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1. Introduction  HPLC-MS/MS methodology achieved its preferred status -Highly selective and effectively eliminated interference -Without cleanup or extraction, no separation, not detect impurities. -Short run time and determination of analytes in complex biological matrixes.  Reliability of quantitative assay and integrity of data may not be absolute. -Lack of selectivity due to suppressions caused by matrix effect, interferences, cross-talk effect -Coeluting, matrix components may reduce or enhance the intensity -Subjects, sampling time

1. Introduction  Need for careful assessment of HPLC-MS/MS assay -But, not sufficiently studied and U.S.FDA do not provide a guidance of matrix effects. -Therefore, a need exists to develop an experimental protocol to demonstrate during assay development and validation the absence or presence of matrix effect in a newly developed bio-analytical method and use this information as guidance -To address this issue, a detailed comparison of the matrix effect under otherwise the same sample extraction and HPLC conditions was made using a heated nebulizer (HN) versus ion spray (ISP) interface.

1. Introduction  Matrix effect -It may originate from the competition between an analyte and the coeluting, undetected matrix components reacting with primary ions formed in the HPLC -MS/MS interface. -To determine an analyte by HPLC-MS/MS, the uncharged molecules of this analyte need to be transformed to ions that are later analyzed by MS/MS according to their mass-to-charge (m/z) ratios. -The rate and efficiency of these reactions are highly dependent on the relative ionization energies, proton affinities, or both of the molecules present in the “reactor” at any given time.

2. Experimental section -Materials, Instrumentation, Standard solutions, Chromatographic conditions, HPLC-MS/MS conditions  Sample preparation -Set 1 : prepared to evaluate the MS/MS response for neat standards of two analytes (1 and 2) injected in the mobile phase. -Set 2: prepared in plasma extracts originating from five different sources and spiked after extraction. -Set 3: prepared in plasma from the same five different sources as in set 2, but the plasma samples were spiked before extraction.

2. Experimental section  Precision, Accuracy, and Recovery -Precision : determined by the replicate analyses (n = 5, set 3) -Accuracy : [(mean observed concentration)/(spiked concentration] X 100] -Recovery : comparing the mean peak areas of 1 and 2 obtained in set 3 to those in set 2.  Matrix effect -Results  Selectivity -The “cross-talk” between MS/MS channels used for monitoring 1 and 2 for both analytes was assessed.

3. Results  Three types of system evaluation -Set 1 : good insight into the overall HPLCMS/MS system reproducibility in measuring the absolute peak areas on consecutive injections, the performance of the detector, and the chromatographic system as a whole. -Set 2: indicative of an effect of sample matrix since analytes at the same concentrations were spiked into plasma extracts. -Set 3: reflect a combined effect of a sample matrix and potential differences in recovery of analytes from different plasma lots.

3. Results neat solution standards in set 1 as A, the corresponding peak areas for standards spiked after extraction into plasma extracts as B (set 2), and peak areas for standards spiked before extraction as C (set 3)  Matrix effect (ME), recovery (RE) of the extraction procedure, and overall “process efficiency” (PE)

3. Results

4. Discussion -The overall precision and accuracy of any bioanalytical method, as determined usually using experimental data obtained in set 3, are dependent on many factors including the overall performance of the chromatographic system, reproducibility of the detector response, reproducibility of sample preparation procedures, consistency of recovery of analytes from different sources of a biofluid, and, finally, absence of a matrix effect on the quantification.

4. Discussion  Matrix effect -Absolute matrix effect, calculated according to eq1 -A value of >100% indicates an ionization enhancement and a value of <100% indicates an ionization suppression. -NH (APCI) < ISP (ESI) -Relative matrix effect, calculated according to eq1 with ratio -CV(%), may may be considered as a measure of the relative matrix effect -NH (APCI) < ISP (ESI)

4. Discussion  Matrix effect -Variable recovery contributions may be assessed by comparing the overall CVs of the 1/2 ratios (set2, 3) -If the range of these two sets of values is similar, the variability in the recovery on the overall method precision may be considered negligible. -Set 2 > Set 3 : variability in recovery, matrix effect -Set 3> Set 2 : variability in recovery

4. Discussion  Recovery vs Process efficiency -Recovery being determined as a value of (C/A X 100) depicted in eq 3. -However, the recovery calculated from eq 3 may not be correct since it does not take into account the matrix effect -Therefore, the recovery (RE) should be determined as a ratio of (C/B X 100) (eq 2), a “true” recovery value that is not affected by the matrix. -NH (APCI) > ISP (ESI)

4. Discussion  Method validation and Matrix effect -The reason for this significant method imprecision (Figure 4) was the presence of high relative matrix effect when the ISP interface was employed, as indicated by the high CV values for peak areas of 1 and 2 in five different plasma lots ( % and %, respectively, Table 4, columns B and F) -The CV values for sets 2 and 3 were high and comparable (14.9 and 13.2%, Table 3, columns B and C), indicating that the overall high variability of the method was due to the matrix effect rather than any potential differences in recoveries between different plasma lots for both 1 and 2.

4. Discussion  Elimination of Matrix Effect -Changing and improving sample extraction procedure and by eliminating undetected matrix interferences -Performing the assay under more efficient chromatographic conditions to separate analytes of interest from undetected endogenous compounds that may affect the efficiency of ionization of analytes -Evaluating and changing the HPLC-MS interface and the mechanism of ionization of analytes