Enzymatic assay of calcium in serum with phospholipase D.

(10) of 2.0 ␮g/L and Holt et al. (12) of Ͻ3.0 ␮g/L. HPLC-MS has no detectable interference with respect to endogenous compounds. The quantification limits based on acceptable imprecision, recovery, and ability to discriminate from potential interferences within the calibration range of the MEIA are 3.0 ␮g/L (the lower limit of quantification) to 22.0 ␮g/L. We have demonstrated that a higher limit of quantification by MEIA (150.0 ␮g/L) can be achieved through dilution, which hedges the HPLC-MS advantage of a wider quantification range. The sirolimus concentrations from a total of 125 blood samples obtained from 25 renal transplant patients receiving sirolimus therapy were measured by both methods and are shown in Fig. 1A. The range of whole blood sirolimus concentrations measured by HPLC-MS was 2.1–25.6 ␮g/L; by MEIA the range was 3.2– 41.6 ␮g/L. Regression analysis of the data yielded the equation for the line of best fit as: MEIA ϭ 1.39 (Ϯ 0.04) ϫ HPLC-MS ϩ 1.30 (Ϯ 0.46) ␮g/L (r ϭ 0.951; S y͉x ϭ 2.46; n ϭ 125). These data were further analyzed by the method described by Bland and Altman (13). A plot of the difference in sirolimus concentrations (MEIA minus HPLC-MS) against the mean sirolimus concentration as measured by both methods is shown in Fig. 1B. The line of best fit and 95% confidence intervals have been shown. The equation for the line of best fit is: MEIA Ϫ HPLC-MS ϭ 0.38 (Ϯ 0.03) ϫ mean concentration ϩ 0.36 (Ϯ 0.39) ␮g/L (r ϭ 0.779; S y͉x ϭ2.02). Based on the 95% confidence intervals, it can be determined that at mean concentrations of 10 and 30 ␮g/L, MEIA will give corresponding estimates of 10.1–18.6 and 37.8 – 46.2 ␮g/L, respectively. The mean bias expressed as a percentage Ϯ the SD between the two methods for renal transplant patient samples was 41.9% Ϯ 15.3%. Unlike HPLC-mass spectrometers, the IMx analyzer is a common instrument in clinical laboratories and is less labor intensive, with shorter sample preparation and analysis time. Thus, if the analytical performance in relation to sirolimus patient samples was similar between the methods, then MEIA could be used as an alternative to HPLC-MS. The acceptable imprecision and recovery data for HPLC-MS for the three quality controls confirm our previous validation (4). Similarly, MEIA had acceptable quality control imprecision and recovery data, which is consistent with reported MEIA studies that were based on a smaller …