RESUMO
OBJECTIVE: Salivary cortisol measurement plays an important role in the evaluation of adrenal function. Its high correlation with free serum cortisol, the easy of sampling and the limited presence of interfering steroids, generated multiple recent studies of its application, in special in the screening of adrenal hyperfunction. In this paper we present our experience in the development of a high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for salivary cortisol and cortisone measurement. MATERIALS AND METHODS: For this study we used 181 saliva samples from our routine diagnostic laboratory. The HPLC-MS/MS method was based on a Waters Quattro Premier tandem mass spectrometer with an electrospray probe. After derivatization with hydroxylamine transitions monitored included cortisol and cortisone. An in-house radioimmunoassay (RIA) was used for salivary cortisol results comparison. RESULTS: Functional sensitivity was 24 ng/dL for cortisol and linearity from 24 to 1929 ng/dL. Saliva cortisol values obtained in the 181 samples presented a median of 52 ng/dL with 5-95% percentile of 24 and 374 ng/dL. With the RIA the results were 86, 25 and 436 ng/dL, respectively, with values for RIA being significantly higher (P<0.0001) and high correlation (r=0.8312, P<0.0001). Cortisone measured in 159 samples showed a median of 278 ng/dL, with 5-95% percentile of 100 and 1,133 ng/dL. Correlation with cortisol values was significant (r=0.820, P<0.0001). CONCLUSION: We conclude that the HPLC-MS/MS method compares favorably with the RIA for salivary cortisol measurement, with the additional possibility of concomitant cortisone measurement and the evaluation of 11ßHSD2 activity.
Assuntos
Cromatografia Líquida de Alta Pressão , Cortisona/análise , Hidrocortisona/análise , Saliva/química , Espectrometria de Massas em Tandem/métodos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Humanos , Radioimunoensaio/métodos , Sensibilidade e EspecificidadeRESUMO
Objective Salivary cortisol measurement plays an important role in the evaluation of adrenal function. Its high correlation with free serum cortisol, the easy of sampling and the limited presence of interfering steroids, generated multiple recent studies of its application, in special in the screening of adrenal hyperfunction. In this paper we present our experience in the development of a high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for salivary cortisol and cortisone measurement. Materials and methods For this study we used 181 saliva samples from our routine diagnostic laboratory. The HPLC-MS/MS method was based on a Waters Quattro Premier tandem mass spectrometer with an electrospray probe. After derivatization with hydroxylamine transitions monitored included cortisol and cortisone. An in-house radioimmunoassay (RIA) was used for salivary cortisol results comparison. Results Functional sensitivity was 24 ng/dL for cortisol and linearity from 24 to 1929 ng/dL. Saliva cortisol values obtained in the 181 samples presented a median of 52 ng/dL with 5‐95% percentile of 24 and 374 ng/dL. With the RIA the results were 86, 25 and 436 ng/dL, respectively, with values for RIA being significantly higher (P<0.0001) and high correlation (r=0.8312, P<0.0001). Cortisone measured in 159 samples showed a median of 278 ng/dL, with 5‐95% percentile of 100 and 1,133 ng/dL. Correlation with cortisol values was significant (r=0.820, P<0.0001). Conclusion We conclude that the HPLC-MS/MS method compares favorably with the RIA for salivary cortisol measurement, with the additional possibility of concomitant cortisone measurement and the evaluation of 11βHSD2 activity. .
Objetivo A dosagem de cortisol salivar é uma metodologia que vem tendo crescente aceitação no estudo da função adrenocortical. Sua alta correlação com a fração livre sérica, facilidade de coleta e presença limitada de interferentes têm originado múltiplas publicações, em especial no screening de pacientes suspeitos de hiperfunção. Neste trabalho apresentamos nossa experiência no desenvolvimento de metodologia baseada em cromatografia líquida e espectrometria de massas (HPLC-MS/MS) para a medida de cortisol e cortisona salivares. Materiais e métodos Para este estudo utilizamos 181 amostras de saliva de nossa rotina diagnóstica. A metodologia de HPLC-MS/MS baseou-se num espectrômetro de massas Waters Quattro Premier. Após derivatização com hidroxilamina, as transições monitoradas incluíram cortisol e cortisona. Um radioimunoensaio (RIE) in house foi empregado para comparação. Resultados A sensibilidade funcional para cortisol foi de 24 ng/dL, com linearidade entre 24 e 1,929 ng/dL. Os valores de cortisol obtidos nas 181 amostras apresentaram mediana de 52 ng/dL, com percentis 5‐95% de 24 e 374 ng/dL. Com o RIE, os resultados foram 86, 25 e 436 ng/L, respectivamente, com os valores obtidos no RIE significativamente mais elevados (P<0,0001), e alta correlação (r=0,8312, P<0,0001). Cortisona, medida em 159 amostras, mostrou mediana de 278 ng/dL, com percentis 5‐95% entre 100 e 1.133 ng/dL. A correlação com os valores de cortisol foi significativa (r=0,820, P<0,0001). Conclusão Concluímos que o método baseado em HPLC-MS/MS compara-se favoravelmente com o RIE para a medida de cortisol salivar, com a possibilidade adicional da medida concomitante de cortisona e avaliação da atividade da enzima 11βHSD2. .