2015. 2. 11
이장우
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
 Matrix effect (ME), recovery (RE) of the extraction procedure, and overall
“process efficiency” (PE)
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)
3. Results
3. Results
3. Results
3. Results
3. Results
3. Results
3. Results
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
(11.6-23.8% and 4.6-11.3%, 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