Validated Method for Macrolide Antibiotics

Extraction of the analytes used an SPE procedure, and these water soluble compounds were obvious candidadtes for analysis by LC-ESI-MS/MS (positive ion mode was used). Considerable pains were taken (Yang 2006) to optimize these steps and these details are not repeated here. However it is worth pointing out that a 3D ion trap was used as the MS/MS detector, certainly advantageous from the viewpoint of capital cost but with the penalty of a much lower duty cycle (Section 6.4.5a) than a triple 

Another positive aspect was the explicit concern for confirmation of analyte identity that used the identification points (IPs) approach (European Contunission 2002, see discussion of Tables 9.1-9.3 in Section 9.3.3b). For low resolution MS/MS detection using an ion trap as was done here, one IP is awarded for the m/z selected precursor ion and 1.5 IPs for each MRM transition monitored, with three IPs required (European Commission 2002) for acceptable confirmation of analyte identity. To qualify for the IPs, at least one ion abundance ratio for two MRM product ions must be measured and fall within tolerance intervals of contemporary values measured for an authentic analytical standard, defined (European Commission 2002) as 20 % for a relative abundance of 50 %, to 25 % for 10-50 % and to 50 % for 10 %, although the US FDA recommends (FDA 2003) that these tolerances should be within 10% regardless of the absolute values of the relative abundances. The present work (Yang 2006) satisfied both tolerance criteria over the calibration range, although it was considered unlikely that they could be satisfied at the MDL. 

2b Extraction/Clean-up for Multi-residue Analysis of Compounds of Widely Different Polarity etc. 

While chromatographic separations and efficient ionization in the mass spectrometer do present difficulties for mixtures of widely different compound types, the most difficult problems in development of multiresidue methods usually concern the need for simultaneous extraction and clean-up of analytes with widely different polarities. With respect to multi-residue extraction and clean-up, SPE approaches (Section 4.3.3c) are the most popular these can use either a single SPE medium in a single extraction step or a combination of two SPE materials used either in series or in parallel, thus delivering the analytes as two or more groups defined by their physico-chemical properties relevant to the SPE process. 

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