Chemistry Reference
In-Depth Information
solutions. Stock solutions should be prepared in an appropriate solvent at known
concentrations. The stability of stock solutions should also be ascertained at room
temperature over at least 6 hours, and storage-condition stability (e.g., in a refrigera-
tor) should be evaluated as well. In addition, because samples commonly will be
left on a bench-top or in an autosampler for some period of time, it is also impor-
tant to establish the stability of processed samples (e.g., drug and internal standard
extracted from sample matrix) over the anticipated run time for the batch of samples
to be processed. Working standards should be prepared from freshly made stock
solutions of the analyte in the sample matrix. Appropriate standard operating proce-
dures (SOPs) should be followed for the experimental studies as well as the poststudy
statistical treatment of the data.
The FDA guidelines recommend a minimum protocol that includes freeze and
thaw stability plus short- and long-term temperature stability. For freeze-thaw sta-
bility, three spiked-matrix sample aliquots at each of the low and high concentrations
should be exposed to three freeze-thaw cycles. The samples should be kept at the
storage temperature for 24 h and then thawed at room temperature (without heating).
When completely thawed, the samples should be refrozen for 12 to 24 h, then thawed
again; this procedure is repeated a third time. Analysis of the sample then proceeds
after completion of the third freeze-thaw cycle.
For short-term temperature stability, three aliquots (at each of the low and high
concentrations) are thawed and kept at room temperature for a period of time that
is equal to the maximum time (e.g., 4-6 h) the samples will be maintained at room
temperature prior to their analysis.
The storage time for a long-term stability evaluation should bracket the time
between the first sample collection and the analysis of the last sample (often 12
months or more); the sample volume reserved should be sufficient for at least three
separate time points. At each time point, at least three aliquots (at each of the low
and high concentrations) stored under the same conditions as the study samples (e.g.,
−20°C or −70°C) should be tested. In a long-term stability study, the concentration of
the stability samples should be determined using freshly made standards. The mean
of resulting concentrations should be reported relative to the mean of the results from
the first day of the study.
7.5.3 r
outIne
A
PPlIcAtIon
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b
IoAnAlytIcAl
m
ethod
Once the bioanalytical method has been validated for routine use, system suitabil-
ity and QC samples are used to monitor accuracy and precision, and to determine
whether to accept or reject sample batches. QC samples are prepared separately and
analyzed with unknowns at intervals according to the number of unknown samples
for a sample batch. Duplicate QC samples (prepared from the matrix spiked with
the analyte) at three concentrations (low, near the LLOQ, midrange, and high) are
normally used. The minimum number of QC samples (in multiples of three—low,
midrange, and high concentration) is recommended to be at least 5% of the number
of unknown samples, or six, whichever is greater. For example, if 40 unknowns are
to be analyzed, 40 × 5% = 2, so 6 QCs are run (2 low, 2 midrange, 2 high); or for 200
samples, 200 × 5% = 10, so 12 QCs are run (4 each, low, midrange, and high). At
