27
Table 2. Calibration summary for all compounds in this application. The mean % deviation refers to the average of the absolute value of the deviations of all points in the curve.
Name
N-desmethyl zopiclone Zopiclone Zolpidem
7-aminoclonazepam Flurazepam
7-aminoflunitrazepam Chlordiazepoxide Midazolam
α-OH midazolam α-OH triazolam α-OH alprazolam Oxazepam Nitrazepam Lorazepam Clonazepam Alprazolam
Nordiazepam Flunitrazepam Temazepam Triazolam Diazepam
R2
0.999 0.998 0.999 1.000 0.998 0.997 1.000 1.000 0.999 1.000 0.999 1.000 0.999 0.999 1.000 0.998 0.999 0.999 0.999 0.999 0.999
Lin/Quad Mean %Dev L L
Q Q Q L
Q Q Q Q Q Q L
Q Q L
Q L
Q Q Q
5.4 5.4 4.1 2.3 4.1 6.2 3.4 4.8 4.0 4.4 9.0 6.2 4.6 4.4 6.2 9.9 6.6 3.9 5.3 4.1 3.7
Six of the curves were fitted with a 1/x weighted line, while 15 were best fit with a 1/x weighted quadratic curve. Figure 4 shows examples of compounds best fit with a line (nitrazepam, alprazolam), and a quadratic curve (diazepam, 7-aminoclonazepam). Regardless of the function used, the fits were excellent and meet the analytical needs of the method. Seventeen compounds had R2 or greater, and the remaining compounds had R2
values of 0.999 values
of 0.997 or greater. Table 2 also shows that the mean % deviations for all compounds from nominal values less than 10%. Tables 3 and 4 show the results of within-batch and between-batch QC results, respectively. The within-batch results show both excellent accuracy and precision. The mean accuracies for all compounds at the four QC levels were 107.8%, 98.5%, 97.5% and 97.5%. For the highest QC values (7.5, 75, and 300 ng/mL) all individual accuracies were within 10% of target values and all %CVs were less than 10%. The between-batch results shown in Table 4 were, if anything, even better. Mean accuracies were 102.1%, 99.3%, 98.2% and 96.8% at the four QC levels. Individual co-efficients of variation (CVs) ranged from 1.1% to 9.0%. These high levels of accuracy and precision demonstrate the consistency and reliability of the Oasis MCX sorbent and extraction technique, and demonstrate that there is no compromise of result quality, even with the in-well hydrolysis and direct sorbent loading used in this method. They also show that the quadratic curves used are fit for purpose and meet the needs of the method.
CONCLUSIONS:
Table 3. Within batch QC results. N=6. Mean values show the average accuracy for each compound and the average accuracy for all compounds at each QC level.
QC 1.5 Name
N-desmethyl Zopiclone Zopiclone Zolpidem
7-aminoclonazepam Flurazepam
7-aminoflunitrazepam Chlordiazepoxide Midazolam
α-OH midazolam α-OH triazolam α-OH alprazolam Oxazepam Nitrazepam Lorazepam Clonazepam Alprazolam
Nordiazepam Flunitrazepam Temazepam Triazolam Diazepam Mean
7.3% 7.4% 6.7% 8.1% 9.0%
QC 7.5 99.3%
10.9% 9.5% 9.9% 8.1% 9.2%
12.6% 6.3% 7.7% 7.2%
10.6% 10.0% 10.1% 8.2% 8.0% 8.4% 7.9%
100.6% 100.1% 96.5% 95.8% 95.8% 97.8% 98.3% 99.5% 95.1% 98.9% 94.6% 96.8% 95.5% 97.2% 99.3%
103.2% 101.4% 102.8% 103.4% 101.5% 98.7%
3.1% 3.2% 2.5% 2.6% 4.5% 4.5% 4.2% 2.3% 2.3% 2.3% 5.2% 3.2% 2.6% 2.0% 3.0% 5.7% 2.4% 2.4% 2.7% 2.5% 2.3%
A rapid and simplified solid phase extraction protocol and LC-MS/MS method for the analysis of urinary benzodiazepines and metabolites was demonstrated. Adopting this multi-analyte method provides quantitative
QC 75 99.8%
100.9% 96.8% 95.4% 96.0% 98.4% 98.5% 98.6% 99.0% 93.1% 94.1% 96.9% 97.0% 96.1% 95.4% 98.7% 99.2% 97.2% 98.5%
101.1% 97.3% 97.5%
2.0% 2.3% 0.9% 1.0% 2.1% 1.7% 1.0% 2.3% 1.6% 1.6% 4.5% 1.4% 0.8% 2.0% 2.0% 4.4% 1.6% 1.9% 1.4% 2.4% 0.8%
QC 300
Mean %CV Mean %CV Mean %CV Mean %CV Mean 103.6% 101.4% 102.7% 102.3% 111.0% 101.9% 100.7% 107.0% 107.4% 109.9% 114.5% 105.4% 108.8% 107.0% 106.7% 116.8% 110.9% 111.1% 110.6% 113.6% 110.3% 107.8%
98.2% 98.1% 93.4% 96.8% 99.2% 97.5%
100.3% 99.4%
101.1% 94.5% 95.4% 95.6% 98.2% 97.4% 94.6%
101.3% 96.3%
100.7% 95.4% 99.4% 95.3% 97.5%
3.9% 4.0% 4.2% 3.5% 4.7% 3.6% 6.1% 2.8% 3.8% 5.4% 8.3% 3.1% 3.5% 4.0% 3.8% 6.1% 3.0% 4.3% 6.0% 1.8% 3.6%
100.2% 100.3% 98.2% 97.8%
100.5% 98.4% 99.3%
100.8% 101.8% 98.1%
100.7% 98.1%
100.2% 99.0% 98.4%
104.0% 102.4% 102.6% 101.8% 104.4% 101.1%
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