A method for the determination of amitriptyline and its metabolites nortriptyline, 10‐hydroxyamitriptyline, and 10‐hydroxynortriptyline in human plasma using stable isotope dilution and gas chromatography‐chemical ionization mass spectrometry (GC‐CIMS)

Abstract

A gas chromatography‐mass spectrometry (GC‐MS) method has been developed to measure amitriptyline and its metabolites nortriptyline, 10‐hydroxyamitriptyline, and 10‐hydroxynortriptyline in human plasma. Deuterated analogs of each compound were synthesized as internal standards. Isobutane was used as both gas chromatography (GC) carrier gas and chemical ionization (Cl) reagent gas. In order to obtain compounds with satisfactory GC and mass spectrometry (MS) properties, the two alcohol metabolites were dehydrated without loss of label during sample preparation. Selective ion monitoring of the MH⁺ ions of the protio‐ and deuterio‐ compounds gave ion ratios which were converted to plasma concentrations using standard curves. For amitriptyline and nortriptyline, which are assayed using multiple deuterated analogs as internal standards, the curves are straight lines. For 10‐hydroxyamitriptyline and 10‐hydroxynortriptyline, which are assayed using monodeuterated analogs as internal standards, the curves are nonlinear and are analyzed using an iterative computer procedure. Assay sensitivity is 0.5 ng/ml for amitriptyline, nortriptyline, and 10‐hydroxyamitriptyline and 1 ng/ml for 10‐hydroxynortriptyline. Assay precision and accuracy in terms of percent error are both < 5%. Following oral administration of a single 75‐mg dose of amitriptyline to two subjects, the mean plasma levels of amitriptyline, nortriptyline, 10‐hydroxyamitriptyline, conjugated 10‐hydroxyamitriptyline, 10‐hydroxynortriptyline, and conjugated 10‐hydroxynortriptyline were 36, 8, 10, 66, 16, and 46 ng/ml, respectively, at 2 hr after dosing and 3, 4, 0.5, 1, 6, and 17 ng/ml, respectively, at 72 hr after dosing. Analyses of plasma samples from 12 subjects who had been receiving 50 mg amitriptyline therapy three times a day for an average ±SD of 32 ± 5 days gave a mean concentration of 81 ± 40 ng/ml for amitriptyline, 71 ± 57 ng/ml for nortriptyline, 12 ± 5 ng/ml for 10‐hydroxyamitriptyline, 91 ± 30 ng/ml for conjugated 10‐hydroxyamitriptyline, 82 ± 27 ng/ml for 10‐hydroxynortriptyline, and 176 ± 64 ng/ml for conjugated 10‐hydroxynortriptyline.