Yuyong Ke (Presenter)
EndoCeutics Inc., 2795 Laurier Blvd, Suite 500, Qu
Bio: Yuyong Ke received his PhD in mass spectrometry from York University, Canada. He is the senior scientist of EndoCeutics Inc. His research interest focuses on LC-MS/MS method development for the accurate measurement of steroids in biological matrices. He has published more than 24 papers in reputed journals and was the winner of first prize of poster presentation at Estrogen Conference in Bethesda, Washington D.C. 2014.
Authorship: Yuyong Ke, Fernand Labrie
EndoCeutics Inc.
| Short Abstract The challenges for LC-MS/MS method development of accurate assays of steroids in human serum are: 1) low ionization efficiency and low concentrations in the circulation; 2) many isobaric isomers; 3) easily interfered by unknown compounds. Three strategies have been proposed and implemented in a fully validated method of simultaneous quantitation of seven androgen- and estrogen-related compounds at the level of pg/mL in postmenopausal serum, and in a fully validated method for the simultaneous quantitation of 7alpha, 7beta OH DHEA and 7 keto DHEA with a novel derivavtization reagent. |
Long Abstract
Introduction
LC-MS/MS has become the standard technology for the accurate and precise assays in bioanalysis due to its high sensitivity, high specificity and the possibility of multiple analytes in complicated matrices. Another advantage is that it usually requires a relatively simple sample preparation procedure. In clinical diagnostics and endocrinology, this technology has been increasingly in need, replacing the conventional immunoassay, with which the results are questionable due to the lack of specificity [1]. In this report, the challenges and strategies of accurate assays of steroids in human serum using LC-MS/MS will be presented and discussed, and accordingly the successful examples will be demonstrated.
Methods
A Shimadzu Nexera (Kyoto, Japan) Qtrap 6500TM (AB Sciex, Concord, Canada) Ultraperformance liquid chromatograph–electrospray ionization–tandem mass spectrometry (UPLC–ESI–MS/MS) system was setup for the steroid measurements, the analytical column is Agilent Poroshell 120 EC-C18 (3.0 x 100 mm, 2.7 mm). The sample preparation is a typical Liquid-liquid extraction, where extracted estradiol sample was derivatized with dansyl chloride while extracted 7a, 7b OH DHEA and 7 keto DHEA were derivatized with 1-amino-4-methyl piperazine.
Results
For accurate measurement of steroids using LC-MS/MS, there are three major challenges: 1) for most steroids such as Estradiol, there is a lack of an ionization efficiency and they are present at very low concentrations in the circulation; 2) there are many isobaric isomers and 3) steroids are easily interfered by unknown compounds and environment. The strategies to circumvent these issues are: a) use the most sensitive instrument and sometimes, design or choose an appropriate derivatization by introducing a large proton affinity group to achieve the required sensitivity; b) use UPLC columns and MRM transitions to optimize the separation of analyte; and d) use an optimal extraction procedure and monitor ion ratios to eliminate the interference during method development.
These strategies have been implemented in a fully validated method of simultaneous quantitation of seven androgen- and estrogen-related compounds at the level of pg/mL in postmenopausal serum, where the low limit of quantitation (LLOQ) of Estradiol is 1 pg/mL and dansyl chloride is used as the derivatization reagent [2]. This method is sensitive, highly specific and robust while the sample preparation procedure is simple. The sensitivity of estradiol has been improved by 10 times with the derivatization. The accuracies and bias of calibration curves and quality controls are less than 10%, thus well meeting the FDA guidelines [3] where a good linearity of designed ranges is observed for all compounds with R > 0.99.
The other example is a fully validated method for the simultaneous quantitation of 7alpha, 7beta OH DHEA and 7 keto DHEA with a novel derivavtization reagent, namely 1-amino-4-methyl piperazine, where the LLOQ of 10 pg/mL is achieved for all three compounds [4]. Compared to underivatized, the sensitivity of all derivatized compounds has been increased by 50 ~ 100 folds. The excess amount of derivatization reagent can be evaporated during the derivatization reaction and thus reducing the potential matrix effect. Within the range of 10 to 2000 pg/mL, a good linearity is observed while the accuracies and bias of the calibration curves and quality controls are less than 10%. With this method, 7alpha, 7beta OH DHEA and 7 keto DHEA are well separated from the isobaric isomers, 11 beta OH DHEA, 16 alpha and 16 beta OH DHEA. There is no significant matrix effect observed for both methods.
In addition, it has been demonstrated [5] that the optimal extraction procedure and ion ratios monitoring are very important in order to eliminate the potential interferences and further improve the specificity for some endogenous compounds during the method development. In the example of accurate assay of endogenous progesterone, it has been found that a threshold of polarity index of extraction solvent exists for obtaining clean progesterone samples, i.e., an optimal value of polarity index range (0–1) of extraction solvent could be appropriate. These methods have been applied to the assay of large series of clinical trial samples [6].
Conclusions
The high sensitivity and specificity are critical for the accurate measurement of steroids in clinical diagnostics and endocrinology. The best sensitivity instrument and derivatization can meet the requirement of sensitivity. The optimal sample preparation procedure can enhance the specificity by eliminating the potential interferences while the powerful UPLC column and derivtization can help resolve the isobaric isomers. The ion ratio monitoring is a very powerful tool to further improve the specificity.
References & Acknowledgements:
References
1. C. Ohlsson, M.E. Nilsson, A. Tivesten, H. Ryberg, D. Mellstrom, M.K. Karlsson, O. Ljunggren, F. Labrie, E.S. Orwoll, D.M. Lee, S.R. Pye, T.W. O'Neill, J.D. Finn, J.E. Adams, K.A. Ward, S. Boonen, G. Bartfai, F.F. Casanueva, G. Forti, A. Giwercman, T.S. Han, I.T. Huhtaniemi, K. Kula, M.E. Lean, N. Pendleton, M. Punab, D. Vanderschueren, F.C. Wu, L. Vandenput, Comparisons of immunoassay and mass spectrometry measurements of serum estradiol levels and their influence on clinical association studies in men, J. Clin. Endocrinol. Metab. 98 (6): E1097–1102, 2013
2. Y. Ke, J. Bertin, R. Gonthier, J.N. Simard and F. Labrie. A Sensitive and Robust LC-MS/MS Method for the Simultaneous Quantification of Seven Androgenic and Estrogenic Steroids in Postmenopausal Serum using a Single Sample Preparation Method. J. Steroid Biochem. Mol. Biol. 144: 523–534, 2014
3. F. Labrie, Y. Ke, R. Gonthier, A. Bélanger. Why both LC-MS/MS and FDA-compliant validation are essential for accurate estrogen assays? J. Steroid Biochem. Mol. Biol. 149: 89-91, 2015
4. Y. Ke, R. Gonthier, J.N. Simard and F. Labrie. A Validated LC-MS/MS Method for the Sensitive Quantitation of Serum 7alpha Hydroxy-, 7beta Hydroxy- and 7keto- Dehydroepiandrosterone using a Novel Derivatization Reagent. Steroids 108: 112-117. 2016
5. Y. Ke and F. Labrie. The importance of optimal extraction to insure the reliable MS-based assays of endogenous compounds. Bioanalysis 8(1): 39–41. 2016
6. Y. Ke, F. Labrie, R. Gonthier, J.N. Simard, D. Bergeron, C. Martel, M. Vaillancourt, M. Montesino, L. Lavoie, D.F. Archer, J. Balser, É. Moyneur et al. Serum Levels of sex steroids and metabolites following 12-weeks of intravaginal DHEA administration. J Ster Biochem Mol Biol. 154:186-196. 2015
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