# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 2 Quantification of Total MC by the MMPB Method
### Limitations and Difficulties Faced during the MMPB Method
#### 2.5.4 Quantification Strategy (Internal vs. External Calibration)  
Different quantification strategies have been set up for the analysis of total MCs after Lemieux oxidation, attempting to compensate for the matrix effects that can be more or less impactful depending on the type of matrix or the analytical instrument used. [PERSON] et al. [87] already listed some of the studies on the MMPB method, including the quantification strategy and the use of an internal standard. This inventory was extended with the objective of gathering studies using the same strategy (or a similar one). The first quantification strategy adopted in MMPB method studies corresponds to an external calibration. The quantification of the MMPB in samples can be performed using an MMPB calibration curve made of an MMPB standard [5,67,29]. Another possibility is using an MMPB calibration curve generated by the oxidation of an MC-LR standard [81]. The disadvantage of these first two strategies is that they do not take into account the presence of the matrix in the samples and, therefore, the matrix effects. Moreover, generating an MMPB standard curve after the oxidation of the different MC-LR concentrations adds an additional bias related to the oxidation efficiency. More common in the studies reviewed is the use of an external matrix-matched calibration. [PERSON] et al. [77], for example, chose to spike the matrix with MMPB after the pH adjustment step, while other studies preferred to use an in situ (here in the sense of calibration curve prepared in the matrix) matrix-matched calibration curve of MC-LR-fortified samples that subsequently went through the oxidation process to generate an MMPB matrix-matched calibration curve [60; 71; 86; 93; 94]. Finally, one study used the standard addition approach, consisting of spiking the analyzed samples at three MC-LR levels prior to oxidation, to quantify the MMPB in the liver samples [78]. Another strategy is to use an internal calibration via an internal standard (IS). For MMPB analysis, two IS can be used, 4-phenylbutyric acid (4-PB) [66; 79; 90] and isotopically-labeled MMPB (d3-MMPB) [84; 88]. The first factor of choice between the two IS is the availability of the product; indeed, while 4-PB is easily available, d3-MMPB is more expensive and more difficult to obtain. On the water matrix, [PERSON] et al. [64] provided a second factor of choice by comparing the deviation between surface water samples and reference HPLC-water samples, both oxidized and spiked with either 4-PB or d3-MMPB. They observed a deviation of 14% for 4-PB and of around 5% for d3-MMPB, concluding that these IS allow a sufficient correction of the matrix effect whatever the IS used. A different quantification comparison was performed by [PERSON] et al. [87] on a fish matrix between a calibration curve using an MMPB standard (not generated from MCs) and an in situ MMPB generated matrix-matched standard curve, both using d3-MMPB as IS. After 2 h of oxidation, the recovery of d3-MMPB was much higher when calculated using the standard curve prepared with the MMPB standard (78.9%) rather than with the generated MMPB (30%), resulting in a lower relative response (MMPB/d3-MMPB) than with a calibration curve using MMPB standard. Thus, according to the tests performed in this study, the MMPB calibration curve generated in situ was three times lower than the one using the MMPB standard. The consequence is an underestimation of the amount of MMPB present in a sample when the curve that is generated in situ is used. It is therefore strongly recommended to use an IS to avoid any bias in the results of the analysis. Some studies also use a dual approach with external and internal quantification [63; 70; 72]. In [PERSON] and [PERSON] [63], for example, the combination of a standard curve of oxidized MC-LR and 4-PB as IS was used for quantification.