1、October 2007DEUTSCHE NORM English price group 12No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13.060.50!$IGy“1383686
2、www.din.deDDIN ISO 20179Water quality Determination of microcystins Method using solid phase extraction (SPE) and high performance liquidchromatography (HPLC) with ultraviolet (UV) detection (ISO 20179:2005)English version of DIN ISO 20179:2007-10Wasserbeschaffenheit Bestimmung von Mikrocystinen Ver
3、fahren mittels Festphasenextraktion (SPE) undHochleistungs-Flssigkeitschromatographie (HPLC) mit ultravioletter (UV) Detektion(ISO 20179:2005)Englische Fassung DIN ISO 20179:2007-10www.beuth.deDocument comprises 22 pages DIN ISO 20179:2007-10 2 Contents Page National foreword 3 National Annex NA (in
4、formative) Bibliography . 4 Introduction 5 1 Scope . 6 2 Normative references . 6 3 Abbreviated terms 6 4 Principle . 7 5 Reagents 7 6 Apparatus 10 7 Procedure 11 8 Method performance characteristics and data. 15 9 Test report . 15 Annex A (informative) Mass spectrometry (MS) as an alternative detec
5、tion . 16 Annex B (informative) Typical chromatogram and absorption spectra . 19 Annex C (informative) Precision data 21 Bibliography . 22 DIN ISO 20179:2007-10 3 This standard is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammunter-suchung Gemeinsam erfassbare Stoff
6、gruppen (Gruppe F) (German standard methods for the examination of water, waste water and sludge Substance group analysis (group F) and describes method F 29. National foreword This standard has been prepared by Technical Committee ISO/TC 147 “Water quality” (Secretariat: DIN, Germany). The responsi
7、ble German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Technical Committees NA 119-01-03-02-12 AK Mikrocystine and NA 119-01-03 AA Wasseruntersuchung. Expert assistance and specialized laboratories will be required to perform the analysi
8、s described in this standard. Existing safety requirements are to be observed. Designation of method F 29 Determination of microcystins Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection: Method DIN ISO 20179 F 29 Standard meth
9、ods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-leaf publication entitled Deutsche Einheits-verfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by Wiley-VCH
10、Verlag and Beuth Verlag GmbH. All standard methods relevant to the Abwasserverordnung (Waste Water Regulation) (AbwV) included in the new Regulation on Section 7a of the Gesetz zur Ordnung des Wasserhaushalts (German Water Management Act together with the Abwasserverordnung and the Gesetz zur Ordnun
11、g des Wasserhaushalts and other valid administrative regulations on waste water have been published as a loose-leaf collection “Analysen-verfahren in der Abwasserverordnung Rechtsvorschriften und Normen”*), with Supplement 1 (DIN Stand-ards), Supplement 2 (DIN EN and DIN EN ISO Standards) and Supple
12、ment 3 (DIN, DIN EN and DIN EN ISO Standards). Standard methods or draft standards bearing the group title “German standard methods for the examination of water, waste water and sludge” are classified under the following categories (main titles): General information (group A) (DIN 38402) Sensory ana
13、lysis (group B) (DIN 38403) Physical and physicochemical parameters (group C) (DIN 38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 38406) Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (group H) (D
14、IN 38409) Biological-ecological methods of analysis (group M) (DIN 38410) Microbiological methods (group K) (DIN 38411) Test methods using water organisms (group L) (DIN 38412) Individual constituents (group P) (DIN 38413) *)Available (in German) from Beuth Verlag GmbH. DIN ISO 20179:2007-10 4 Sludg
15、e and sediments (group S) (DIN 38414) Bio-assays with microorganisms (group T) (DIN 38415) In addition to the methods described in the DIN 38402 to DIN 38415 series of standards, there are a number of European and International Standards available as DIN EN, DIN EN ISO and DIN ISO Standards, which a
16、lso form part of the collection of German standard methods. Information on Parts of these series of standards that have already been published can be obtained from the offices of the Normenausschuss Wasserwesen, telephone +49 30 2601-2448, or from Beuth Verlag GmbH, Burggrafenstrae 6, 10787 Berlin.
17、This standard contains in addition to the legal units also the unit “psi” to facilitate business (import/export) with countries where this unit is used. Users of this standard should note that the Gesetz ber Einheiten im Messwesen (German Law on units in metrology) prohibits the use of the unit “psi
18、” for official and commercial purposes in Germany. The DIN Standards corresponding to the International Standards referred to in the EN are as follows: ISO 3696 DIN ISO 3696 ISO 5667-3 DIN EN ISO 5667-3 ISO 5667-4 DIN 38402-12 ISO 5667-5 DIN 38402-14 ISO 5667-6 DIN 38402-15 ISO 5725-1 DIN ISO 5725-1
19、 ISO 5725-2 DIN ISO 5725-2 National Annex NA (informative) Bibliography DIN 38402-12, German standard methods for the examination of water, waste water and sludge General information (group A) Sampling from barrages and lakes (A 12) DIN 38402-14, German standard methods for the examination of water,
20、 waste water and sludge General information (group A) Sampling of untreated water and drinking water (A 14) DIN 38402-15, German standard methods for the examination of water, waste water and sludge General information (group A) Sampling of flowing waters (A 15) DIN EN ISO 5667-3, Water quality Samp
21、ling Part 3: Guidance on the preservation and handling of water samples DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2, Acc
22、uracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method Introduction The user should be aware that particular problems could require the specification of additional conditions.
23、5 DIN ISO 20179:2007-10Water quality Determination of microcystins Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection WARNING The method requires use of microcystin-containing solutions. Microcystins are highly hepatotoxic to h
24、umans. Laboratory wastes of microcystins shall be collected separately and disposed as highly toxic chemical waste. Long-term decontamination with concentrated sodium hypochlorite (NaClO) solution is also possible. Persons using this International Standard should be familiar with normal laboratory p
25、ractice. This standard 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 essen
26、tial that tests conducted according to this standard be carried out by suitably trained staff. 1 Scope This International Standard specifies a method for the determination and quantification of microcystins in raw water (containing biomass) and treated water, such as tap water. The method described
27、is validated for MCYST-RR, MCYST-YR, and MCYST-LR. It is also applicable for the determination of several structure variants1of these microcystins, but an unambiguous identification cannot be made due to the lack of commercially available standards and due to co-elution. The threshold value of 1 g/l
28、 of MCYST-LR in water, proposed by the World Health Organization, can be followed after microcystin enrichment using solid phase extraction (SPE). 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition
29、cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 5667-4, Water quality Sampling Part 4: Guidance on sampling from lakes, natural and man-made
30、ISO 5667-5, Water quality Sampling Part 5: Guidance on sampling of drinking water from treatment works and piped distribution systems 3 Abbreviated terms For the purposes of this document, the following abbreviated terms apply. APCI atmospheric pressure chemical ionization MCYST microcystin MCYST-LR
31、 microcystin containing leucine (L) and arginine (R) 6 DIN ISO 20179:2007-10MCYST-RR microcystin containing two arginine (R) units MCYST-YR microcystin containing tyrosine (Y) and arginine (R) SIM selected ion monitoring SEC size exclusion chromatography 4 Principle Water samples containing cyanobac
32、terial material (biomass) shall be filtered first. The biomass is extracted separately with a solvent (methanol/water). The extract is filtered, diluted and a solid phase extraction (SPE) is applied for sample clean-up. The filtrate is treated as a pure water sample (see below). Pure water samples s
33、uch as tap water are enriched using SPE. The microcystins are eluted from the SPE cartridges with methanol/water 90/10 by volume containing 0,1 % by volume of trifluoroacetic acid (TFA). Microcystins are quantified by reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet/d
34、iode array detection at 238 nm. 5 Reagents Use only reagents of recognized analytical grade and water complying with grade 3 as specified in ISO 3696:1987, unless otherwise specified. 5.1 Methanol, CH3OH, HPLC grade. 5.2 Acetonitrile, CH3CN, HPLC grade. 5.3 Trifluoroacetic acid, TFA, CF3COOH. 5.4 St
35、andard dilution solution, SPE rinsing solvent, and re-dissolving solvent. Methanol/water 20/80 by volume. 5.5 Extraction solution Methanol/water 75/25 by volume. 5.6 SPE elution solution Methanol/water 90/10 by volume containing 0,1 % by volume TFA. 5.7 Sodium thiosulfate, solution. Dissolve 1 g of
36、sodium thiosulfate Na2S2O3(anhydrous or with 5 H2O) in 100 ml of water. The final concentration is = 10 g/l (63 mmolar in case of anhydrous Na2SO3). 5.8 Ammonium hydroxide solution Commercially available 1 mol/l of ammonium hydroxide solution, NH4OH. 5.9 Solid phase extraction cartridges (SPE) for m
37、icrocystin enrichment The column shall have a minimum capacity (amount of analyte to be retained by the column) of not less than 100 g of each microcystin and shall give a recovery of not less than 80 % for MCYST-LR and not less than 70 % for MCYST-RR and MCYST-YR when applied as a standard solution
38、 in water containing 0,05 g of each microcystin. 7 DIN ISO 20179:2007-10NOTE The recovery strongly depends on the SPE cartridge material/brand, material specifications such as carbon load, particle size etc. The recovery data are based on C-18 cartridges determined by a single measurement. The mater
39、ial should have the following material specifications: carbon load (16,9 %), particle diameter (54 m), surface coverage (333 g/m2based on % C) cartridge volume (3 ml), material per cartridge (500 mg). If the above required recovery values can not be reached, changing the brand of the SPE cartridge i
40、s recommended. Disk-type SPE cartridges may also be used for the microcystin enrichment from water samples 2. 5.10 HPLC mobile phase solution (A) To a 1 000 ml volumetric flask, add 800 ml of acetonitrile (5.2) and 500 l of TFA (5.3) and bring to volume with acetonitrile. Transfer this solution in a
41、 HPLC-eluent bottle. Degas the solution before use. This solution is stable at room temperature for about 3 weeks. 5.11 HPLC mobile phase solution (B) To a 1 000 ml volumetric flask, add 800 ml of water and 500 l of TFA (5.3) and bring to volume with water. Transfer this solution in a HPLC-eluent bo
42、ttle. Degas this solution before use. This solution is stable at room temperature for about 2 weeks. 5.12 HPLC mobile phase gradient (an example) Table 1 HPLC mobile phase gradient Time HPLC mobile phase solution (A) Acetonitrile with 0,05 % TFA (5.10) HPLC mobile phase solution (B); water with 0,05
43、 % TFA (5.11) Total volume flow rate, depending on the columnmin % % ml/min 0 30 70 0,3 to 1,0 10 35 65 0,3 to 1,0 40 70 30 0,3 to 1,0 42 100 0 0,3 to 1,0 44 100 0 0,3 to 1,0 46 30 70 0,3 to 1,0 55 30 70 0,3 to 1,0 5.13 Microcystins, commercially available film in ampoules. NOTE The quality of comme
44、rcially available microcystins is very variable. Thus, it is important to follow the procedure given in 5.14. 5.14 Microcystin stock solutions To determine the exact concentration of microcystins, dissolve in each stock solution the individual microcystin delivered from the supplier in pure methanol
45、 (5.1). Record the absorption curve between 220 nm and 250 nm in 1 cm quartz glass cells in a spectrophotometer with methanol (5.1) in the reference cell. 8 DIN ISO 20179:2007-10Calculate the mass concentration of each microcystin, i, in micrograms per millilitre, g/ml, using Equation (1): max iii10
46、00AMd=(1) where Amaxis the absorbance determined at the maximum of the absorption curve; Miis the molar mass of each microcystin, in grams per mol; g/mol; iis the molar absorptivity of each microcystin in methanol (5.1), in litres per (mole centimetre), l/(mol cm); d is the optical path length of th
47、e cell, in centimetres, cm; 1 000 is a calculation factor to achieve the final unit micrograms per millilitre, g/ml. Miand iare tabulated in Table 2. Table 2 Molar mass and molar absorptivity of MCYST-LR, -YR, and -RR (in methanol, at 238 nm) Microcystin Miig mol1l mol1cm1-LR 994 39 800 -YR 1 044 39
48、 800 -RR 1 037 39 800 NOTE Data taken from Reference 1. For further details refer to this reference. For further HPLC analysis, the solvent methanol/water ratio for the MCYST-LR, -YR, and -RR standards can be adjusted to 20/80 by volume (i.e. to the standard dilution solution described in 5.4), by a
49、dding water and allowing a concentration of 10 g/ml for each microcystin. 5.15 Mixed microcystin stock solutions Prepare a standard solution containing 2,5 g/ml each of MCYST-LR, -YR, and -RR in the standard dilution solution (5.4). Store it below 16 C. To avoid incorporation of water by condensation, do not open the vial until its contents have reached room temperature. If the solution is to be stored for a long period, use a hermetic vial. In case of doubt, weigh the vial and r
