The Second SeaWiFS HPLC Analysis Round-Robin Experiment (SeaHARRE-2)

Abstract

Eight international laboratories specializing in the determination of marine pigment concentrations using high performance liquid chromatography (HPLC) were intercompared using in situ samples and a variety of laboratory standards. The field samples were collected primarily from eutrophic waters, although mesotrophic waters were also sampled to create a dynamic range in chlorophyll concentration spanning approximately two orders of magnitude (0.3 25.8 mg m-3). The intercomparisons were used to establish the following: a) the uncertainties in quantitating individual pigments and higher-order variables (sums, ratios, and indices); b) an evaluation of spectrophotometric versus HPLC uncertainties in the determination of total chlorophyll a; and c) the reduction in uncertainties as a result of applying quality assurance (QA) procedures associated with extraction, separation, injection, degradation, detection, calibration, and reporting (particularly limits of detection and quantitation). In addition, the remote sensing requirements for the in situ determination of total chlorophyll a were investigated to determine whether or not the average uncertainty for this measurement is being satisfied. The culmination of the activity was a validation of the round-robin methodology plus the development of the requirements for validating an individual HPLC method. The validation process includes the measurements required to initially demonstrate a pigment is validated, and the measurements that must be made during sample analysis to confirm a method remains validated. The so-called performance-based metrics developed here describe a set of thresholds for a variety of easily-measured parameters with a corresponding set of performance categories. The aggregate set of performance parameters and categories establish a) the overall performance capability of the method, and b) whether or not the capability is consistent with the required accuracy objectives.