LC-MS-based bioanalytical assays
REGRESSION, SEPARATION
The amount of nelfinavir in mouse lung homogenate and plasma was measured using an AB Sciex API 4000 Q Trap mass spectrometer (Toronto, Canada), interfaced with a Turbo V ion source and an Agilent 1290 Infinity II LC (Waldbronn, Germany). Ten volumes of methanol (three volumes of ethanol was used instead for rhesus macaques plasma samples obtained from SARS-CoV-2 model) were used to precipitate the samples, followed by a further centrifugation. With a 5 μm Agilent ZORBAX Eclipse Plus C18 column (50 mm × 2.1 mm i.d.; Santa Clara, CA, USA), chromatographic separation was accomplished using a mobile phase (delivered at 0.3 mL/min) that contained (A) water (containing 9 mmol/L formic acid) and (B) acetonitrile (containing 9 mmol/L formic acid). A 6-min gradient program that included 0–1.0 min, at 10% B; 1.1–2.5 min, at 65% B; 2.6–4.0 min, at 100% B; 8.5–10 min, at 100% B; and 4.1–6.0 min at 10% B was employed. The positive ion mode of operation was used for the mass spectrometer. Optimal precursor-to-product ion pairs for multiple reaction monitoring of nelfinavir and the internal standard reserpine were m/z 568 → 330 (collision energy, 45 V) and 609 → 195 (48 V), respectively. Weighted (1/X) linear regression of the peak areas (Y) of the analytes against the corresponding nominal analytes’ concentrations (X; 4.12, 12.4, 37.0, 111, 333, 1000, and 3000 nmol/L) was used to construct matrix-matched calibration curves with reference standards, and the curves demonstrated strong linearity (