Biomedical Engineering Reference
In-Depth Information
sample and reduce matrix effects. Plasma and serum are the specimens of choice
in TDM environments, and it is therefore not surprising that the majority of the
published methods for the analysis of APs use plasma. In a postmortem setting,
whole blood is routinely used for analysis, as plasma cannot be obtained in the
majority of cases due to postmortem lysis and other forms of decomposition
[
38
]. One needs to be aware that once a method has been validated for use in a
particular matrix (e.g., plasma) it cannot be transferred to another matrix (e.g.,
whole blood) without sufficient cross-validation, as factors such as matrix effects
are likely to vary.
Whilst there has been a large number of single analyte procedures published over
the last decade, multianalyte procedures targeting APs are still limited. To the
authors' knowledge, only three currently published methods include at least 15 APs
[
39-
41
] . Kratzsch et al. [
40
] include 15 APs and three metabolites in their analytical
method using plasma. Method validation has been carried out; however, matrix
effects were not investigated in this method published in 2003, as they were not
considered an essential part of method validation at the time. Kirchherr et al. [
39
]
cover 22 APs and over 20 antidepressants in their LC-MS/MS method suitable for
detection and quantification of APs in serum. The only drawback of this compre-
hensive method is the use of therapeutic drugs (and metabolites) as internal standard
(IS). This is not good practice and can cause significant problems including overes-
timation of drug concentrations, which is discussed later in this chapter. A compre-
hensive method for the detection and quantification of APs was published in 2010
by Saar et al. [
41
]. This fully validated multianalyte procedure covers 30 APs. It will
therefore be used as an example method to highlight the important components of
AP analysis methods and potential pitfalls.
TDM laboratories largely focus on targeted APs and therefore only include
relevant APs of interest in their respective methods. However, in a patient-
population where noncompliance is a problem and polypharmacy is a common
occurrence, it is advisable to include more APs in the analytical methodology. In
the case of a prescribed drug not being present or at a subtherapeutic concentra-
tion, it may be helpful to ensure no other APs (or indeed other relevant drugs) are
present in the patient's sample.
Sample volumes ranging from 25 m L [
31
] at the lower end up to 1 mL of speci-
men are commonly used. Saar et al. [
41
] chose a volume of 100 mL as this provided
easy handling, and was likely to be still obtained in cases where only limited sample
volume was available but was also sufficient to reach the LLOQ for all drugs incor-
porated in the method.
An important step in AP analysis is appropriate sample preparation. Frequently
used extraction techniques for the analysis of APs include liquid-liquid extraction
(LLE) and solid-phase extraction (SPE). Whilst SPE procedures show the advan-
tage of being more automated and requiring less time of the analyst, this can be
outweighed by technical issues such as blockage, causing delays especially when
dealing with more complex matrices like postmortem whole blood. Less common
approaches include protein-precipitation [
39,
42,
43
] and direct injection [
27,
44
] .
In order to manage large volumes of samples, several authors describe sample
