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Method Validation
Accuracy and precision were tested by using five replicates
of three levels of quality controls over four days and
quantitating them using calibration curves at the
beginning and end of the batch run. The fourth day of
accuracy and precision was performed in real urine to
cross-verify the use of real matrix. Carryover was
calculated by dividing the total analyte signal of the lower
limit of quantitation (LLOQ) by the total analyte signal
found in the matrix blank after the upper limit of
quantitation (ULOQ). This number could not exceed 20%
of the total LLOQ signal. Additionally, autosampler
stability (24 hours at 4 °C) was determined by running
QC samples that were refrigerated overnight in the
autosampler to a new calibration curve the following day.
Results and Discussion
The assay precision had %RSD values that were within
20.0% at the LLOQ and low QC, and within 15.0% for
all other QC and calibration standard levels. Additionally,
accuracy was within 20.0% at the LLOQ and low QC,
and within 15% for all other QC and calibration standard
levels. All of these results are shown in Table 3.
The short 4.25 minute analytical method provided ample
resolution for all isobaric compounds. All the analytes
passed acceptance criteria for carryover, matrix effects,
and autosampler stability. Example chromatograms for
each of the compounds are shown in Figure 2.
Table 3. Accuracy and precision results
Analyte
Accuracy
Precision (%RSD)
Intra-Assay
Inter-Assay
Normorphine
94.6
<14.3
<5.7
Dihydromorphine
102
<14.1
<8.2
Morphine
99.2
<8.8
<4.8
Oxymorphone
103
<10.3
<3.5
Hydromorphone
102
<14.1
<5.8
Norcodeine
98.6
<9.6
<4.1
Dihydrocodeine
99.5
<11.1
<5.3
Codeine
99.2
<13.6
<5.7
Norhydrocodone
98.2
<13.5
<9.2
Oxycodone
99.4
<14.1
<5.8
Noroxycodone
100
<11.6
<10.4
Hydrocodone
95.2
<7.4
<5.0
6-Acetylmorphine
103
<9.7
<4.4
Codeine 6B glucuronide
102
<8.5
<4.1
Oxymorphone 3B glucuronide
100
<14.4
<4.4
Hydromorphone 3B glucuronide
108
<7.9
<5.7
Morphine 3B glucuronide
98.5
<14.9
<4.1
Morphine 6B glucuronide
99.0
<10.8
<3.7
6-Acetylcodeine
102
<6.1
<6.9