| Title | The proposed work item is the standardisation of an analytical method applied for measurement of MEL, AMN, AMD, CYA, CYRO, and HMMM in aqueous matrices. Quantification of the substances is achieved by calibration with analytical standards in drinking water. For all substances except for HMMM, isotope-labelled internal standards are applied to correct for intensity variations. The method can be applied for various water matrices, including drinking water, surface water, and wastewater.
For most samples, the method can be used with direct injection, or, for wastewater, after dilution of samples. In this case, water samples only have to be diluted with 90% acetonitrile prior to analysis to create the necessary matrix. If lower limits of quantification (LOQs) need to be achieved, pre-concentration by evaporation can be applied. However, it should be noted that in wastewater, HMMM should only be measured with direct injection, due to significant matrix effects during evaporation. In undiluted water samples, the following LOQs apply: With direct injection MEL 0.10 µg/L, AMN 0.2 µg/L, AMD 0.1 µg/L, CYA 1.0 µg/L, CYRO 0.10 µg/L, and HMMM 0.10 µg/L ; with evaporation MEL 0.010 µg/L, AMN 0.010 µg/L, AMD 0.010 µg/L, CYA 0.050 µg/L, CYRO 0.010 µg/L, and HMMM 0.010 µg/L.
Measurements are performed by high-performance liquid chromatography (HPLC) and MS/MS detection. A hydrophilic interaction liquid chromatography (HILIC) column (Luna HILIC (150 x 2 mm, 3 µm, Phenomenex) with HILIC security guard cartridge (4 x 2 mm, Phenomenex)) is applied on an HPLC Infinity 1290 System (Agilent Technologies). An injection volume of 10 µL and a column temperature of 30 °C is used. Acetonitrile and ultrapure water with addition of 10 mM ammonium acetate and 0.1% formic acid are applied as eluents with isocratic elution of 90% acetonitrile and a flow rate of 0.4 mL/min. The mass spectrometric detection is performed on an API5500 Triple Quad (Sciex) after electrospray ionisation (ESI). All analytes are detected in multiple reaction monitoring (MRM) detection mode, with MEL, AMN, CYRO, and HMMM measured in positive mode, and AMD and CYA detected in negative mode. The analytes show retention times between 1.2 min (HMMM) and 3.4 min (AMN), while the chromatographic run is continued until 10 min to avoid carryover.
The method has been validated concerning recovery of evaporation, calibration linearity, matrix effects, and reproducibility. Stability tests showed no analyte degradation in glass, polyethylene, or polystyrene containers over 28 days, regardless of the storage temperature. Recommendations for sampling strategies of different water matrices and sample storage were also derived from the conducted tests as well as from established sampling standards.
During preparation of the European standard, a round robin test with several laboratories participating in the application of the analytical method should be conducted. |
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| Scope | The proposed work item is the standardisation of an analytical method applied for measurement of MEL, AMN, AMD, CYA, CYRO, and HMMM in aqueous matrices. Quantification of the substances is achieved by calibration with analytical standards in drinking water. For all substances except for HMMM, isotope-labelled internal standards are applied to correct for intensity variations. The method can be applied for various water matrices, including drinking water, surface water, and wastewater.
For most samples, the method can be used with direct injection, or, for wastewater, after dilution of samples. In this case, water samples only have to be diluted with 90% acetonitrile prior to analysis to create the necessary matrix. If lower limits of quantification (LOQs) need to be achieved, pre-concentration by evaporation can be applied. However, it should be noted that in wastewater, HMMM should only be measured with direct injection, due to significant matrix effects during evaporation. In undiluted water samples, the following LOQs apply: With direct injection MEL 0.10 µg/L, AMN 0.2 µg/L, AMD 0.1 µg/L, CYA 1.0 µg/L, CYRO 0.10 µg/L, and HMMM 0.10 µg/L ; with evaporation MEL 0.010 µg/L, AMN 0.010 µg/L, AMD 0.010 µg/L, CYA 0.050 µg/L, CYRO 0.010 µg/L, and HMMM 0.010 µg/L.
Measurements are performed by high-performance liquid chromatography (HPLC) and MS/MS detection. A hydrophilic interaction liquid chromatography (HILIC) column (Luna HILIC (150 x 2 mm, 3 µm, Phenomenex) with HILIC security guard cartridge (4 x 2 mm, Phenomenex)) is applied on an HPLC Infinity 1290 System (Agilent Technologies). An injection volume of 10 µL and a column temperature of 30 °C is used. Acetonitrile and ultrapure water with addition of 10 mM ammonium acetate and 0.1% formic acid are applied as eluents with isocratic elution of 90% acetonitrile and a flow rate of 0.4 mL/min. The mass spectrometric detection is performed on an API5500 Triple Quad (Sciex) after electrospray ionisation (ESI). All analytes are detected in multiple reaction monitoring (MRM) detection mode, with MEL, AMN, CYRO, and HMMM measured in positive mode, and AMD and CYA detected in negative mode. The analytes show retention times between 1.2 min (HMMM) and 3.4 min (AMN), while the chromatographic run is continued until 10 min to avoid carryover.
The method has been validated concerning recovery of evaporation, calibration linearity, matrix effects, and reproducibility. Stability tests showed no analyte degradation in glass, polyethylene, or polystyrene containers over 28 days, regardless of the storage temperature. Recommendations for sampling strategies of different water matrices and sample storage were also derived from the conducted tests as well as from established sampling standards.
During preparation of the European standard, a round robin test with several laboratories participating in the application of the analytical method should be conducted. |
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