1、 Reference number ISO 20179:2005(E) ISO 2005INTERNATIONAL STANDARD ISO 20179 First edition 2005-10-01 Water quality Determination of microcystins Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with ultraviolet (UV) detection Qualit de leau Dosage des micr
2、ocystines Mthode utilisant lextraction en phase solide (SPE) et la chromatographie en phase liquide haute performance (CLHP) avec dtection dans lultraviolet (UV) ISO 20179:2005(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may b
3、e printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobes licensing policy. The ISO Central Secretariat accepts
4、 no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the f
5、ile is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. ISO 2005 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any for
6、m or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09
7、47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2005 All rights reservedISO 20179:2005(E) ISO 2005 All rights reserved iii Contents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Abbreviated terms 1 4 Principle. 2 5 Reagents 2 6 Apparatus 5 7 Proc
8、edure 6 8 Method performance characteristics and data 10 9 Test report . 10 Annex A (informative) Mass spectrometry (MS) as an alternative detection . 11 Annex B (informative) Typical chromatogram and absorption spectra . 14 Annex C (informative) Precision data. 16 Bibliography . 17 ISO 20179:2005(E
9、) iv ISO 2005 All rights reservedForeword 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 inte
10、rested 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 Electrotechnica
11、l Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the t
12、echnical 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. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. I
13、SO shall not be held responsible for identifying any or all such patent rights. ISO 20179 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical, chemical and biochemical methods. ISO 20179:2005(E) ISO 2005 All rights reserved v Introduction The user should be awa
14、re that particular problems could require the specification of additional conditions. INTERNATIONAL STANDARD ISO 20179:2005(E) ISO 2005 All rights reserved 1 Water quality Determination of microcystins Method using solid phase extraction (SPE) and high performance liquid chromatography (HPLC) with u
15、ltraviolet (UV) detection WARNING The method requires use of microcystin-containing solutions. Microcystins are highly hepatotoxic to humans. Laboratory wastes of microcystins shall be collected separately and disposed as highly toxic chemical waste. Long-term decontamination with concentrated sodiu
16、m hypochlorite (NaClO) solution is also possible. Persons using this International Standard should be familiar with normal laboratory practice. 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 app
17、ropriate safety and health practices and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted according to this standard be carried out by suitably trained staff. 1 Scope This International Standard specifies a method for the determi
18、nation and quantification of microcystins in raw water (containing biomass) and treated water, such as tap water. The method described is validated for MCYST-RR, MCYST-YR, and MCYST-LR. It is also applicable for the determination of several structure variants 1of these microcystins, but an unambiguo
19、us identification cannot be made due to the lack of commercially available standards and due to co-elution. The threshold value of 1 g/l of MCYST-LR in water, proposed by the World Health Organization, can be followed after microcystin enrichment using solid phase extraction (SPE). 2 Normative refer
20、ences The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laborat
21、ory use Specification and test methods ISO 5667-4, Water quality Sampling Part 4: Guidance on sampling from lakes, natural and man-made 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 pu
22、rposes of this document, the following abbreviated terms apply. APCI atmospheric pressure chemical ionization MCYST microcystin MCYST-LR microcystin containing leucine (L) and arginine (R) ISO 20179:2005(E) 2 ISO 2005 All rights reservedMCYST-RR microcystin containing two arginine (R) units MCYST-YR
23、 microcystin containing tyrosine (Y) and arginine (R) SIM selected ion monitoring SEC size exclusion chromatography 4 Principle Water samples containing cyanobacterial material (biomass) shall be filtered first. The biomass is extracted separately with a solvent (methanol/water). The extract is filt
24、ered, 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 such as tap water are enriched using SPE. The microcystins are eluted from the SPE cartridges with methanol/water 90/10 by volume containing
25、 0,1 % by volume of trifluoroacetic acid (TFA). Microcystins are quantified by reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet/diode array detection at 238 nm. 5 Reagents Use only reagents of recognized analytical grade and water complying with grade 3 as specified i
26、n ISO 3696:1987, unless otherwise specified. 5.1 Methanol, CH 3 OH, HPLC grade. 5.2 Acetonitrile, CH 3 CN, HPLC grade. 5.3 Trifluoroacetic acid, TFA, CF 3 COOH. 5.4 Standard dilution solution, SPE rinsing solvent, and re-dissolving solvent. Methanol/water 20/80 by volume. 5.5 Extraction solution Met
27、hanol/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 sodium thiosulfate Na 2 S 2 O 3(anhydrous or with 5 H 2 O) in 100 ml of water. The final concentration is = 10 g/l (63 mmolar in case
28、 of anhydrous Na 2 SO 3 ). 5.8 Ammonium hydroxide solution Commercially available 1 mol/l of ammonium hydroxide solution, NH 4 OH. 5.9 Solid phase extraction cartridges (SPE) for microcystin enrichment The column shall have a minimum capacity (amount of analyte to be retained by the column) of not l
29、ess 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 in water containing 0,05 g of each microcystin. ISO 20179:2005(E) ISO 2005 All rights reserved 3 NOTE The recovery stro
30、ngly 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 material should have the following material specifications: carbon load (16,9 %), particle diameter
31、 (54 m), surface coverage (333 g/m 2based 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 is recommended. Disk-type SPE cartridges may also be used for the microcystin enrichment from
32、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 HPLC-eluent bottle. Degas the solution before use. This solution is stable at room temperatu
33、re 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 bottle. Degas this solution before use. This solution is stable at room temperature for about 2
34、 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 % TFA (5.11) Total volume flow rate, depending on the column min % % ml/min 0 30 70 0,3 to 1
35、,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 commercially available microcystins is very variable. Thus, it is important to follow the procedu
36、re 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 (5.1). Record the absorption curve between 220 nm and 250 nm in 1 cm quartz glass cells in
37、a spectrophotometer with methanol (5.1) in the reference cell. ISO 20179:2005(E) 4 ISO 2005 All rights reservedCalculate the mass concentration of each microcystin, i , in micrograms per millilitre, g/ml, using Equation (1): max i i i 1000 AM d = (1) where A maxis the absorbance determined at the ma
38、ximum of the absorption curve; M iis 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 the cell, in centimetres, cm; 1 000 is a calculation factor
39、 to achieve the final unit micrograms per millilitre, g/ml. M iand iare tabulated in Table 2. Table 2 Molar mass and molar absorptivity of MCYST-LR, -YR, and -RR (in methanol, at 238 nm) Microcystin M i ig mol 1l mol 1cm 1-LR 994 39 800 -YR 1 044 39 800 -RR 1 037 39 800 NOTE Data taken from Referenc
40、e 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 adding water and allowing a concentration of 10 g/ml
41、 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 rea
42、ched 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 record any changes in mass during storage. 5.16 Mixed microcystin standard solutions Pipette the volumes of microcystin stock solutions (5.14) given in Table 3 into 1 m
43、l vials. To each vial, add the volume of the standard dilution solution (5.4) given in Table 3 to achieve a final volume of 1 000 l, and shake well. ISO 20179:2005(E) ISO 2005 All rights reserved 5 Table 3 Pipetting scheme for the mixed microcystin standard solutions Standard solution Withdrawal vol
44、ume from each microcystin stock solution MCYST- LR, -YR, -RR (5.14) Volume to add from the standard dilution solution (5.4) to achieve a final volume of 1 000 l Mass concentration of standard solution l l g/ml MCYST-LR MCYST-YR MCYST-RR 1 20 940 0,20,2 0,2 2 40 880 0,4 0,4 0,4 3 100 700 1,0 1,0 1,0
45、4 200 400 2,0 2,0 2,0 5 300 100 3,0 3,0 3,0 5.17 Spiking solution for method control Prepare a spiking solution by pipetting 200 l of the mixed microcystin stock solution (prepared according to 5.15) into a 500 ml volumetric flask. Dilute it to the mark with water (tap-water or blank water from a na
46、tural lake), and shake well. The concentration of this spiking solution is 1 g/l for MCYST-LR, -YR, and -RR. 6 Apparatus 6.1 General The laboratory glassware and equipment to be used is not specified in this International Standard, as the choice of apparatus will depend on the specific applications
47、and circumstances. Avoid the use of plastics whenever possible. This is necessary because the use of plastics (e.g. plastic pipettes, plastic tubing or plastic cartridges) may cause losses of microcystins through absorption on the surface walls. 6.2 Adjustable horizontal shaker, needed only for the
48、analysis of samples containing phytoplankton. 6.3 Glass microfibre filter paper, retention size 1 m to 2 m. The maximum diameter of the filter should be 47 mm. Filtration is needed only for the analysis of samples containing phytoplankton. 6.4 SPE reservoir, 500 ml with connector for cartridges. 6.5
49、 Vacuum pump for SPE 6.6 Laboratory centrifuge, W 4 000 min 1 , relative centrifugal force (RCF) W 10 000 g. The use of an explosion-proof centrifuge is strongly advised due to the use of inflammable extraction solvents. 6.7 Ultrasonic probe, with characteristics of 60 W, 20 kHz. 6.8 Ultrasonic bath ISO 20179:2005(E) 6 ISO 2005 All rights reserved6.9 Heating block with temperature control and nitrogen gas delivery unit, with the following
