To specify the objective of an analytical method transfer.

1. TRANSFER OF A MULTIDIMENSIONAL ON-LINE SPE-LC-ECD METHOD FOR THE DETERMINATION OF THREE MAJOR CATECHOLAMINES IN NATIVE HUMAN URINE. E. Rozet 1, R. Morello 2, W. Dewe 3, P. Chiap 4, B. Boulanger 3, K. S. Boos 2, J. Crommen4 and Ph. Hubert 1 1 Laboratory of Analytical Chemistry and 4 Laboratory of Analytical Pharmaceutical Chemistry, Bioanalytical Chemistry Research Unit, ULg, B 36, B-4000 Liège, Belgium. 2 Laboratory of BioSeparation, Institute of Clinical Chemistry, University Hospital Grosshadern, D-81366 Munich, Germany. 3 Lilly Development Centre, Statistical department, rue Granbompre, 11, B-1348 Mont-Saint-Guibert, Belgium. TRANSFER SETUP OBJECTIVE Method transfer is the last step before implementation of the method in routine use at the receiving laboratory. The design used to assess the acceptation of the transfer must reflect the future use of the method. At the receiver the analytical method will be used in routine by only 1 operator (B) and on 1 LC equipment (II). Therefore this setup was used during the transfer study. Another point is the choice of the number of series and repetitions per series to perform in each laboratory. This choice is based on simulations using the results of the validation of the method (bias, RSD of repeatability and intermediate precision) made by the sender to achieve the best power for the transfer. Table 1, illustrates the experimental design applied in each laboratory. • To specify the objective of an analytical method transfer. • To recommend the use of a total error based criterion for the decision of accepting/rejecting an analytical method transfer. • To demonstrate the applicability of this decision rule with the real transfer of a multidimensional on-line SPE-LC-ECD method for the quantitative determination of norepinephrine (NE), epinephrine (E) and dopamine (DA) in native human urine. ANALYTICAL METHOD TRANSFER: DEFINITION AND OBJECTIVE • Definition: An analytical method transfer consists in transferring a previously validated analytical method from a sending laboratory (called sender) mastering this method, to a receiving laboratory (called receiver) after having experimentally demonstrated that the receiver also masters the method. • The Objective of a transfer is therefore: To provide users the guarantee that each measurement (xi) on unknown sample is close enough to the true value (mT). • Enough, means for example less than 15% away from sample true unknown value (l = acceptance limits, e.g. 5% (dosage form), 10% (impurities) or 15% (bio-analysis)). • Guarantee, means that it is very likely that whatever the measurements, it will be close enough from the unknown true value (1-β = risk), e.g. 5%, 20% or 33%. Table 1. Experimental design performed in both laboratories for the method transfer. The standards used to assess the transfer were prepared in native human urine at 3 concentration levels: 50, 250 and 500 µg/L. RESULTS Results of the transfer are shown in Figure 1 as risk profiles for NE, E and DA at each concentration level evaluated during method transfer. The transfer is rejected if the observed risk is above the maximum risk accepted. As can be seen, the risk is smaller than the maximum risk of 5% for all the catecholamines studied irrespective of the concentration levels. Therefore, the risk of having results out of specification during routine use of the method for the receiver is of maximum 2.5%, 3.4% and 3.1% for NE, E, DA respectively. TOTAL ERROR BASED APPROACH • A new total error based approach is proposed to correctly assess the acceptation of a transfer [1,2]. • Compute the confidence interval of the mean of the sender results: [LS;US]. • Compute the β-expectation tolerance interval of the receiver results: [LR;UR], i.e. the interval in which one can expect that at least a proportion β (e.g. 95%) of future individual results will lay. • Compute the decision interval: • Compute the probability P of having results falling outside the pre-specified acceptance limits λ and compare it with the maximum tolerated risk 1- β. • If P 1- β the transfer is accepted • If P >1- β the transfer is rejected Fig. 1. Transfer results. DESCRIPTION OF THE TRANSFERRED METHOD CONCLUSIONS The objective of an analytical method transfer is to provide users guarantee in order to minimize the risks to have future results out of specifications. The total error approach achieve this by computing the risk of having future individual results outside the acceptance limits. This new approach was applied to the transfer of a SPE-LC-ECD method for the quantitative determination of three major catecholamines in native human urine. The risk approach gives the guarantee that the receiver masters the analytical method and furthermore allow to manage the risks of having results out of specifications during routine use [3]. • The transferred method is an on-line SPE-LC-ECD method for the quantitative determination of NE, E and DA in native human urine. • Mobile phases: • Sample clean up: 0.2M (NH4)2HPO4, 3.72g/L EDTA, methanol, 95/5, v/v, pH 8.7 • Chromatographic separation: 50mM KH2PO4, 2.5mM sodium octanesulfonate, 0.1g/L EDTA, acetonitrile, 96.5/3.5, v/v, pH 3.5 • SPE column: Restricted Access Material SPE column modified with • nitrophenylboronic acid as affinity ligand. • Analytical column: Zorbax bonus RP-C18 (150 x 4.6 mm i.d., 5 µm particle • size) • Temperature: 30°C • Detection: ECD at 600 mV AKNOWLEDGMENTS Research grant from the Walloon Region and the European Social Fund to one of the author (E. Rozet) is gratefully acknowledged (First Europe Objective 3 project n°215269). REFERENCES PRE-SPECIFIED PARAMETERS [1]. Dewé, W. and al. Chemom. Intell. Lab. Syst. Accepted for publication. [2]. Rozet E. and al. J. Pharm. Biomed. Anal. In press. [3]. FDA, Process Analytical Technology (PAT) Initiative, 2004. • Maximum risk tolerated: 1- β = 5% • Acceptance limits: l = ± 15%