English Version. Sludge, treated biowaste and soil - Determination of pharmaceutical products

Transcription

1 TECHNICAL SPECIFICATION SPÉCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS February 2012 ICS English Version Sludge, treated biowaste and soil - Determination of pharmaceutical products Boues, bio-déchets traités et sols - Détermination des produits pharmaceutiques Schlamm, behandelter Bioabfall und Boden - Bestimmung pharmazeutischer Produkte This Technical Specification (CEN/TS) was approved by CEN on 24 April 2011 for provisional application. Dit document mag slechts op een stand-alone PC worden geinstalleerd. Gebruik op een netwerk is alleen. toestaan als een aanvullende licentieovereenkomst voor netwerkgebruik met NEN is afgesloten. This document may only be used on a stand-alone PC. Use in a network is only permitted when a supplementary license agreement for us in a network with NEN has been concluded. 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 worldwide for CEN national Members. Ref. No. CEN/TS 16178:2012: E

2 Contents Page Foreword...3 Introduction Scope Principle Reagents Chemicals Pharmaceutical standards and internal standards for calibration Preparation of stock solutions Preparation of working solutions Apparatus Sample pretreatment Extraction and clean-up Extraction of a dry sample Concentration Clean-up Procedure Blanks Injection blank Extraction blank Calibration Control solution Analysis Calculation and expression of results Calibration Calculation Test report Annex A (informative) Examples of chromatograms Bibliography

3 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. 3

4 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. 4

5 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. Steroid hormones Table 1 Details to the pharmaceutical compounds of interest CAS-RN a Purity % Temperature preservation Formula Molar mass Abbreviation g/mol Estrone > C C 18 H 22 O 2 270,4 E1 17β-Estradiol > C C 18 H 24 O 2 272,4 E2 17α-Estradiol C C 18 H 24 O 2 272,4 α-e2 Ethinylestradiol > C C 20 H 24 O 2 296,4 EE2 Estrone d4 Estradiol d5 Ethinylestradiol d4 Estrone- 2,4,16,16-d4 17b-Estradiol- 2,4,16,16,17-d5 17α- Ethinylestradiol- 2,4,16,16-d > C C 18 H 18 O 2 D 4 274,4 E1 d > C C 18 H 19 O 2 D 5 277,4 E2 d > C C 20 H 20 O 2 D 4 300,4 EE2 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 18 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. 5

6 3 Reagents 3.1 Chemicals Solvents used for extraction and clean-up shall be of HPLC grade or equivalent quality and checked for blanks Water, H 2 O Methanol, CH 3 OH Acetonitrile, C 2 H 3 N Ammonia, NH 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 Paracetamol-d4 Not available Diclofenac Diclofenac-d4 Not available Ketoprofen Naproxen Not available Ibuprofen Phenazone Phenazone-d Beta-blocker Metoprolol Propanolol Propanolol-d Anti-epileptic Carbamazepine Primidone Lipid regulator Bezafibrate Gemfibrozil Gemfibrozil-d NOTE It is preferable to get one internal standard per molecule. If it is not possible, the recovery should be checked. 6

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