ISO/DIS 11027: 2025(en)

ISO TC 34/SC 7

Date: 2025-12-27

Secretariat: BIS

Pepper and pepper oleoresins – Determination of piperine content – Method using high-performance liquid chromatography

Poivres, oléorésines de poivres — Détermination de la teneur en pipérine — Méthode par chromatographie en phase liquide à haute performance

© ISO 2025

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of the requester.

ISO copyright office

CP 401 • Ch. de Blandonnet 8

CH-1214 Vernier, Geneva

Phone: +41 22 749 01 11

Email: copyright@iso.org

Website: www.iso.org

Published in Switzerland

Contents

This template allows you to work with default MS Word functions and styles. You can use these if you want to maintain the Table of Contents automatically and apply auto-numbering.

To update the Table of Contents please select it and press "F9".

Foreword iv

1 Scope (mandatory) 1

2 Normative references (mandatory) 1

3 Principle 1

4 Reagents 2

5 Appartus 2

6 Calibration method…………………………………………………………….………………………………………..3

7 Sampling ………………………………………………………………………………………………………………………4

8 Preparation of test sample ………………………………………………………………………………………….4

9 Procedure…………………………………………………………………………………………………………………….4

10 Calculation …………………………………………………………………………………………………………………..5

11 Repeatability ……………………………………………………………………………………………………………….6

12 Test report …………………………………………………………………………………………………………………..6

Annex A (informative) Typical chromatograms.…………………………….………………………………………………………………………………………….7

Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing international Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electro Technical Commission (IEC) on all matters of electro technical standardization.

Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.

International Standard ISO 110027 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Sub-Committee SC 7, Spices and condiments.

Annex A of this International Standard is for information only.

Any feedback or questions on this document should be directed to the user’s national standards body. A complete listing of these bodies can be found at www.iso.org/members.html.

Pepper and pepper oleoresins – Determination of piperine content – Method using high-performance liquid chromatography

This International Standard specifies a method for the determination, by high-performance liquid chromatography, of piperine content of peppers (Piper nigrum Linnaeus), whole or powdered, as well as their extracts (Oleoresins).

This method also enables the separation and, if necessary, the determination of the other alkaloids of pepper (isochavicine, isopiperine, and piperittin).

The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards.

ISO 2825:21981, Spices and condiments — Preparation of a ground sample for analysis

ISO 5564:1982, Black pepper and white pepper, whole or ground — Determination of piperine content — Spectrophotometric method

3.1

Ground pepper

Extraction with ethanol under reflux, followed by the determination of piperine by high-performance liquid chromatography (HPLC), in accordance with the procedure described in this International Standard.

3.2

Whole pepper

Preparation by grinding the sample, then extraction of the powder obtained, followed by determination of piperine by HPLC, in accordance with the procedure described in this International Standard.

3.3

Oleoresins of pepper

Dilution of the oleoresin in ethanol, then determination of piperine by HPLC, in accordance with the procedure described in this International Standard.

Use only reagents of recognized analytical or HPLC grade and distilled or demineralized water or water of equivalent purity.

Piperine of at least 98 % purity, determined by the spectrometric method described in ISO 5564.

WARNING – This product should be handled with care as it is strongly irritating.

4.2.1 Ethanol, 96 % (v/v).

4.2.2 Acetonitrile HPLC Grade

WARNING – This product should be handled with care as it is lachrymatory.

4.2.3 Acetic acid, 1 % (v/v) aqueous solution.

Mix 52 volumes of acetic acid solution (4.2.3) and 48 volumes of acetonitrile (4.2.2).

Usual laboratory equipment and, in particular, the following.

5.1 Pipettes, of 2 ml, 5 ml, and 10 ml capacity.

5.2 Volumetric flasks, of 25 ml, 50 ml, and 100 ml capacity (made of antiactinic glass or wrapped in aluminum foil).

5.3 Filtration diaphragms, non-adsorbing.

5.4 Separation system

5.4.1 HPLC with an isocratic or binary pumps and degassing systems, for solvents.

5.4.2 Suitable detector system, adjustable to a wavelength of 343 nm.

5.5 Recorder or integrator, the performance of which is compatible with the apparatus as a whole.

5.6 Column

Material: stainless steel

Length: 10 to 25 cm

Inside diameter: 0,4 cm to 0,5 cm

Stationary phase: grafted silica with derived octadecyl functional group, C₁₈, particle size 5 µm max.

5.7 Injection system

Injection valve with 10 0r 20 µm loop or any other system giving the same injection accuracy.

5.8 Tet sieve, of aperture size 500 µm.

WARNING – Throughout the operation, it is imperative that the solutions are not exposed to light (for instance wrap the flasks in aluminium foil) and are used as quickly as possible because of the instability of the piperine solutions.

Prepare for immediate use a 1 g/l stock solution of the piperine (4.1) in ethanol (4.2.1).

From the reference solution (6.1), prepare at least five standard solutions of piperine with concentrations ranging from 0,05 g/l to 0,2 g/l. Inject each solution into the HPLC (5.4.1). Repeat the determination at least once. Measure the peak areas and repeat the tests if the results deviate by more than 5 %.

Plot the calibration curve, i.e. the mass of piperine injected versus the peak area.

Define the measured slope of the curve.

Calculate the mean response factor K from the calibration data using the following formulae:

where

It is important that the laboratory receives a sample that is truly representative and has not been damaged or changed during the transport of storage.

Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 948¹).

WARNING – It is imperative to operate away from light because of the instability of the piperine solutions.

8.1.1 Check that all of the test sample passes through the 500 µm sieve (5.8). If this is not the case, grind the powder in accordance with ISO 2825 until the required particle size is achieved.

8.1.2 From the test sample prepared as in 8.1.1, weigh, to the nearest 0,01 g, 0,5 g to 1 g, and transfer it to a 100 ml round-bottom flask (5.2) fitted with a reflux condenser. Add 50 ml of ethanol (4.2.1) and bring it to the boil. Maintain for 3 h under reflux.

Allow the solution to cool. Filter the solution and collect it in a 100 ml one-mark volumetric flask (5.2). Rinse the extraction flask and filter with successive portions of ethanol (4.2.1). Add the wash liquids to the 100 ml volumetric flasks and makeup to the mark with ethanol.

8.2.1 Grind the test sample in accordance with ISO 2825 until the powder obtained passes entirely through the 500 µm sieve (5.8).

8.2.2 Using the test sample prepared as in 8.2.1 proceed as described in 8.1.2.

Thoroughly homogenize the test sample of oleoresin.

Take with the pipette (5.1) 10 ml of the extraction solution, obtained as described in 8.1.2 or 8.2.2, and transfer it to a 25 ml one-mark volumetric flask (5.2). Makeup to the mark with the elution solvent (4.3).

NOTE 1 This dilution may be modified, if necessary, by the analyst depending on the assumed piperine content of the sample and the sensitivity of the detector.

Weigh into a 100 ml one-mark volumetric flask, to the nearest 0,1 mg, 0,2 g of oleoresin (see 8.3), and make up to the mark with ethanol (4.2.1).

Pipette 10 ml of this mixture into a 50 ml one-mark volumetric flask and makeup to the mark with the elution solvent (4.3).

NOTE 2 This dilution may be modified, if necessary, by the analyst depending on the assumed piperine content of the sample and the sensitivity of the detector.

The final dilution shall be clear. If this is not the case, filter it on the filtration diaphragm (5.3). Inject the test solution into the chromatograph (5.4.1).

On the chromatogram obtained, measure the peak of the piperine (see typical chromatograms given in annex A).

Carry out two tests and repeat the determination if the results deviate by more than 5 %.

Calculate the piperine content, as a percentage by mass, using the following formula:

where

The piperine content can also be calculated directly from the calibration curve using the HPLC softwares after incorporating the dilution factors.

NOTE 3 The ratio 25/10 is related to the dilution proposed in 9.1.1 and should be modified accordingly when another dilution is used.

Calculate the piperine content, as a percentage by mass, using the following formula:

Where A, m_x and K have the same meaning as in 10.1.

The piperine content can also be calculated directly from the calibration curve using the HPLC softwares after incorporating the dilution factors.

NOTE 4 The ratio 50/10 is related to the dilution proposed in 9.1.2 and should be modified accordingly when another dilution is used.

The repeatability of the analysis in terms of piperin content should be less than or equal to 5 % RSD of a minimum of three determinations.

The test report shall include the following information:

a) reference to this International Standard;

b) the apparatus system;

c) the characteristics of the column (material, dimensions, packing, stationary phase);

d) the characteristics of the detector (optional) and the operating conditions;

e) the characteristics of the mobile phase (flow rate and nature);

f) identification of the test sample (quantity injected and final dilution);

g) results obtained.

1. 
   (informative)
   
   Typical chromatograms

Figure A.1 shows typical chromatograms, taken under the operating conditions given in this International Standard.

Figure A.1