Validation of a Quantitative Method

The most ideal internal standard is a stable isotope-labeled analog of the analyte because its chemical and physical properties are virtually identical to those of the analyte. Its cost can, however, be prohibitive. Internal standards labeled with H, 0, and Cl have been used in quantitative assays. The 

The use of deuterated internal standards has been reported for the quantification of endogenous peptides, prostaglandins, pharmaceutical drugs, and the drugs of abuse. In the quantification of methionine enkephalin [1,5-7] and P-endorphin [1,5-7], the deuterium was incorporated in the phenyl ring of the phenylalaline and Ile residues, respectively. The phenyl ring-deuterated IS was also used in the quantification of p.-opioid receptor agonist Tyr-D-Arg-Phe-Lys-NH2 from ovine plasma [8], 

The process of method validation (i.e., evaluation of the assay) affects the quality of the quantitative data directly [9 A Guide to Analytical Method Validation, Waters Corporation]. Through method validation, it is assured that the method developed is acceptable. Issues involved in the validation of a mass spectrometry method for quantitative analysis are similar to those in any other analytical technique. The validation involves undertaking a series of studies to demonstrate the limit of detection G OD) limit of quantitation (LOQ) linear range specificity within-day precision and accuracy and day-to-day precision and accuracy, specificity, and robustness of the method. All of these parameters must be determined with those commonly accepted good laboratory practices criteria that are applicable in the vafidation of analytical methods. 

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In a series of papers, the group of Volmer [130-132] studied the analysis of azaspiracid biotoxins. Ultrafast and/or high-resolution LC of azaspiracids on monohthic LC columns was evaluated [130]. Chromatograms of five azaspiracids on a 100-mm and a 700-mm monolithic column are shown in Figure 14.11. Fragmentation of azaspiracids in MS-MS on ion-trap and triple-quadrupole instruments was studied as well [131]. The interpretation was confirmed using accurate-mass data from a Q-TOF instrument. Validation of a quantitative method for AZA-1 was also reported [132]. The LOQ was 5 and 50 pg/ml extract using a triple-quadrapole in SRM mode and an ion-trap instrument, respectively. 

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