CEN/TS 16178:2012
(Main)Sludge, treated biowaste and soil - Determination of pharmaceutical products
Sludge, treated biowaste and soil - Determination of pharmaceutical products
This Technical Specification specifies a method to analyse pharmaceutical compounds in solid matrices. Pharmaceuticals analysis has been carried out on a LC/MS-MS quantum. The main difficulty in this project is the lack of sample certified in researched analytes. Even with spiked solid matrices it is still delicate to correctly verify the impact of extraction step, because it does not reproduce a real sample.
What is proposed here is an analytical method on pharmaceuticals products on sludge, soils and sediments.
This document provides a final protocol on the extraction and purification tested on sludge, soils and sediments spiked with pharmaceutical compounds.
Schlamm, behandelter Bioabfall und Boden - Bestimmung pharmazeutischer Produkte
Diese Technische Spezifikation legt ein Verfahren zur Analyse pharmazeutischer Verbindungen in festen Matrices fest. Analysen von Pharmazeutika wurden an einem LC/MS-MS-Quantum durchgeführt. Die größte Schwierigkeit dieses Projekts ist der Mangel an zertifizierten Proben der untersuchten Analyten. Sogar mit aufgestockten festen Matrices ist es noch schwierig, den Einfluss des Extraktionsschrittes korrekt zu verifizieren, da das Ergebnis nicht auf echte Proben übertragen werden kann. Dieses Dokument liefert ein abschließendes Protokoll zur Extraktion und Reinigung, das mit Schlamm, Boden und Sedimenten, die mit den in Tabelle 1 aufgeführten pharmazeutischen Verbindungen aufgestockt wurden, geprüft wurde.
Boues, biodéchets traités et sols - Détermination des produits pharmaceutiques
La présente Spécification technique définit une méthode pour analyser les composés pharmaceutiques dans
les boues, les biodéchets traités et les sols. L'analyse des produits pharmaceutiques a été effectuée selon
une technique CL/MS-MS quantum. La principale difficulté pour l’analyse réside dans l'absence de tout
échantillon certifié pour les analytes cibles. Même avec des matrices solides dopées, il est toujours délicat de
vérifier correctement l'impact de l'étape d'extraction, car celle-ci n’est pas transférable à un échantillon réel.
Ce document fournit un protocole final pour l'extraction et la purification, testé sur des boues, des sols et des
sédiments dopés par les composés pharmaceutiques énumérés dans le Tableau 1.
Blato, obdelani biološki odpadki in tla - Določevanje ostankov farmacevtskih sredstev
Ta tehnična specifikacija določa metodo za analizo farmacevtskih spojin v blatu, obdelanih bioloških odpadkih in tleh. Farmacevtska analiza je bila izvedena na kvantu LC/MS-MS. Glavna težava za analizo je pomanjkanje vzorca, potrjenega za ciljne analize. Tudi pri primešanih trdnih matrikah je še vedno težko pravilno preveriti vpliv postopka ekstrakcije, ker ga ni mogoče prenesti na realni vzorec.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-junij-2012
%ODWRREGHODQLELRORãNLRGSDGNLLQWOD'RORþHYDQMHRVWDQNRYIDUPDFHYWVNLK
VUHGVWHY
Sludge, treated biowaste and soil - Determination of pharmaceutical products
Schlamm, behandelter Bioabfall und Boden - Bestimmung pharmazeutischer Produkte
Boues, bio-déchets traités et sols - Détermination des produits pharmaceutiques
Ta slovenski standard je istoveten z: CEN/TS 16178:2012
ICS:
13.030.20 7HNRþLRGSDGNL%ODWR Liquid wastes. Sludge
13.080.10 .HPLMVNH]QDþLOQRVWLWDO Chemical characteristics of
soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
TECHNICAL SPECIFICATION
CEN/TS 16178
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
February 2012
ICS 13.030.01
English Version
Sludge, treated biowaste and soil - Determination of
pharmaceutical products
Boues, bio-déchets traités et sols - Détermination des Schlamm, behandelter Bioabfall und Boden - Bestimmung
produits pharmaceutiques pharmazeutischer Produkte
This Technical Specification (CEN/TS) was approved by CEN on 24 April 2011 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16178:2012: E
worldwide for CEN national Members.
Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Principle .5
3 Reagents .6
3.1 Chemicals .6
3.2 Pharmaceutical standards and internal standards for calibration .6
3.3 Preparation of stock solutions .7
3.4 Preparation of working solutions .7
4 Apparatus .7
5 Sample pretreatment .8
6 Extraction and clean-up .8
6.1 Extraction of a dry sample .8
6.2 Concentration .8
6.3 Clean-up .9
7 Procedure .9
7.1 Blanks .9
7.1.1 Injection blank .9
7.1.2 Extraction blank .9
7.2 Calibration .9
7.3 Control solution .9
7.4 Analysis .9
8 Calculation and expression of results . 10
8.1 Calibration . 10
8.2 Calculation . 10
9 Test report . 11
Annex A (informative) Examples of chromatograms . 12
Bibliography . 18
Foreword
This document (CEN/TS 16178:2012) has been prepared by Technical Committee CEN/TC 400 “Project
Committee - Horizontal standards in the fields of sludge, biowaste and soil”, the secretariat of which is held by
DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
The preparation of this document by CEN is based on a mandate by the European Commission (Mandate
M/330), which assigned the development of standards on sampling and analytical methods for hygienic and
biological parameters as well as inorganic and organic determinants, aiming to make these standards
applicable to sludge, treated biowaste and soil as far as this is technically feasible.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy,
Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia,
Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Introduction
Drugs absorbed by the organism after intake are subject to metabolic reactions, such as hydroxylation or
cleavage. However, a significant amount of the original or metabolised substance leaves the organism via
urine or faeces. The contamination concerns ground and surface water, but also waste water and the solid
matrices like sludge or soils.
Due to their polarity, persistence and water solubility, some drugs and metabolites are able to pass through
the wastewater treatment plants. Their low adsorption on sludge and soils may cause the contamination of
surface and ground water. It is therefore necessary to analyse these molecules through an analytical SPELC-
MS/MS method as presented in this document. The method allows the identification of twelve molecules
belonging to the four predominant therapeutic classes in France: analgesics/anti-inflammatories, lipid
regulators, beta-blocker and anti-epileptics.
WARNING — Persons using this Technical Specification should be familiar with usual laboratory
practice. This Technical Specification does not purport to address all of the safety problems, if any,
associated with its use. It is the responsibility of the user to establish appropriate safety and health
practices and to ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this Technical
Specification be carried out by suitably trained staff.
1 Scope
This Technical Specification specifies a method to analyse pharmaceutical compounds in sludge, treated
biowaste and soil. Pharmaceuticals analysis has been carried out on a LC/MS-MS quantum. The main
difficulty for analysis is the lack of sample certified for target analytes. Even with spiked solid matrices it is still
delicate to correctly verify the impact of extraction step, because it is not commutable to a real sample.
This document provides a final protocol on the extraction and purification tested on sludge, soils and
sediments spiked with the pharmaceutical compounds listed in Table 1.
Table 1 — Details to the pharmaceutical compounds of interest
a
Steroid CAS-RN Purity Temperature Formula Molar mass Abbreviation
hormones preservation
% g/mol
Estrone 53-16-7 > 99 25°C C H O 270,4 E1
18 22 2
17β-Estradiol 50-28-2 > 98 25°C C H O 272,4 E2
18 24 2
17α-Estradiol 57-9 99 25°C C H O 272,4 α-E2
18 24 2
Ethinylestradiol 57-63-6 > 98 25°C C H O 296,4 EE2
20 24 2
Estrone d4
53866-34-5 > 95 25°C C H O D 274,4 E1 d4
18 18 2 4
Estrone-
2,4,16,16-d4
Estradiol d5 221093-45-4 > 98 25°C C H O D 277,4 E2 d5
18 19 2 5
17b-Estradiol-
2,4,16,16,17-d5
Ethinylestradiol 350820-06 > 98 25°C C H O D 300,4 EE2 d4
20 20 2 4
d4
17α-
Ethinylestradiol-
2,4,16,16-d4
a
Chemical Abstracts Service Registry Number
2 Principle
After pretreatment the freeze-dried sample is extracted by ultrasonication with an appropriate solvent. Then
the extract is purified on a suitable cartridge. The extract is analysed by high-performance liquid
chromatography (HPLC) on a C column and detected by mass spectrometry.
The identification is based on the retention times of the analytes and on the mass-spectrometric detection.
The detection is conducted in the MS/MS-mode in order to avoid interferences and over-quantification.
3 Reagents
3.1 Chemicals
Solvents used for extraction and clean-up shall be of HPLC grade or equivalent quality and checked for
blanks.
3.1.1 Water, H O.
3.1.2 Methanol, CH OH.
3.1.3 Acetonitrile, C H N.
2 3
3.1.4 Ammonia, NH .
3.2 Pharmaceutical standards and internal standards for calibration
Pharmaceutical standards shall be of analytical grade (> 90 %).
For example, analysis can be undertaken with compounds given in Table 2.
Table 2 — Proposed pharmaceuticals for use as internal standards
Therapeutic group Compound CAS-RN
Analgesics/anti-inflammatories Paracetamol 103-90-2
Paracetamol-d4 Not available
Diclofenac 15307-79-6
Diclofenac-d4 Not available
Ketoprofen 22071-15-4
Naproxen Not available
Ibuprofen 15687-27-1
Phenazone 60-80-0
Phenazone-d3 65566-62-3
Beta-blocker Metoprolol 56392-17-7
Propanolol 318-98-9
Propanolol-d7
344298-99-3
Anti-epileptic Carbamazepine 298-46-4
Primidone 125-33-7
Lipid regulator Bezafibrate 49562-28-9
Gemfibrozil 25812-30-0
Gemfibrozil-d6 25812-30-0
NOTE It is preferable to get one internal standard per molecule. If it is not possible, the recovery should be checked.
3.3 Preparation of stock solutions
3.3.1 Solution 1
Prepare a stock solution containing all pharmaceuticals at 100 mg/l in methanol (3.1.2) (solution 1). Store this
solution in dark glass bottles at –20 °C no longer than one month.
3.3.2 Solution 2
Prepare all deuterium-labelled substances at a concentration of 100 mg/l in methanol (3.1.2) (solution 2).
Store this solution in dark glass bottles at –20 °C.
3.4 Preparation of working solutions
Prepare the working solutions by dilution of stock solutions (3.3). Working solutions shall be prepared on the
day of analysis.
NOTE 1 Some pharmaceutical compounds deteriorate in solution in less than 24 h [3].
Prepare working solution at 1 mg/l (solution 3) by dilution of solution 1 (3.3.1) in methanol (3.1.2)
Prepare a mix of the internal standards at 100 µg/l (solution 4) by dilution of solution 2 (3.3.2) in a
water:methanol mixture (95:5).
Prepare calibration standards with appropriate amounts of the working solutions. For example, concentrations
for the calibration should be between 5 ng/ml and 500 ng/ml and concentrations for internal standards should
be 100 ng/ml.
A point of control is necessary to follow the performance of the chromatographic system. Prepare this solution
at 1 mg/l (solution 5) by dilution of solution 1 in methanol (3.1.2).
NOTE 2 That allows the comparison of the value of internal standard in the control sample with the value of internal
standard in
...
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