ISO 20179 pdf download – Water quality – Determination ofmicrocystins – Method using solidphase extraction (SPE) and highperformance liquid chromatography(HPLC) with ultraviolet (uv) detection.
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 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 such as tap water are enriched using SPE The microcystins are etuted from the SPE cartridges with methanol/water (90(10 by volume) containing 0,1 % by volume of trifluoroacelic 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 analylical grade and water complying with grade 3 as specified In
ISO 3696:1987, unless otherwise specified.
5.1 Methanol. CH3OH. HPIC grade.
5.2 Acetonitnle CH3CN, HPLC grade.
5.3 Trifluoroacetlc acid, TFA, CF3COOH,
5.4 Standard 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 sodium thiosulfate Na2S2O3 (anhydrous or with 5 H20) In 100 ml of water. The final concentration Is p 10 g/l (63 mmolar in case of anhydrous Na2SO3).
5.8 Ammonium hydroxide solution
Commercially available — 1 moth of ammonlum hydroxide solution. NH4OH.
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 less than 100 pg of each microcystin and shall give a recovery of not less than 80 % for MCYST.LR and no4 less than 70 % for MCYST-RR and MCYST.YR when applied as a standard solution In water containing 0.05 pg of each microcystin.
NOTE mere is a possdility that various filter matenals may retain microcysbns. Optionally a microfuge may be used
in order to avoàd losses.
6.11 Sampling bottles, dark glassware, sterile and pra-cleaned.
6.12 HPLC system. consisting of the following.
6.12.1 Binary HPLC pump, suitable for volume flow rates between 0.3 mI/mm and 1,0 mI/mm.
6.12.2 HPLC column oven, with temperature control unit (35 C).
6.12.3 lnI.ctlon system, with Injection volume range from 5 p1 to 20 p1.
6.12.4 HPLC column (..g. C18 column), packed with material of partide size 3 pm to 5 pm and mner diameter 2 mm to 4.6 mm, length 250 mm, to ensure baseline resolution of MCYST-LR. -YR. and -RR standards.
A suitable guard-column should be used. Pressure range should be 70,000 hPa to 200,000 hPa (1 015 psi to 2 900 psi).
6.12.5 UVlphoto diode array (PDA) detector, with a wavelength of A = 238 nni ricluding background correction.
The wavelength range of the POA shall be 200 nm to 300 om.
The linit of detection (LOO) for the system should be 0,1 ngipl (signal-to-noise-ratio = 3). and the limit of quantification (LOQ) should be 0.2 ng/pl (signal-to-noise-ratio =6) for each microcystin (using a standard solution).
7 Procedure
7.1 Sampling and conditioning
Collect water samples as specified in ISO 5667-4 and ISO 5667-5 and store the samples (not longer than 48 h) in a cool (4 C to 8 C) and dark place.
Prior to conditioning, adjust the SPE cartridge (5.9) to room temperature.
For conditioning, refer to the suppliers’ recommendation. If this is not indicated, first pass 4 ml of methanol (5.1) through the Cartridge. Then pass 4 ml of water ttvough the cartndge. Let the solvents pass at a rate
-: 10 mI/mm through the column, and make sure that a small portion of the solvent remains on top of the column until the sample solution Is applied.
7.2 Sample preparation
7.2.1 Treated water/tap water
Concentrate microcystins in water samples using solid phase extraction (7.4).
ISO 20179 pdf download – Water quality – Determination ofmicrocystins – Method using solidphase extraction (SPE) and highperformance liquid chromatography(HPLC) with ultraviolet (uv) detection
