1、September 2017 English price group 14No part of this translation 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!%mMk“2744272www.din.
2、deDIN ISO 16308Water quality Determination of glyphosate and AMPA Method using high performance liquid chromatography (HPLC) with tandem mass spectrometric detection (ISO 16308:2014),English translation of DIN ISO 16308:2017-09Wasserbeschaffenheit Bestimmung von Glyphosat und AMPA Verfahren mittels
3、HochleistungsFlssigkeitschromatographie (HPLC) mit tandemmassenspektrometrischer Detektion (ISO 16308:2014),Englische bersetzung von DIN ISO 16308:2017-09Qualit de leau Dtermination du glyphosate et de lAMPA Mthode par chromatographie en phase liquide haute performance (CLHP) avec dtection par spect
4、romtrie de masse en tandem (ISO 16308:2014),Traduction anglaise de DIN ISO 16308:2017-09www.beuth.deDocument comprises 28 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.08.17 Foreword 6Introduction 71 Scope . 82 Normative refe
5、rences 83 Principle 84 Interferences 95 Reagents 96 Apparatus 117 Sampling . 128 Procedure 128.1 Pre-treatment (Suspended particular matter) . 128.2 Chelate break and derivatization . 128.3 Pre-concentration 128.4 Chromatographic determination 138.5 Identification and confirmation of the analytes 14
6、8.6 Blank control monitoring 149 Calibration . 149.1 Concentration ranges . 149.2 Matrix-matched calibration 149.3 Internal standard calibration 1510 Expression of results .1611 Test report 16Annex A (informative) Performance data 17Annex B (informative) Examples of chromatographic conditions 20Anne
7、x C (informative) Examples of chromatograms 21Annex D (informative) Analysis of gluphosinate .22Annex E (informative) Pre-treatment of hard water samples .26Bibliography .28DIN ISO 16308:2017-09 2Contents PageNational foreword 3 National Annex (informative) Bibliography 5 .A comma is used as the dec
8、imal marker.National foreword The text of this document (ISO 16308:2014) has been prepared by Technical Committee ISO/TC 147 “Water quality” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was DIN-Normenausschuss Wasserwesen (DIN Standards Committee Water Practic
9、e), Working Committee NA 119-01-03 AA “Water examination (CEN/TC 230, ISO/TC 147) , Working Group NA 119-01-03-02-16 AK “LC-MS/MS methods”. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. DIN shall not be held responsible for iden
10、tifying any or all such patent rights. Designation of the method: Determination of glyphosate and AMPA Method using high performance liquid chromatography (HPLC) with tandem mass spectrometric detection (F 45): Method DIN ISO 16308 F 45 The DIN Standards corresponding to the International Standards
11、referred to in this document are as follows: ISO 3696 DIN ISO 3696 ISO 5667-3 DIN EN ISO 5667-3 ISO 5725-2 DIN ISO 5725-2 ISO 6058 DIN 38406-3 ISO 6059 DIN 38406-3 DIN ISO 16308:2017-09 3”DIN ISO 16308:2017-09 4Expert assistance and specialized laboratories will be required to perform the analyses d
12、escribed in this standard. Existing safety requirements are to be observed. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whether and to what extent additional conditions will have to be specified. This standard contains a standard method for the exa
13、mination of water, waste water and sludge, that has been developed by DIN-Normenausschuss Wasserwesen (DIN Standards Committee Water Practice) in collaboration with the Wasserchemische Gesellschaft (Water Chemistry Society), a division of the Gesellschaft Deutscher Chemiker (German Chemical Society)
14、. The standard method is: Determination of glyphosate and AMPA Method using high performance liquid chromatography (HPLC) with tandem mass spectrometric detection (F 45). Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The
15、 standard methods included in the loose-leaf publication entitled Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published by Beuth Verlag GmbH and Wiley-VCH Verlag GmbH however, the determination by HPLCESIMS/MS can be much more specific (unambiguous i
16、dentification) and more sensitive (limits of quantification of approximately 30 ng/l for both glyphosate and AMPA). This International Standard is based on this analytical technique and is intended for laboratories involved in the regulatory control of the aquatic environment. Many such laboratories
17、 are now equipped with this kind of apparatus.DIN ISO 16308:2017-09 7 WARNING Persons using this International Standard should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated with its use. It is the r
18、esponsibility 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 in accordance with this International Standard be carried out by suitably qualified staff.1 Scope
19、This International Standard specifies a method for the determination of dissolved fraction of glyphosate and its major metabolite, aminomethylphosphonic acid (AMPA), in drinking water, ground water, and surface water at concentrations of 0,03 g/l to 1,5 g/l. It does not apply to marine or salty wate
20、r. This method can be applicable to other types of waters, provided the method is validated for each case.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition
21、cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 3696, Water for analytical laboratory use Specification and test methodsISO 5667-3, Water quality Sampling Part 3: Preservation and handling of water samplesISO 8466-1, Water q
22、uality Calibration and evaluation of analytical methods and estimation of performance characteristics Part 1: Statistical evaluation of the linear calibration function3 PrincipleGlyphosate and AMPA (dissolved fraction after filtration) are derivatized using 9-fluorenylmethylchloroformate (FMOC-Cl) (
23、5.17) to lower their polarity and increase the retention of compound in a separation on a reverse phase column (e.g. C18) as well as to improve the mass spectrometric detection. If the mass spectrometer has sufficient detection capability, it is possible to omit the solid phase extraction and to ana
24、lyse the analytes by direct injection (see 8.2.1).The derivatized sample is purified by liquid/liquid extraction and then concentrated by solid phase extraction (SPE).The analysis is performed by high performance liquid chromatography coupled with tandem mass spectrometry via an electrospray source
25、(HPLCESIMS/MS), using matrix-matched calibration.DIN ISO 16308:2017-09 8 Table 1 Substances addressedName FormulaMolecular mass g/molCAS-RNaGlyphosateN-(phosphonomethyl)glycineC3H8NO5P 169,1 1071836AMPAAminomethylphosphonic acidCH6NO3P 111,0 1066519aCAS-RN Chemical Abstracts Service Registry NumberN
26、OTE Gluphosinate, belonging to the aminophosphonate family, can be determined simultaneously, provided it can be demonstrated that there is no interference with the sample (matrix) subject to analysis.4 InterferencesThis method is validated for hard water containing up to 3,2 mmol/l of the sum of ca
27、lcium and magnesium. For waters with a higher calcium and magnesium content, it may be necessary to increase the concentration of EDTA-Na2(5.16) at the derivatization step (see Annex D).It can prove necessary to include the acidification step described in Annex D even for some water types below 3,2
28、mmol/l of the sum of calcium and magnesium. The laboratory shall check the necessity of this procedure for its routine samples.The presence of free chlorine, e.g. in treated waters, can cause losses of glyphosate by oxidation. Consequently sodium thiosulfate shall be used (see Clause 7).5 ReagentsUn
29、less otherwise stated, all reagents and solvents shall be of sufficient purity, e.g. “for trace analysis”.5.1 Deionized water.5.2 Ultra-pure water, complying with grade 1 of ISO 3696.5.3 Nitrogen, N2, purity 99,996 % volume fraction.5.4 Laboratory detergent, alkaline.5.5 Sodium thiosulfate, Na2S2O3.
30、5.6 Acetonitrile, C2H3N, HPLC grade.5.7 Methanol, CH4O, HPLC grade.5.8 Ethanol, C2H6O, 95 %, HPLC grade mass fraction.5.9 Ethyl acetate, C4H8O2, HPLC grade.5.10 Ammonium acetate, C2H7O2N.5.11 Triethylamine, C6H15N.5.12 Ammonium hydroxide, NH4OH,28 % mass fraction.DIN ISO 16308:2017-09 9 5.13 Formic
31、acid, CH2O2.5.14 Hydrochloric acid, HCl, 300 g/l.5.15 Glacial acetic acid, C2H4O2.5.16 Ethylenediaminetetraacetic acid (EDTA), disodium salt dihydrate, C10H14N2O8Na22H2O, with a minimum purity of 99 % mass fraction.5.17 9-Fluorenylmethyl chloroformate,(FMOC-Cl), C15H11ClO2, with a minimum purity of
32、97 % mass fraction.FMOC-Cl is used to prepare the derivatization reagent, FMOC-Cl solution, 50 mg/ml, in acetonitrile (5.6). This solution can be stored at 18 C 3 C for 6 months.For direct injection (8.2.1), use a FMOC-Cl solution of 0,5 mg/ml in acetonitrile.5.18 Reference substances, according to
33、Table 1.5.18.1 Glyphosate, N-(phosphonomethyl)glycine, C3H8NO5P, purity 98 % mass fraction.5.18.2 AMPA, aminomethylphosphonic acid, CH6NO3P, purity 98 % mass fraction.5.18.3 1,2-13C2,15N-labelled glyphosate, surrogate standard, purity 98 % mass fraction.5.18.4 13C,15N-labelled AMPA, surrogate standa
34、rd, purity 98 % mass fraction.5.19 Calibration solutions.Individual stock solutions of glyphosate (5.18.1) and AMPA (5.18.2), 100 mg/l, prepared in ultra-pure water (5.2). These solutions can be stored at 4 C 3 C for 1 month.Individual stock solutions of 1,2-13C2,15N-labelled glyphosate (5.18.3) and
35、 13C,15N-labelled AMPA (5.18.4) 100 mg/l, prepared in ultra-pure water (5.2). These solutions can be stored at 4 C 3 C for 1 month.Multi-substance working solution of surrogates: 1,2-13C2,15N-labelled glyphosate and 13C,15N-labelled AMPA, 20 g/l, prepared in ultra-pure water (5.2). This solution can
36、 be stored at 4 C 3 C for 1 month.NOTE Stock and calibration solutions can be stored longer, provided the adequate justifications are given regarding stability.5.20 Triethylammonium acetate buffer, 0,1 % triethylamine (5.11) solution adjusted to pH 9,5 with glacial acetic acid (5.15) (mobile phase).
37、5.21 Sodium tetraborate, decahydrate, Na2B4O710H2O.5.22 Borate-Na buffer, 0,05 mol/l; pH = 9,2.Dissolve 19 0,1 g of sodium tetraborate (5.21), decahydrate in 1 l of water (5.1). This solution can be stored for approximately 1 mo at 4 C 3 C.5.23 Mineral water, containing less than 3,2 mmol/l divalent
38、 cations (Mg2+and total Ca2+), for preparing matrix-matched calibration.DIN ISO 16308:2017-09 10 6 ApparatusThe material or any parts that are likely come into contact with the sample shall be free from any residue that could cause unacceptable interference in the blanks.Glass and plastics container
39、s can be used for sampling and for all steps before derivatization. Glass vials (6.10) and glass test tubes (6.11) shall be used after the derivatization step.6.1 Usual laboratory glassware, or apparatus and in particular the following.6.2 Glass, polyethene (PE) or polypropene (PP) bottles, minimum
40、50 ml, for sampling.6.3 Glass, polyethene (PE) or polypropene (PP) syringe, 50 ml, for sample filtration.6.4 Single use filter for syringe, diameter 25 mm, with a hydrophilic membrane, 0,45 m, e.g. from regenerated cellulose.6.5 Glass, or single use PE or PP conical bottomed tubes, approximately 50
41、ml, for derivatization.6.6 Micropipettes, adjustable from 100 l to 500 l.6.7 pH-meter.6.8 SPE cartridges, e.g. Oasis HLB1)Waters, 60 mg, 3 ml, or equivalent.6.9 Centrifugation device, capable of 6 500 m1.6.10 Glass vials, suitable for autosampler, with caps and polytetrafluoroethene (PTFE) or silico
42、n rubber septa.6.11 Glass test tube, 15 ml or smaller.6.12 Reversed phase column, e.g. XBridge C181)column Waters, 50 mm 2,1 mm internal diameter (i.d.) 2,5 m, column with guard column (Waters, 10 mm 2,1 mm i.d. 2,5 m).A column whose stationary phase is alkali proof (pH 9 to pH 9,5) is highly recomm
43、ended.NOTE A Gemini-NX1)column (Phenomenex) with similar dimensions is also suitable. Other columns can be used, provided the separation conditions are adjusted.6.13 High performance liquid chromatograph (HPLC), consisting of 6.13.1 to 6.13.5.6.13.1 Injector, manual or automated.6.13.2 Gradient pump
44、.6.13.3 Thermoregulation oven, for HPLC column.6.13.4 Mass spectrometer, with triple quadrupole analyser and an electrospray source.1) Oasis HLB, XBridge C18and Gemini-NX are examples of suitable products available commercially. This information is given for the convenience of users of this document
45、 and does not constitute an endorsement by ISO of these products.DIN ISO 16308:2017-09 11 6.13.5 Data acquisition and processing system.7 SamplingIf the sampling bottles are not for single use, rinse the sample bottles (6.2) with deionized water (5.1), then clean with a laboratory detergent (5.4). R
46、inse with water (5.1), then with ultra-pure water (5.2), and finally with 95 % ethanol (5.8).Perform the sampling in accordance with ISO 5667-3 in these bottles (6.2) (approximately 50 ml).For samples suspected of containing free chlorine, add about 2 mg of sodium thiosulfate (5.5) or any other chlo
47、rine reducing agent per 100 ml of sample.Store the samples according to ISO 5667-3 at 3 C 2 C and pre-treat within 24 h.8 Procedure8.1 Pre-treatment (Suspended particular matter)Place filter (6.4) on syringe (6.3). Rinse with 5 ml of ultra-pure water (5.2).Filter the sample (approximately 50 ml), di
48、scard the first 5 ml. Collect the filtrate in a conical bottomed tube (6.5).Store the filtered samples at 4 C 3 C for one week maximum before the derivatization step.8.2 Chelate break and derivatization8.2.1 GeneralAdapt the derivatization step to the kind analytical step which follows derivatization: using a mas
