1.2.5.
Specific performance criteria for the determination of an analyte using liquid chromatography with detection techniques other than mass spectrometry
For authorised substances only, the following techniques can be used as alternative for mass spectrometry based methods, provided that the relevant criteria for these techniques are fulfilled:
1.
full-scan diode array detection spectrophotometry (DAD) in case used with HPLC;
2.
fluorescence detection spectrophotometry (FLD) in case used with HPLC.
Liquid chromatography with UV/VIS detection (single wavelength) is not suitable on its own for use as a confirmatory method.
1.2.5.1. Performance criteria for full-scan diode array spectrophotometry
The performance criteria for chromatographic separation included in Chapter 1.2.3 shall be fulfilled.
The absorption maxima in the UV spectrum of the analyte shall be at the same wavelengths as those of the calibration standard in matrix within a maximum margin, which is determined by the resolution of the detection system. For diode array detection, this maximum margin is typically within ± 2 nm. The spectrum of the analyte above 220 nm shall, for those parts of the two spectra with a relative absorbance greater than or equal to 10 %, not be visibly different from the spectrum of the calibration standard. This criterion is met when firstly the same maxima are present and secondly when the difference between the two spectra is at no point greater than 10 % of the absorbance of the calibration standard. In the case computer-aided library, searching and matching are used, the comparison of the spectral data in the official samples to that of the calibration solution has to exceed a critical match factor. This factor shall be determined during the validation process for every analyte on the basis of spectra for which the criteria described above are fulfilled. Variability in the spectra caused by the sample matrix and the detector performance shall be checked.
1.2.5.2. Performance criteria for fluorescence detection spectrophotometry
The performance criteria for chromatographic separation included in Chapter 1.2.3 shall be fulfilled.
The selection of the excitation and emission wavelengths in combination with the chromatographic conditions shall be done in such a way to minimise the effects of interfering components in blank sample extracts. There should be a minimum of 50 nanometres between the excitation and emission wavelength.
The nearest peak maximum in the chromatogram shall be separated from the designated analyte peak by at least one full peak width at 10 % of the maximum height of the analyte peak.
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