Biomedical Engineering Reference
In-Depth Information
analysis, testing results (purity or strength, moisture, impurities, etc.), storage
conditions, and expiration or retesting date. More detailed information on reference
standard can be found in USP34-NF29 and International Organization for
Standardization (ISO) Guides 30-35.
4.3
Standardization of Methodology
In general, the analytical methodology evaluates and defines bioanalytical/analyti-
cal procedures using a number of parameters, including selectivity or specificity,
accuracy, precision, matrix effect, recovery, calibration model (linearity) and range,
sensitivity (limit of quantitation—LOQ, and limit of detection—LOD), sample sta-
bility, ruggedness and robustness [
87
]. The USP and ICH guidelines listed in Table
4
focus on the role of analytical methodology in quality control and compliance of
drug substances and drug products in pharmaceutical industry. The IUPAC, ISO,
and NIST 1297 guidelines emphasize on the definition and evaluation of trueness
and uncertainty in analytical methodology. The FDA guideline is the most direct
guidance on bioanalysis for human and nonhuman (animal and biological) studies
in pharmacology, toxicology, pharmacokinetics, and drug metabolism. Typical bio-
analytical methodology guidelines and LC-MS/MS methods for steroid hormone
analyses are shown in Table
6
.
To standardize the steroid hormone analyses, standard operation procedures
(SOPs) should be generated for all laboratory functions, e.g., facilities, instrument,
reference standards, samples, procedures, data collection and processing, documen-
tation, etc., following GLP, reference standard, and the methodology guidelines. All
the laboratory activities should follow these SOPs. Furthermore, the experimental
procedures or techniques, e.g., deconjugation, extraction (LLE or SPE), derivatiza-
tion, isotope dilution, instrument setting (e.g., LC-MS/MS modes), etc., should also
be standardized. It is a challenge to ask different laboratories meet the same criteria,
e.g., sensitivity and precision, using different models of instruments, and to make
the results comparable and reliable for clinical diagnosis, due to the significant dif-
ferences in LC-MS/MS hardware (configuration, ionization modes, and parameter
setting) and software (data acquisition speed and processing) from different vendors
and different models.
Standardization of GC-MS methodology for steroid hormones is relatively
straightforward, because most of the unconjugated steroid hormones have a hydroxyl
group(s) at C
3
, C
11
, C
17
, or C
21
position, which may be easily derivatized with a trim-
ethylsilylation agent, e.g., MSTFA, and derivatives may be well separated by GC,
and detected by MS using EI
+
mode [
6
]. Similarly, standardization of LC-MS/MS
methodology for estrogens and metabolites is also not very complicated, because all
unconjugated estrogens and metabolites have phenolic hydroxyl group(s) at C
3
, and
C
2
or C
4
positions, which may be derivatized with a sulfonyl chloride, e.g., dansyl
chloride, and analyzed LC-MS/MS using ESI
+
[
8,
84,
88
] . The other derivatization
agents, e.g., hydrazide and hydroxylamine, may react only with those ketolic
