2ISA, Food Quality Laboratory, Boulevard Vauban 48, F-59046 Lille Cedex, France

3Institute of Chemistry, University of Silesia, 9 Szkolna Street, 40 006 Katowice, Poland

Corresponding Author: Eveline De Mey

E-mail address: eveline.demey@kuleuven.be

Tel: +32(0)9 265 86 10

Fax: +32(0)9 265 87 24

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Abstract

The N-nitrosopiperidine formation in blends of spices and nitrite curing salt was investigated

in relation with the piperine and piperidine contents in spices. Firstly, two analytical methods

were developed. Piperine was extracted with dichloromethane by means of accelerated

solvent extraction, and determined by HPLC-DAD (λ=343 nm). A selective hydroextraction

of piperidine using ASE and its quantification by HPLC-ELSD was applied. Both methods

were sufficiently sensitive and accurate (limit of detection, limit of quantification and

recovery: 0.28 µg, 0.84 µg, and 98.9±2.6% for piperine, and 5.76 µg, 17.45 µg, and

95.9±2.9% for piperidine, respectively). Secondly, both compounds were quantified in

commercial samples (black and white pepper, paprika, chili pepper, allspice and nutmeg). The

maximum amount of piperine (21.12 mg g-1) was found in pepper, while the other spices

contained only traces. Piperidine was detected mainly in the pepper samples, whereby the

highest concentration was found in the white pepper extract (11.42 mg g-1). Thirdly, during

the storage of spices blended with nitrite curing salt, the N-nitrosopiperidine content was

determined, using a gas chromatograph coupled with a thermal energy analyzer. Against our

expectations, no N-nitrosopiperidine formation was observed in the curing mixture which

contained white pepper extract. This result remains in contrast with the white pepper mixture,

in which the N-nitrosopiperidine content significantly increased from not detected to

9.80±0.41 ng g-1 after the two months storage period. In conclusion, high amounts of piperine

or piperidine in spices do not systematically result in the formation of N-nitrosopiperidine,

when blended with nitrite curing salt.

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Key words: piperyol-piperidine, piperidine, N-nitrosopiperidine, accelerated solvent

extraction, spices, nitrite curing mixtures.

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1. Introduction

Piperidine is a cyclic secondary amine, which can be considered as a parent molecular

structure for many plant alkaloids, and piperine (1-piperyol-piperidine) is the main pungent

compound of pepper (Piper nigrum, Piperaceae) (Figure 1). White and black pepper,

produced from the dried fruit without the pericarp and the dried whole, unripe berry,

respectively [1], are used for many culinary purposes. The pungency, and thus the quality, of

pepper (P. nigrum) are related to the amount of piperine [2]. However, this quality can be

influenced by the hydrolysis of piperine, resulting in the cleavage of the piperidine ring [3].

In food (e.g, in spice premixes and the meat products), simultaneous presence of nitrite

(as a curing agent) and pepper can give rise to the formation of the carcinogenic N-

nitrosopiperidine (NPIP). This formation can occur either by the oxidative cleavage of the

amide bond of piperine and a subsequent nitrosation of piperidine [4], or by a direct

nitrosation of the already available piperidine [5,6]. In order to evaluate a risk of N-

nitrosamine formation, it would be interesting to determine the piperine and piperidine

contents in spices.

Normally, piperine and piperidine are extracted from ground pepper with use of

organic solvents (e.g., methanol, ethanol, hexane, and dichloromethane). However, large

quantities of these volatile solvents are consumed during the long extraction [7,8]. An

optimized Soxhlet apparatus technique was proposed [9], but several alternative techniques

have also been developed. For instance, high solubility of piperine in glacial acetic acid can

accelerate the extraction [10], while the microwave-assisted extraction (MAE) enhances

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solvent penetration by breaking the cell structure of pepper [11]. Furthermore, ionic liquids

can be used in ultrasonic assisted extraction (UAE) [12]. Although accelerated solvent

extraction (ASE) is known to be a powerful technique to reduce the time and solvent

consumption, reports concerning the extraction of piperidine alkaloids from spices are scarce.

In fact, Rajopadhye et al. [13] described an ASE procedure for the extraction of piperine from

the roots of Piper longum, while no reports were found about an application of this technique

to isolation of piperidine from P. nigrum.

The most frequently used analytical techniques are thin-layer chromatography (TLC)

and high performance liquid chromatography (HPLC). TLC is carried out on silica plates,

occasionally treated with silver nitrate, and usually with n-hexane or ethyl acetate as eluents

[14,15]. Although TLC is a relatively cheap method, it is less sensitive, as compared with

HPLC. For the separation of the piperine isomers by HPLC, a C18 column is recommended

[16]. Piperine analogues can directly be detected by the UV absorption spectroscopy.

However, piperidine is often derivatized with ninhydrin [17] or dansyl-chloride [18,19], due

to the lack of a proper chromophoric group.

The aim of this study was threefold. Firstly, the methods for the determination of

piperine and piperidine in spices were developed, in order to assess the quality of the

commercial pepper samples. Secondly, the piperine and piperidine concentrations in a

selection of culinary spices were measured. And finally, the N-nitrosopiperidine formation

was evaluated during the storage of certain spices in combination with the nitrite curing salt.

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In that way, possible risk of the N-nitrosopiperidine formation was assessed, caused by

piperine and piperidine contained at different concentration levels in spices.

2. Experimental

2.1 Spices and nitrite curing mixtures

Different quality grades of the white and black pepper samples (P. nigrum) were selected.

They also differed in particle diameter (e.g., the whole peppercorns, the cracked pepper with

the 12 mm particle diameter, and the pepper powder with the ca. 0.3 mm average particle

diameter were analyzed). The commercial powder was either produced by the fine grinding,

or by evaporating ethanol from the spice extract. Besides of the pepper samples, some other

spices were also purchased, namely paprika (Capsicum annuum) and chili pepper (Capsicum

frutescens), both belonging to the plant family of the nightshades (Solanaceae), and also

allspice (Pimenta dioica, Myrtaceae) and nutmeg (Myristica fragans, Myristicaceae). The

spice samples were purchased either from the local supermarket (the black pepper powder and

peppercorns came from Delhaize, Brussels, Belgium), or directly from the two suppliers.

Nutmeg powder, white pepper powder and white pepper extract came from Raps (Kulmbach,

Germany). The paprika, chili and allspice powder, and also the cracked white pepper and the

Muntok white peppercorns originated from Rejo (Nazareth-Eke, Belgium).

In order to evaluate the risk of the N-nitrosopiperidine formation, meat curing mixtures were

prepared. These premixes consisted of 3 g of one of the above mentioned spices in 100 g

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nitrite curing salt (NaCl + 0.6% NaNO2, Rejo). These mixtures were vacuum packed and

stored in the darkness at 25°C. For tracing the N-nitrosopiperidine formation, the mixtures

were sampled (n=3) after one day, one week, and two months storage period.

2.2 Chemicals and standards

Hydrochloric acid (37% HCl), potassium hydroxide pellets (KOH), and antifoam silicone

were all obtained from VWR International (West Chester, PA, USA). Methanol and

dichloromethane (DCM) were purchased from Merck (Darmstadt, Germany). All chemicals

were of analytical grade. Water used in the experiment was de-ionized and double distilled in

our laboratory by means of the Elix Advantage model Millipore system (manufactured in

Molsheim, France). In order to prepare 3M KOH, a suitable amount of potassium hydroxide

(KOH) was dissolved in de-ionized water. The 6M HCl solution was prepared by diluting a

suitable volume of hydrochloric acid (HCl) with de-ionized water.

The piperine (≥97%), piperidine (≥99%), N-nitrosopiperidine (NPIP), and N-

nitrosodipropylamine (NDPA) standards were purchased from Sigma-Aldrich (St Louis, MO,

U.S.A.). For the purpose of the HPLC analyses, piperine was dissolved in DCM at the

concentration of 0.10 mg mL-1, and piperidine was dissolved in water at the concentration of

1.00 mg mL-1. NPIP was analyzed with a gas chromatograph coupled with a thermal energy

analyzer (GC-TEA). For this purpose, the NPIP standard was dissolved and diluted in DCM

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to obtain a concentration of 0.25 µg mL-1. Quantification was carried out using NDPA as an

internal standard (IS) at a concentration of 1 µg mL-1 DCM.

2.3 Accelerated solvent extraction

Prior to the HPLC analysis of piperine and piperidine, the ASE 200 model extractor

(manufactured by Dionex, Sunnyvale, CA, U.S.A.) was used to carry out the accelerated

solvent extraction on the spice samples. A 0.5-g portion of the dry spice was carefully

weighed and placed in the stainless steel cell and underwent three consecutive extraction runs.

A pressure of 100 atm was applied. The remaining working parameters were, as follows: the

solvent volume, 24 mL; static time, 15 min; number of extraction cycles within one extraction

run, 2. Three 24-mL portions of extract obtained from a single spice sample were merged in a

100-mL calibrated volumetric flask and filled up to 100 mL with DCM or water, respectively.

These solutions were used for the chromatographic quantification of piperine and piperidine

in the investigated spices.

In order to optimize the extraction temperature for piperine with DCM as an extraction

medium, the white pepper powder from Raps (containing a high level of piperine) was used as

a model spice at six different working temperatures (50, 60, 65, 70, 75 and 80°C). For the

isolation of piperidine, the temperature of hydroextraction was optimized and this time, the

white pepper extract from Raps (containing a high level of piperidine) was used as a model

spice at six different working temperatures (40, 45, 50, 55, 60, and 65°C).

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2.4 Recovery study

In order to evaluate the efficiency of the ASE procedure, a recovery study was performed.

Therefore, the ground black pepper fruit was used as a model sample for the preparation of

the blank plant matrix sample. In order to exhaustively extract piperine and piperidine from

this sample, the aforementioned ASE procedure with DCM and H2O, respectively, as a

solvent was repeated six consecutive times.

One series of the blank matrix samples was spiked with piperine (and extracted with DCM),

and the other series of the blank matrix samples was spiked with piperidine (and extracted

with H2O). The obtained concentrations per one gram of the blank plant matrix were specified

in Table 1. From each dried and spiked matrix, four 1-gram portions were weighed 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.

2.5 Determination of piperine and piperidine by HPLC

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The HPLC analyses were carried out using a Varian model 920 liquid chromatograph (Varian,

Harbor City, CA, USA) equipped with a Varian 900-LC model autosampler, a gradient pump,

a Varian model 330 DAD detector, a Varian 380-LC model ELSD detector, and the Galaxie

software for data acquisition and processing. The analyses were carried out in the isocratic

mode, using a Pursuit 5 C18 (5 µm particle size) column (250 mm × 4.6 mm i.d.; Varian). As

mobile phase, we employed methanol at a flow rate of 0.5 mL min-1 in the isocratic mode.

The retention times (tR) of piperine and piperidine were equal to 5.9 and 10.9 min,

respectively.

The presence of an aromatic chromophore in piperine offers a possibility of using a diode

array detector (DAD). For the calibration curve purpose, the peak heights at the wavelength of

343 nm (i.e., at the absorption maximum of piperine) were employed. The calibration curve

for piperine was obtained for the aliquots ranging from 0.20 to 1.50 µg piperine (in the

intervals of 0.10 µg). The chromatographic peak heights were plotted against the microgram

amounts of piperine injected on to the column. For each individual aliquot, six repetitions

were performed (n=6). The limit of detection (LOD) and the limit of quantification (LOQ)

were calculated using the formulas LOD = 3.3 × SD/a, and LOQ = 10 × SD/a, where SD is

standard deviation of the peak height (n=6) taken as a measure of noise, and a is the slope of

the corresponding calibration curve (y = ax + b).

The HPLC results valid for piperidine were derived from the ELSD detector. The calibration

curve for piperidine was obtained for the aliquots ranging from 2 to 7 μg piperidine (in the

intervals of 1 μg piperidine). This time, the chromatographic peak heights were plotted

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against the microgram amounts of piperidine. For each individual aliquot, six sampling

repetitions were performed (n=6). The calibration curve and the LOD and LOQ values were

determined, as described earlier.

2.6 Determination of NPIP by GC-TEA

In order to determine the contents of N-nitrosopiperidine, the method according to Drabik-

Markiewicz et al. [20] was used, with some modifications. The spice samples (in the 5-g

portions) were spiked with 500 µL NDPA, and mixed with 200 mL 3 M KOH and 4 mL

antifoam. The volatile N-nitrosamines were extracted from the spices by means of vacuum

distillation from the alkaline solution. Subsequently, the distillates were extracted with DCM

and washed with 6 M HCl. Finally, the extract was concentrated to 100 µL in a Kuderna-

Danish apparatus (Sigma Aldrich). For the detection and quantification, a GC-TEA (Thermo

Electron Cooperation, Rodano, Italy) was used. The 5-µL aliquots of the concentrated extracts

were injected on a packed column (10% Carbowax 20M + 2% KOH on Chromosorb WAW,

80/100 mesh, 1.8m, 2mm i.d. Varian, Middelburg, The Netherlands) and a chromatographic

separation was carried out with use of argon as a carrier gas (25 mL min -1). The injection port

was set at 175°C and the oven temperature was increased from 110°C to 180°C at the 5°C

min-1 rate. The temperature of the interface and the pyrolizer of TEA were set at 250°C and

500°C, respectively. The retention times (tR) of NDPA and NPIP were equal to 6.3 and 10.4

min, respectively. The calibration curves for NPIP were obtained from 7 aliquots ranging

from 0.625 to 12.5 ng with NDPA as an internal standard. The relative chromatographic peak

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areas (area NPIP/area NDPA) were plotted against the relative amounts of NPIP (µg NPIP/µg

NDPA). For each individual aliquot, six repetitions were performed (n=6). The limit of

detection (LOD) and the limit of quantification (LOQ) were 0.68 and 2.04 ng, respectively.

2.7 Statistical analyses

The results were expressed as the means ± standard deviation. The linear least-square

regression was used to calculate the intercepts (a), slopes (b) and coefficients of

determinations (r2) of the respective calibration curves (Microsoft Office Excel 2007,

Microsoft Corporation, Redmond, WA). Significant differences among the extraction

temperatures were assessed with a one-way analysis of variance (ANOVA, PASW Statistics

19.0.0, SPSS Inc.). Piperine and piperidine contents of the spices and the accumulation of the

NPIP content in the blends were also subjected to one-way ANOVAs. Tukey’s honestly

significant difference criterion (p<0.05) was used to compare the means.

3. Results and discussion

3.1 Evaluation of the HPLC procedure

The chemical structure of the two investigated alkaloids proved a decisive factor for the

choice of the most suitable detection and thus, the quantification mode also. The presence of

an aromatic chromophore in piperine allowed the use of a diode array detector (DAD). As a

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result, good sensitivity was obtained and since the wavelength of 343 nm was proven to be

very selective, no other interfering peaks could be seen in the chromatograms (Figure 2).

The lack of a chromophore in the chemical structure of piperidine excludes a possibility of

using DAD, and enforces an employment of the universal ELSD detector, which is sensitive

to the analyte’s molecular weight. In that way, an additional derivatization step, as applied by

several authors [17-19] could be avoided. Although the LOD and LOQ values for piperidine

are by one magnitude order higher than those valid for piperine, it can be concluded that all

the results concerning method characteristics are satisfactory from the analytical point of view

(see Table 2).

Most frequently, piperine and piperidine are extracted from the plant material by the Soxhlet

extraction. In this study, ASE was applied as an alternative extraction technique, in order to

reduce the analysis time and the solvent consumption. As it can be seen from Figure 3, the

extraction yield of piperine (measured after a single extraction run) increased, when elevating

the working temperature of the ASE extraction. At the temperatures higher than 70°C, no

more significant improvement of the extraction yield could be observed, so it was decided to

employ the temperature of 70°C for the DCM extraction of piperine. Due to an excellent

solubility of piperidine and practical insolubility of piperine (and the other alkaloids) in water,

a selective hydroextraction method for the isolation of piperidine was elaborated. Complete

extraction of piperidine was observed over the whole range of the employed temperatures

(results not shown). For practical reasons, the temperature of 50°C was chosen for the

hydroextraction of piperidine (as the lowest working temperature of the ASE 200 apparatus is

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set on 40°C). Thus, the hydroextraction of piperidine could be done at a temperature

considerably lower than that employed for the extraction of piperidine with DCM.

In order to evaluate the accuracy and precision of both extraction procedures, the recovery

studies were performed as well. For that purpose, a blank plant matrix was prepared from the

black pepper powder by thoroughly extracting piperine with DCM and piperidine with water,

using the ASE technique. After the six consecutive runs, neither of the two compounds could

be chromatographically detected in the final extract. Hence, after drying the extracted plant

material, it could be used as a blank plant matrix for the recovery study. The obtained results

showed an excellent performance of the ASE extraction system and practically full recovery

of the two compounds of interest from the spiked plant matrix (Table 1). Moreover, the

recovery precision was confirmed by a good repeatability of the recovery results (RSD of ca.

23%). This successful outcome allowed using the calibration curves derived for the piperine

and piperidine standards from the HPLC quantification experiments (without a tedious

spiking of the blank plant matrix, in order to prepare the plant-material-based calibration

curves).

3.2 Piperine and piperidine content in spices

In Table 3, the results of the piperine and piperidine analyses for the commercially available

spices are given. In the white and black pepper samples (P. nigrum), a maximum of 21.12 mg

g-1 piperine was measured, although in the literature, the piperine contents usually show

within the range of 3080 mg g-1 [21,22]. Nevertheless, the lower amounts have been 14

reported as well, especially due to the ageing of the pepper samples [2]. In the white pepper

extract, a significantly lower amount of piperine was found, although it was much higher than

the amounts extracted from the whole peppercorns. In the course of the ASE procedure, the

compounds present inside the cells of the non-ground botanical matrix do not come in a

sufficient contact with the extraction solvent, which results in the lower extraction yields. In

fact, the whole peppercorns certainly contain higher levels of piperine than those measured in

this study. Nevertheless, these results are interesting since they suggest a possibility of a

reduced release of piperine from the non-ground spices in the curing mixtures and / or in the

cured meat products. In the other investigated spices, only the trace amounts (± 2 mg g -1) of

piperine were detected, and no significant difference among these samples could be observed.

In the pepper samples (P. nigrum), the piperidine content was in most cases a fraction of the

piperine content, except for the black peppercorns, in which the piperidine level exceeded the

low piperine content. An industrial extract of white pepper contained the highest level of

piperidine among all tested spices, while its piperine content was rather low. Although no

information is available about an industrial production of the spice extract, it is clear that the

composition of the white pepper extract is affected by the industrial extraction process. The

piperine content apparently decreased, while the piperidine content was remarkably higher in

the white pepper extract than in the pulverized white pepper samples. Considering the

reversed ratio of the two alkaloids, it can be expected that piperine degrades during the

production of the extract, e.g., under the influence of heat [23].

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In the other spices, only traces (paprika and chili), or very low amounts (allspice and nutmeg)

of piperidine were detected. As checked in the literature, no written evidence could be found

on any meaningful amounts of piperidine or piperidine alkaloids contained in the spices from

the families Solanaceae, Myrticaceae and Myristicaceae, so that our results remain in

conformity with the literature data.

3.3 Evaluation of the NPIP formation in the nitrite curing mixtures

Four spice samples were then tested for the risk of the NPIP formation. As it can be seen from

Table 4, in none of these spices NPIP could initially be detected, which suggested their safe

use in the food products. After one day from the preparation of the nitrite curing salt blended

with these spices, no detectable amount of NPIP was found in any of them. After one week,

however, the curing mixture containing the white pepper powder showed a NPIP

contamination, which significantly increased after the two months storage period. In an

agreement with the results of the earlier studies [24,25], the presence of the secondary amines

(such as piperidine), can cause a risk of the N-nitrosamines formation. For this reason, the

FDA strongly discourages combination of nitrites and nitrates with spices [26]. Although

NPIP was easily formed in the curing mixture containing the white pepper powder, all the

other curing mixtures remained free from NPIP. As anticipated, no NPIP was formed in the

mixtures prepared with nutmeg and paprika, due to very low concentrations of piperine and

piperidine in these spices. However, based on an elevated piperidine level, the NPIP

formation was expected in the mixture produced with use of the white pepper extract.

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Probably in that case, the lack of the NPIP formation can be linked with an altered

composition of white pepper in the course of the industrial extraction process [27]. It is

known from the literature that many spices contain antioxidants, such as polyphenols and the

nitrite scavenging compounds [28,29], and the activity of these compounds is higher in the

extracts than in the unprocessed spices [30]. In that case, antioxidants and the nitrite

scavenging compounds can probably inhibit the N-nitrosamines formation [30]. Finally, it has

to be emphasized that no information is available on the production process of the white

pepper extract and on the polyphenolic composition contained therein, and a further research

is needed to reveal the inhibitory effects of this extract.

4. Conclusions

Rapid and accurate accelerated solvent extraction procedures were developed to facilitate

quantification of piperine and piperidine in spices by means of HPLC. For the determination

of piperine, high selectivity was obtained by using the DAD detector at the specific

wavelength of 343 nm, after the ASE extraction with DCM (70°C) as an extraction solvent.

For the determination of piperidine and due to the lack of a chromophoric group, the universal

ELSD detector was used. In that case, the selectivity was obtained by an application of a

hydroextraction at 50°C. For both alkaloids, good recovery was obtained by means of ASE.

The piperine contents in the pepper samples were considerably higher than the trace levels in

the spices belonging to the botanical plant families of Solanaceae, Myrtaceae, and

Myristicaceae. The piperidine content in the extract of the white pepper powder was 17

considerably higher than that found in the pulverized samples. As predicted by the low

piperine and piperidine levels, nitrite curing mixtures prepared with nutmeg and paprika did

not show any NPIP contamination. Against our expectations, the use of the white pepper

extract, containing a high piperine concentration, did not induce the NPIP formation. In

contrast, the use of the white pepper powder in the curing mixtures caused an increase of the

N-nitrosopiperidine content from not detected to 9.80±0.41 ng g-1 after the two month storage

period. In conclusion, high amounts of piperine and piperidine in the spices do not necessarily

result in the NPIP formation, which depends on the total composition and the industrial

processing of the spices and extracts.

Acknowledgements

This work was performed in the framework of the MeCagrO2 project “Safe products,

sustainable processes and employment increased attractiveness for companies from the 2 Seas

agro food Area”. Note: “The document reflects the authors’ views. The interreg IVA 2 Seas

Programme Authorities are not liable for any use that may be made of the information

contained therein.”

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Table 1 The recovery (T%) and the repeatability (r) expressed as RSD (%) of piperine

and piperidine 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=4).

Compound Spiked amount

(mg/g)

Recovery

(mg/g)

T (%) RSD (%)

Piperine 0.100 0.098 98.4 2.80

0.200 0.198 98.8 2.76

0.500 0.496 99.2 2.89

1.000 0.989 99.2 2.11

Piperidine 0.100 0.092 92.3 3.54

0.200 0.189 94.5 3.11

0.500 0.491 98.2 2.74

1.000 0.985 98.5 2.79

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Table 2 Calibration curves obtained for piperine and piperidine by HPLC, and the respective

LOD and LOQ values (n=6)

Compound Calibration curve

y= ax + b

r2 SD LOD

(g)

LOQ

(g)

Piperine y =554.296x–08.602 0.9565 46.7470 0.28 0.84

Piperidine y=40.145x64.001 0.9835 70.0700 5.76 17.45

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Table 3 Contents (mg g-1) piperine and piperidine in the spice samples (mean±standard

deviation; n=6)

Spice sample Piperine Piperidine

White pepper (P. nigrum)

powder (Raps) 20.65±0.84c,d 3.70±0.11c

cracked peppercorns (Rejo) 19.18±0.66c 0.06±0.00a

powder, Muntok (Rejo) 20.80±0.52d 5.83±0.17e

peppercorns, Muntok (Rejo) 2.51±0.10a 0.08±0.00a

extract (Raps) 12.12±0.31b 11.42±0.29g

Black pepper (P. nigrum)

powder (Delhaize) 21.12±0.75d 6.49±0.21f

peppercorns (Delhaize) 3.32±0.12a 4.22±0.18a

Paprika (C. annuum)

powder (Rejo) 1.96±0.06a 0.02±0.00a

Chili pepper (C. frutescens)

powder (Rejo) 1.97±0.08a 0.04±0.00a

Allspice (P. dioica)

powder (Rejo) 2.11±0.08a 0.66±0.03b

Nutmeg (M. fragrans)

powder (Raps) 2.17±0.06a 0.69±0.03b

Different letters in the same column indicate significant differences at p<0.05 (Tukey's post

hoc test)

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Table 4 Contents (ng g-1) NPIP in the spice samples and during the storage of the nitrite curing

mixtures (mean±standard deviation; n=3)

Spice Curing mixture

Spice sample 1 day 1 week 2 months

White pepper (P. nigrum)

powder (Raps) nd nd 3.28±0.19a 9.80±0.41b

extract (Raps) nd nd nd nd

Paprika (C. annuum)

powder (Rejo) nd nd nd nd

Nutmeg (M. fragrans)

powder (Raps) nd nd nd nd

Different letters in the same row indicate significant differences at p<0.05 (Tukey's post hoc

test)

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Captions to figures

Fig. 1 Chemical structure of piperine (A) and piperidine (B)

Fig. 2 HPLC-DAD chromatogram of piperine extracted from the white pepper powder

sample, detected at 343 nm

Fig. 3 Effect of the extraction temperature on ASE extraction of piperine with DCM from the

white pepper powder sample (n=3). Data points with different letters are significantly

different at p<0.05 (Tukey's post hoc test)

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