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.

Role of the Mevalonate Pathway in Adrenocortical Tumorigenesis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme of the mevalonate pathway, which generates cholesterol and non-sterol compounds such as isoprenoid, which are involved in key steps of tumorigenesis such as cell growth and proliferation. Our aim was to evaluate the role of the mevalonate pathway in adrenocortical tumors (ACTs). Expression pattern of HMGCR, FDFT1, LDLR, SCARB1, StAR, TSPO, CYP11A1, CYP11B1, CYP17A1, CYP21A1, and HSD3B1 genes, involved in the mevalonate pathway and steroidogenesis, was quantified by real-time RT-PCR in 46 ACT [14 adenomas (ACA) and 11 carcinomas (ACC) from adults and 13 ACA and 8 ACC from pediatric patients]. Effects of the mevalonate pathway inhibition on NCI-H295A cell viability was assessed by colorimetric assay. HMGCR was overexpressed in most adult ACT. The expression of TSPO, STAR, CYP11B1, CYP21A1, and HSD3B1 in adult ACC was significantly lower than in ACA (p<0.05). Regarding pediatric ACT, the expression of genes involved in steroidogenesis was not different between ACA and ACC. Inhibition of isoprenoid production significantly decreased the viability of NCI-H295A cells (p<0.05). However, cholesterol synthesis blockage did not show the same effect on cell viability. Low expression of TSPO ,: StAR, CYP11B1, CYP21A1, and HSD3B1 characterized a signature of adult ACCs. Our data suggest that HMGCR overexpression in adult ACC might lead to intracellular isoprenoid accumulation and cell proliferation. Therefore, the mevalonate pathway is a potential target for ACC treatment.

[Analysis of an iodide radioimmunoassay for 11-deoxicortisol measurement]. / Análise de um radioimunoensaio iodado para determinação de 11-deoxicortisol.

OBJECTIVE: Our aim was to correlate 11-deoxycortisol levels obtained by two currently available techniques for 11-deoxycortisol measurement: radioimmunoassay, and high performance liquid chromatography followed by tandem mass spectrometry (MS/MS). The latter is the gold standard method for steroid hormone measurement. MATERIALS AND METHODS: We selected 88 samples and the results of these two methods were compared by Deming regression. RESULTS: The analytical sensitivity of the RIA was 0.30 ng/mL, with inadequate linearity and inadequate precision profile (34% of the samples had a CV ≥ 20%). From the selected samples, 54 had measurable levels of 11-deoxycortisol in both methods and were used in the comparison. The comparison of RIA with LC-MS/MS showed an overestimation of the results by RIA. The correlation coefficient was 0.610; linear regression slope was 3.751; and the intercept was 0.145, indicating a poor correlation between the two methods. CONCLUSION: We concluded that 11-deoxycortisol measured by radioimmunoassay, despite a good analytical sensitivity, showed very low specificity, precluding its use as a reliable method for 11-deoxycortisol measurement.

Análise de um radioimunoensaio iodado para determinação de 11-deoxicortisol / Analysis of an iodide radioimmunoassay for 11-deoxicortisol measurement

Objetivo : Nosso objetivo foi comparar duas técnicas de dosagem do 11-desoxicortisol: a técnica de radioimunoensaio iodado, a qual foi validada neste trabalho, e a cromatografia líquida de alta performance seguida por espectrometria de massa em tandem (LC-MS/MS), sendo a última considerada o padrão-ouro para dosagem dos hormônios esteroides. Materiais e métodos : Para a comparação entre os resultados de 11-desoxicortisol, foram selecionadas 88 amostras. Resultados : A sensibilidade analítica do radioimunoensaio foi de 0,30 ng/mL, com linearidade e perfil de precisão inadequado (34% das amostras com CV ≥ 20%). Das 88 amostras selecionadas, apenas 54 apresentaram resultados mensuráveis em ambos os métodos. A comparação desses resultados, por meio da regressão de Deming, resultou em um coeficiente de correlação de 0,610, inclinação de 3,751, intercepção de 0,145, evidenciando a pobre correlação entre os resultados e a superestimação dos resultados pelo RIA. Conclusão : Concluímos que o método de dosagem de 11-desoxicortisol por radioimunoensaio iodado apresentou resultados inadequados nos diversos parâmetros avaliados, inviabilizando sua utilização como método de dosagem do 11-desoxicortisol. Arq Bras Endocrinol Metab. 2014;58(3):232-6 .

Objective : Our aim was to correlate 11-deoxycortisol levels obtained by two currently available techniques for 11-deoxycortisol measurement: radioimmunoassay, and high performance liquid chromatography followed by tandem mass spectrometry (MS/MS). The latter is the gold standard method for steroid hormone measurement. Materials and methods : We selected 88 samples and the results of these two methods were compared by Deming regression. Results : The analytical sensitivity of the RIA was 0.30 ng/mL, with inadequate linearity and inadequate precision profile (34% of the samples had a CV ≥ 20%). From the selected samples, 54 had measurable levels of 11-deoxycortisol in both methods and were used in the comparison. The comparison of RIA with LC-MS/MS showed an overestimation of the results by RIA. The correlation coefficient was 0.610; linear regression slope was 3.751; and the intercept was 0.145, indicating a poor correlation between the two methods. Conclusion : We concluded that 11-deoxycortisol measured by radioimmunoassay, despite a good analytical sensitivity, showed very low specificity, precluding its use as a reliable method for 11-deoxycortisol measurement. Arq Bras Endocrinol Metab. 2014;58(3):232-6 .