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Calibration curves were made to determine method linearity by using analyte-spiked charcoalated plasma samples. A blank sample, a zero sample (blank + IS) and 10 non-zero concentrations were prepared to construct each calibration curve. Each back calculated was at a standard concentration with ± 15% deviation from the nominal value except at LLOQ which was set at ± 20% for acceptance.

Two zero samples were injected directly after injecting an HQC sample to check the carry-over. The response of the first zero sample was required to be <20% of the response of a processed LLOQ sample to conclude no carry-over.

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