Guidance on quality control for spectral data and methods of analysis

Transcription

1 Guidance on quality control for spectral data and methods of analysis 1. Background If a manufacturer or an importer places a new chemical substance on the European market this substance has to be notified in accordance with Directive 67/548/EEC on the classification, packaging and labelling of dangerous substances. In February 1997 the European Commission published this Technical Guidance for the completion of a summary notification dossier for a new chemical substance utilising the structured notification interchange format (SNIF). This document outlines which information is required to fill in the various sections in the SNIF. In addition to the SNIF the notifier has to submit spectral data to confirm the identity of the notified substance. Section of this Technical Guidance outlines the requirement for submission of spectral data. Over the years the CAs have encountered problems with the submission of spectral data, in particular with the standard and the quality of the submitted data. This might due to the fact that that the Technical Guidance is lacking in quality criteria for submission of spectral data. This is in contrast to tests conducted to evaluate physicochemical properties, toxicity and ecotoxicity endpoints of the new chemical substances, which must be performed in compliance with Annex V to Directive 67/548/EEC and/or OECD Test Guidelines. 2. Introduction The aim of this document is to outline more in detail the criteria that are needed for the assessment of spectral data, in particular Ultraviolet and Visible Absorption Spectroscopy (UV/VIS), Infrared Spectroscopy (IR) Nuclear Magnetic Resonance Spectroscopy (NMR) and Mass spectroscopy (MS). Furthermore, this document will outline some requirements regarding chromatographic methods, such as Gas Chromatography (GC) and High-Performance Liquid Chromatography (HPLC). 3. General remarks All spectroscopic and analytical methods used to identify the new substance should be carried out on the same batch (otherwise it should be indicated). This batch should be prepared by the manufacturer and used for the various spectroscopic analysis performed on the substance as notified. The same batch should be used for the determination and detection of the substance and impurities. The batch number and the purity of the substance should be listed in each single report. The amount and identity of impurities or residual solvents should be indicated (if known). If more than one batch is used, for each batch a certificate of analysis should be submitted. All solvents should be spectroscopically pure. Instruments on which the measurements are performed should be calibrated periodically using suitable references. The spectrum obtained must be in accordance with the structure and composition of the notified substance. Each of the reports should state the laboratory, the head of laboratory, the responsible technician and the date the spectrum or chromatogram was obtained and should be written in English. It should be noted that the minimum spectral data package for most organic substances should contain UV/VIS, IR and 1 H-NMR spectra and an analytical method such as HPLC, GC or other. If these techniques cannot be used, a justification should be given. Additional information, such as 13 C-NMR, MS etc, is required if the other

2 methods are not sufficient to identify the substance. However, they can be used to obtain additional information if also submitted. The quality of the various spectra and chromatograms is very important and therefore: Abscissa and ordinate should be properly marked. Changes of the scale should be clearly indicated as well as any amplifications or changes of the base line. If possible, the original spectra should be submitted. Photocopies should be of good quality. If spectra are electronically available they should be submitted as pdf-file in addition to the paper copy. 4. UV/VIS Spectroscopy 4.1 Identity of the substance: 4.2 Test conditions: spectrometer (name and type), slit width, cell type (e.g. silica), path length (e.g cm) solvent (e.g. water, methanol, acetonitrile) concentration of test substance (e.g molar solution or less) test temperature (e.g ºC) ph environments used: neutral (ph = 7), acid (ph < 2) and alkaline (ph > 10) (for substances which react with hydrochloric acid or sodium hydroxide, a UV/VIS spectrum should be run only at ph = 7).

3 4.3 Measurements: Each spectrum will be measured over at least the wavelength (λ) range 200 to 750 nm with a wavelength accuracy of +/- 0.5 nm in a suitable solvent at the various ph values. The scan speed should be reported. If there is any significant deviation from zero absorbance a blank spectrum should be run. Evidence of regular wavelength/absorbance accuracy checks should be briefly provided. 4.4 Spectra: The test substance spectra can be annotated as necessary. The spectra can be plotted as a log 10 (I 0 /I) ordinate and λ abscissa or log ε versus λ. I 0 is the transmitted light through the solvent only and I is the transmitted light through the substance in solution. For each spectral maximum, the extinction coefficient ε value and the wavelength λ in nm will be calculated with the maximum wavelength, and should be reported in tabular form. 5. IR Spectroscopy 5.1 Identity of the substance: 5.2 Test conditions: spectrometer (name and model, e.g. FT-IR) operating temperature concentration of the test substance (if appropriate) method of preparation of the sample (KBr disc, liquid film, etc.) reference solvent (if appropriate) 5.3 Measurement: The spectrum will be measured over at least the range from 600 to 4000 cm -1, typically with a resolution of 2 cm -1 or at least of 4 cm -1. The instrument accuracy should be checked regularly with, e.g. polystyrene standard. It shall be +/- 4 cm -1 in the range cm -1 and +/- 2 cm -1 in the range cm -1. In case that some of the bands are too intense, hence run off-scale, an additional IR spectrum should be run at a lower concentration. 5.4 Spectrum: The main bands should be indicated in the spectrum and a tentative assignment of the structurally most significant bands should be provided. A short summary of the results should be given. 6. NMR Spectroscopy

4 6.1 Identity of the substance: 6.2 Test conditions: spectrometer (name and model) operating temperature operating frequency in MHz (e.g. 200, 400 or 500 MHz) nucleus (e.g. 1 H, 19 F, 13 C 31 P, etc.) concentration of the test substance (e.g. 1 H-NMR: 1-10 mg and 13 C-NMR: mg) solvent (e.g. CDCl 3, DMSO, D 2 O, CD 3 CN etc.) instrument settings: acquisition, processing and plot parameters internal standard (e.g. TMS) 6.3. Measurement: The instrument sensitivity (signal/noise ratio) and resolution (line width of standard substance at mid-height) must be checked regularly and specified. The solvent used should not obscure important substance-related signals. 1 H-NMRs should be recorded between 0 and 10 ppm (in some cases 0-15 ppm, e.g. carbonic acids, aldehydes). TMS should be used as internal standard and set to 0 ppm. Each individual signal should be integrated. For clarification of overlapping signals, doublets, triplets, quartets, multiplets, should be expanded. If the structure contains e.g. OH-group(s) attached to CH- or CH 2 -, the prepared sample should be shaken in D 2 O and run again. 13 C-NMRs should be recorded between 0 and 250 ppm. TMS should be used as internal standard and set to 0 ppm. 13 C- 1 H-splitting should be suppressed by decoupling. In case of complicated structures, additional spectra, such as APT (Attached Proton Test), DEPT (Distortionless Enhancement by Polarisation Transfer), COSY (Correlated spectroscopy, 2D-NMR), NOESY (Nuclear Oberhauser and Exchange Spectroscopy, 2D-NMR) or others could be run. If the structure contains fluorine or phosphorus atoms a 19 F or 31 P-NMR can give additional useful information. 6.4 Spectrum: NMR spectra in the range as mentioned above and for further clarification expanded 1 H-NMR spectra should be submitted. The intensity of the signals should be as such that singlets, doublets, triplets etc. can be identified.

5 All signals should be indicated (preferred in ppm) and for 1 H-NMR spectra their integrated value should be given A list of signals with the integration and assignment of the signals All relevant information concerning interpretation of result In case where signals are missing an explanation should be given, e.g. due to complex structure or paramagnetic effect etc. 7. Mass spectroscopy 7.1 Identity of the substance: 7.2 Test conditions: Spectrometer (name and model) specification (low or high resolution) ionization mode: electron impact (EI) or chemical ionisation (CI), Desorption ionisation (laser desorption (LD), plasma desorption (PD), fast atom bombardement (FAB) or secondary ion mass spectroscopy (SIMS)) Characteristics of the source: ionizing energy (e.g. 70 ev) ionizing current ion accelerating voltage pressure, temperature sample inlet system: direct inlet, GC/MS interface, or LC/MS interface type of analyser (magnetic, quadrupole, etc) calibration measurements: name of reference 7.3 Measurements: The mass spectrum should be recorded from zero to the main fragment/molecular mass (m/z) of the tested substance or additional 20% of the substance s molecular mass. The relative abundance should be given from 0 to Mass spectrum: Mass of molecular ion, most important fragments, and typical background fragments should be indicated and fragments assigned. A possible fragmentation of the molecule should be given. In cases where isotopes, such as chlorine ( 35 Cl and 37 Cl, bromine ( 79 Br and 81 Br), sulphur ( 32 S, 33 S, 34 S), are present, the masses quoted should be those for the fragment with the most abundant isotope (e.g. for chlorine, 37 Cl). 8. High Performance Liquid Chromatography (HPLC)

6 8.1 Identity of the substance: 8.2 Test conditions: The analytical method should include: type of spectrometer column (type, length, diameter) temperature mobile phase concentration of standard solutions flow detection, eg. UV injection volume run time The analytical method should be validated for: repeatability of injections stability of the chromatographic system stability of the standard solutions linearity limit of detection 8.3 Measurement: The test substance should be run with different concentrations in order to show the response of the test substance as a function of its concentration. In addition to the test substance a blank solution should be run to show the linearity of the base line. Depending on the retention times of the substance and impurities the chromatogram should be run at least 5minutes longer than the retention time of the last signal. It should be noted components and impurities might have different response factors. This should be taken into account when calibrating. Sometimes it might be necessary to run measurements for the components and other s for the impurities in order to determine the percentage of each individual In case of stereoisomers, a reverse phase column should be used in order to separate the isomers. The measurement with a reverse phase column should be done in addition. 8.4 Results: The results of repeatability of injections, the stability of the chromatographic system, the stability of the standard solutions, the linearity and the limit of detection should be summarised in tables. A chromatogram of the blank solution, at least one chromatogram of the substance with a print-out of all integrated signals with their retention times and area

7 percentages and the graph of the regression line, hence the response of the substance as function of its concentration should be submitted. The substance and the main impurities should be indicated and if possible identified. 9. Gas Chromatography (GC) 9.1 Identity of the substance: 9.2 Test conditions: The analytical method should include: type of spectrometer column (type, length, diameter) detector, e.g. FID column temperature detector temperature injector temperature temperature program carrier gas flow internal standard injection volume run time The analytical method should be validated for: repeatability of injections stability of the chromatographic system stability of the internal standard solutions column resolution variation in the capacity factor linearity limit of detection 9.3 Measurement: The test substance should be run with different concentrations in order to show the response of the test substance as a function of its concentration. In addition to the test substance a blank solution should be run to show the linearity of the base line. Depending on the retention times of the substance and impurities the chromatogram should be run at least 5minutes longer than the retention time of the last signal.

8 9.4 Results: The results of repeatability of injections, the stability of the chromatographic system, the stability of the internal standard solution, the linearity and the limit of detection should be summarised in tables. A chromatogram of the blank solution, at least one chromatogram of the substance with a print-out of all integrated signals with their retention times and area percentages and the graph of the regression line, hence the response of the substance as function of its concentration should be submitted. The substance and the main impurities should be indicated and if possible identified. 10. Other techniques Depending on the nature of the substance the above mentioned methods might not always be the best practice. In case of inorganic substances other methods, such as x- ray diffraction or atomic absorption spectroscopy (AAS) might be more appropriate. Therefore other methods that might be more suitable to identify the substance can be used. These could be ion chromatography (IC), wet chemistry titration, gravimetrical analysis, elemental analysis and others that are not listed in this document. Please note that this document will not be automatically updated when a new method has been developed.