Biomedical Engineering Reference
In-Depth Information
precise and true. Only the smallest possible laboratory errors—negligible imprecision
and small inaccuracy—permit dosage adjustments at a narrow therapeutic target
range. When systematic (e.g. calibration bias) and intra-individual (biologic) variabil-
ity are added to this measurement dispersion entirely common in the practice, analysis
platforms of this kind are no longer able to meet clinical requirements [ 63, 64 ] . The
fact that modern therapeutic schemes (e.g. combination therapies, dosage reduction in
cases of partial immunologic tolerance) [ 65, 66 ] mean an additional demand to mea-
sure lower drug levels more correctly than in the past, represents a challenge to be met
by all commercial providers and manufacturers of “in-house assays” alike.
During the last years two distinctly different LC-MS/MS instrument con fi gurations
have emerged as suitable for immunosuppressant TDM. In each case sample prepa-
ration by precipitation of cellular components and serum proteins precedes chro-
matographic analysis. Similar to some immunoassays, a mixture of an aqueous zinc
sulphate solution with methanol is used in many cases. The subsequent LC separa-
tion of the analytes also serves to purify the sample separating the analytes from
hydrophilic non-precipitated matrix components (e.g. zinc sulphate) as well as from
lipophilic matrix components (e.g. phospholipids). Sufficiently good assay results
can be achieved with only one single, simple chromatographic step (LC-MS/MS) as
long as important details are taken into account.
As stated further above, under all circumstances trueness, accuracy and sensitiv-
ity of the assay should be demonstrated on a sufficient number of patient samples.
In our view, however, the use of an additional, also easily realizable chromato-
graphic dimension (online-SPE-LC-MS/MS) [ 67, 68 ] without a doubt represents
the analytical “state of the art” in immunosuppressant TDM. Nowadays tandem MS
instruments are used almost exclusively for the detection and quantification of ana-
lytes. The detection of analytes is generally performed in the “selected reaction
monitoring” (SRM, synonym MRM) mode. Depending on which instrumentation is
used, an analysis can be completed within two to four minutes.
In spite of all the efforts with assay establishment described in the previous para-
graphs and even taking into account the limitation of the method LC-MS/MS is an
entirely valuable alternative to immunoassays. The statistics of the UK-NEQAS
proficiency testing (PT) scheme ( http://www.bioanalytics.co.uk ) , the largest and
most significant PT scheme in immunosuppressant TDM, show that there are at
least one hundred operational and active LC-MS/MS installations, corresponding to
20-50 % of the respective total [ 69 ]. The extraordinarily high number of challenges
(per year 12 for cyclosporine, tacrolimus and sirolimus, 6 for everolimus, 4 for
MPA) and the regular inclusion of patient samples (pool samples) make the
UK-NEQAS results a valuable database to allow a critical evaluation of the capa-
bility of the analysis platforms. Since in the PT evaluation the current immunoas-
says as well as the LC-MS/MS platforms are gathered in separate sub-groups, such
comparisons can be easily performed. The trueness analysis (comparison to the
weighted analyte amount in standard addition samples or comparison to substance-
specific measuring LC-MS/MS group) as well as the analysis of dispersion within
one group (assessment of assay inaccuracy) permit valuable conclusions on the
capability of measurement systems under routine conditions.
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