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Surrogate matrix: opportunities & challenges for tissue sample analysis Review


(Equation 1, Box 1). The other method requires deter- mining the responses of extracted incurred tissue or control matrix in addition to the pre- and postsamples. Recovery is calculated by subtracting the response of analyte in the tissue or control matrix from that of pre- and postsamples first and then comparing the responses (Equation 2, Box 1). Since the amount of analyte in the tissue or control matrix is inherent to the sample and not spiked into the sample, one can argue that Equation 2 generates more accurate recovery results [13]. Of course, if there is no inherent amount of analyte in the tis- sue or control matrix, then, the Equation 2 becomes Equation 1. The %RE are calculated for analyte and IS separately using peak area or peak height [10,13,16,23,26] as well as peak area ratios [21,22]. The %PE can be calculated using peak response


or concentration (Equations 3 & 4 in Box 1, respec- tively) [23,26,53]. Both calculations have approximately equal occurrences.


Matrix effects Matrix effects in both surrogate and in tissue should be evaluated because they can be different. The impact of either signal suppression or enhancement to the quan- titation needs to be assessed. This principle is dem- onstrated by the case of quantitation of tacrolimus in human kidney biopsy using rat kidney as surrogate [25]. The analyte showed about 19% signal enhancement in human kidney. Luckily, the IS also showed a signal increase which led to acceptable results. For qualitative evaluation, postcolumn infusion


is used to ensure the absence of matrix effects at the retention time of the peak of interest [3,10,27,54]. For quantitative determinations, the postextraction spike method is used, with which the response of postextrac- tion spiked sample is compared with that of neat solu- tion (Equation 5, Box 1). The quantitative determina- tion of matrix effects is often done at 3 concentrations with 3–6 replicates at each concentration [4,14,22,28]. To understand lot-to-lot variations, the matrix effects are evaluated using two or more lots of tissues [22]. Two variations of Equation 5 were reported for con-


veniently determining matrix effects when solvent is used as surrogate matrix. In variation 1, standards are prepared in neat solution and QC samples are prepared in tissue. The accuracy of QC samples is indicative of matrix effect. This experiment combines the precision and accuracy assessment with the matrix effect assess- ment. In variation 2, the calibration curves are pre- pared using solvent surrogate and reconstituted matrix extract, the matrix effects are determined by statistically comparing the slopes of the standard curves [17,29]. This approach is the same as described earlier as method 1 for validating the suitability of surrogate matrix, except


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it calculates the matrix effects by dividing the slope of matrix curve by that of solvent curve.


Stability Due to restrictions of time and resource, comprehensive stability evaluation is not always possible. In fact, it is relatively rare. More often than not, only a few selected kinds of stability are evaluated. In many papers, sta- bility evaluations are not mentioned [10–11,17,24–26,28–29]. In some cased, the stability for plasma is evaluated but not for tissue [4]. An example of rather exten- sive stability evaluation can be found in works of Teunissen et al. [27]. Analyte stability in surrogate and tissue samples


can be different [13,16]. Schmidt et al. find retinoic acid (RA) is more subject to isomerization in real tissue samples than in surrogate, which is a mixture of meth- anol and phosphate buffered saline (PBS) buffer [16]. In their case, the difference is relatively small, about 5%. Nevertheless, it illustrates analyte stability difference between surrogate and tissue can lead to bias during quantitation. Table 3 lists typical types of stability for a surrogate


matrix tissue method. They can be divided into four categories: stability in solution (stock and working solution), stability in surrogate and tissue homogenate, stability in extract (for both surrogate and tissue) and stability in tissue. In most literature, the acceptance criteria for stability are not mentioned, except for one group, who considered the analyte is to be stable if the analyzed results are within the prescribed accu- racy (85–115% of the nominal value) and precision (15% RSD) limits [3]. For stability in surrogate and tissue homogenates,


only stability in tissue homogenate (i.e., authentic matrix) needs to be evaluated if standards (in surro- gate matrix) are prepared fresh. Since preparing fresh standards for every analysis is not desirable, stability in surrogate matrix is often evaluated, also. In principle, the stability of both in surrogate and in tissue homog- enate should be evaluated as they can be different [7,16]. In practice, some evaluated stability in both [7], but others only evaluated in either surrogate or in tissue homogenate [3,6]. For stability in tissue homogenate, both spiked QC samples and real samples have been used [22,23]. In addition to the bench-top, freeze–thaw and long-term stability, photo and thermal stability in surrogate and tissue homogenate may also need to be evaluated if the analyte is suspected of instability under those conditions. An example can be found in the work of Neuschwander-Tetri and Roll, who evaluated the photo and thermal stability of glutathione derivative in liver homogenate under dark condition at 5 and 25°C and under light condition at 25°C [53].


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