Development and validation of a liquid chromatography coupled to a diode array detector (LC-DAD) method for measuring mitotane (DDD) in plasma samples.

INTRODUCTION: Mitotane (o,p'-DDD) is the drug of choice for Adrenocortical Carcinomas (ACC) and its measurement in plasma is essential to control drug administration. OBJECTIVE: To develop and validate a simple, reliable and straightforward method for mitotane determination in plasma samples. METHOD: Drug-free plasma samples were collected in potassium-ethylenediamine tetraacetate (K-EDTA) tubes and spiked with 1.0, 2.5, 10.0, 25.0 and 50.0 µg/mL of mitotane (DDD). The p,p'-DDD was used as an Internal Standard (IS) and was added at 25.0 µg/mL concentration to all samples, standards and controls. Samples were submitted to protein precipitation with acetonitrile and then centrifuged. 50 uL of the supernatant was injected into an HPLC system coupled to a Diode Array Detector (DAD). DDD and IS were detected at 230 nm in a 12 min isocratic mode with a solvent mixture of 60 % acetonitrile and 40 % formic acid in water with 0.1 % pump mixed, at 0.6 mL/min flow rate, in a reversed-phase (C18) chromatographic column kept at 28°C. The sensitivity, selectivity, precision, presence of carry-over, recovery and matrix-effect, linearity, and method accuracy were evaluated. RESULTS: The present study's method resulted in a symmetrical peak shape and good baseline resolution for DDD (mitotane) and 4,4'-DDD (internal standard) with retention times of 6.0 min, 6.4 mim, respectively, with resolutions higher than 1.0. Endogenous plasma compounds did not interfere with the evaluated peaks when blank plasma and spiked plasma with standards were compared. Linearity was assessed over the range of 1.00-50.00 µg/mL for mitotane (R2 > 0.9987 and a 97.80 %‒105.50 % of extraction efficiency). Analytical sensitivity was 0.98 µg/mL. Functional sensitivity (LOQ) was 1.00 µg/L, intra-assay and inter-assay coefficient of variations were less than 9.98 %, and carry-over was not observed for this method. Recovery ranged from 98.00 % to 117.00 %, linearity ranged from 95.00 % to 119.00 %, and high accuracy of 89.40 % to 105.90 % with no matrix effects or interference was observed for mitotane measurements. Patients' sample results were compared with previous measurements by the GC-MS method with a high correlation (r = 0.88 and bias = -10.20 %). CONCLUSION: DDD determination in plasma samples by the developed and validated method is simple, robust, efficient, and sensitive for therapeutic drug monitoring and dose management to achieve a therapeutic index of mitotane in patients with adrenocortical cancer.

Development of a high-performance liquid chromatography method with fluorescence detection for the routine quantification of tamoxifen, endoxifen and 4-hydroxytamoxifen in plasma from breast cancer patients.

To date, several methods for the quantification of tamoxifen and its metabolites have been developed, most of which employ liquid chromatography tandem-mass spectrometry (LC-MS/MS). These methods are highly sensitive and reproducible, but are also time-consuming and require expensive equipment; one of their main disadvantages is matrix ionization effects. A more viable option, particularly in developing countries, is high-performance liquid chromatography coupled with UV or fluorescence detection. We developed and validated a method for simultaneous quantification of tamoxifen, endoxifen and 4-hydroxytamoxifen based on high-performance liquid chromatography with fluorescence detection in a reverse-phase column. The method is rapid (16 min plus 5 min of column re-equilibrium), accurate (80-100%) and precise (0.23-6.00%), and does not require any additional irradiation process. Sample pretreatment consists of protein precipitation with acetonitrile under alkaline conditions, employing only 200 µL plasma. The validated method's wide range allowed quantification of steady-state levels in patients under standard tamoxifen treatment (20 mg/day). This assay is ready for application in clinical studies and routine quantification of tamoxifen, endoxifen and 4-hydroxytamoxifen in healthcare institutions.

Sensitive HPLC-PDA determination of tamoxifen and its metabolites N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma.

A highly sensitive HPLC-UV method for the simultaneous determination of tamoxifen, N-desmethyltamoxifen, 4-hydroxytamoxifen and endoxifen in human plasma samples was developed and validated. The method employs a two step liquid-liquid extraction and a reversed phase separation on a Hypersil Gold(®) C18 column (150mm×4.6mm, 5µm) with isocratic elution. Mobile phase was a mixture of triethylammonium phosphate buffer 5mM pH 3.3 and acetonitrile (57:43, v/v). Total analytical run time was 16min. Precision assays showed CV % lower than 10.53% and accuracy in the range of 93.0-104.2%. The lower limits of quantification (0.75-8.5ngml(-1)) are adequate to measure clinically relevant concentrations in plasma samples. The method was successfully applied to 110 clinical plasma samples. Median plasma levels and interquartile range were: tamoxifen 55.77ngml(-1) (38.42-83.69ngml(-1)), N-desmethyltamoxifen 124.83ngml(-1) (86.81-204.80ngml(-1)), 4-hydroxytamoxifen 1.09ngml(-1) (0.76-1.53ngml(-1)) and endoxifen 6.18ngml(-1) (4.17-8.22ngml(-1)). The procedure has adequate analytical performance and can be employed in therapeutic drug monitoring of tamoxifen or pharmacokinetics studies.