Abstract
Eleven steroid hormones (SHs: androstene-3,17-dione, estrone, β-estradiol, α-estradiol, testosterone, dehydroepiandrosterone, 17á-hydroxyprogesterone, medroxyprogesterone, megestrol acetate, progesterone, and androsterone) were detected from New Zealand deer (Cervus elaphus var. scoticus) velvet antler (NZA, 鹿茸). A method for the quantification of eleven SHs was established by using ultraperformance liquid chromatography (UPLC)-MS/MS. The linearities (R2 > 0.991), limits of quantification (LOQ values, 0.3 ng/mL to 23.1 ng/mL), intraday and interday precisions (relative standard deviation: RSD<2.43%), and recovery rates (97.3% to 104.6%) for all eleven SHs were determined. In addition, a method for the quantification of three 7-oxycholesterols (7-O-CSs: 7-ketocholesterol, 7α-hydroxycholesterol, and 7β-hydroxycholesterol) in the NZA was established by using an HPLC-photodiode array (PDA) method. The linearities (R2 > 0.999), LOQ values (30 ng/mL to 350 ng/mL), intraday and interday precisions (RSD < 1.93%), and recovery rates (97.2% to 103.5%) for the three 7-O-CSs were determined. These quantitative methods are accurate, precise, and reproducible. As a result, it is suggested that the five steroid compounds of androstene-3,17-dione, androsterone, 7-ketocholesterol, 7α-hydroxycholesterol, and 7β-hydroxycholesterol could be marker steroids of NZA. These methods can be applied to quantify or standardize the marker steroids present in NZA.
REFERENCES
(1). Hattori M., Yang X. W., Kaneko S., Nomura Y., Namba T.Jap. Soc. Pharmacog. 1989; 43:173–176.
(2). Sunwoo H. H., Nakano T., Hudson R. J., Sim J. S. J.Agric. Food Chem. 1995; 43:2846–2849.
(3). Lee N. K., Shin H. J., Kim W. S., Lee J. T., Park C. K.Nat. Prod. Sci. 2014; 20:160–169.
(4). Chang I. M.Treatise on Asian Herbal Medicines I; Haksulpyunsukwan press, Korea. 2003; 90. 111, 184.
(5). Ker D. F. E., Wang D., Sharma R., Zhang B., Passarelli B., Neff N., Li C., Maloney W., Quake S., Yang Y. P.Stem Cell Res. Ther. 2018; 31:292–306.
(6). Lu C., Wang M., Mu J., Han D., Bai Y., Zhang H.Food Chem. 2013; 141:1796–1806.
(7). Griffiths W. J., Abdel-Khalik J., Crick P. J., Yutuc E., Wang Y. J.Steroid Biochem. Mol. Biol. 2016; 162:4–26.
(8). Lund B. C., Bever-Stille K. A., Perry P. J.Pharmacotherapy. 1999; 19:951–956.
(9). Frye C. A.Minerva Ginecol. 2009; 61:541–562.
(10). Funder J. W., Krozowski Z., Myles K., Sato A., Sheppard K. E., Young M.Recent Prog. Horm. Res. 1997; 52:247–260.
(11). Gleixner A., Meyer H. H. D.Fresenius J. Anal. Chem. 1997; 357:1198–1201.
(12). Gupta B. B. P., Lalchhandama K.Current Sci. 2002; 83:1103–1111.
(13). Jeanneret F., Tonoli D., Rossier M. F., Saugy M., Boccard J., Rudaz S. J.Chromatogr. A. 2016; 1430:97–112.
(14). McDonald J. G., Smith D. D., Stiles A. R., Russell D. W. J.Lipid Res. 2012; 53:1399–1409.
(15). Rodriguez I. R., Alam S., Lee J. W.Invest. Ophthalmol. Vis. Sci. 2004; 45:2830–2837.
(16). International Conference on Harmonization ICH (Q2) R1. Validation of Analytical Procedures Test and Methodology; ICH;Switzerland: 2005. p. 6–13.
(17). Kaminski R. M., Marini H., Kim W. J., Rogawski M. A.Epilepsia. 2005; 46:819–827.
(18). Yang Y., Shao B., Zhang J., Wu Y., Duan H. J.Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877:489–496.
Fig. 1.
The total ion chromatograms for the eleven standard steroid hormones (1. androstene-3, 17-dione; 2. estrone; 3. β-estradiol; 4. α-estradiol; 5. testosterone; 6. dehydroepiandrosterone; 7. 17α-hydroxyprogesterone; 8. medroxyprogesterone; 9. megestrol acetate; 10. progesterone; 11. androsterone) (A) and New Zealand deer antler's 70% ethanol fraction (B) by UPLC-MS/MS in multiple reaction monitoring mode.
![nps-25-49f1.tif](/upload/SynapseXML/0228nps/thumb/nps-25-49f1.gif)
Fig. 2.
The chromatograms for (A): the standard of three 7-oxycholesterols (7-ketocholesterol, 7α-hydroxycholesterol, and 7β-hydroxycholesterol); (B): the 70% ethanol fraction (mixture of tip, upper, middle, and base) of New Zealand deer antler; (C): the chromatograph of 100 times the height of (B) by HPLC-PDA.
![nps-25-49f2.tif](/upload/SynapseXML/0228nps/thumb/nps-25-49f2.gif)
Table 1.
UPLC-MS/MS detection conditions for the eleven SHs
Table 2.
Regression equation, correlation coefficients (R2), linear range, the LODs, and the LOQs for eleven SHs and three 7-O-CSs
Table 3.
Intraday and interday precisions of eleven SHs and three 7-O-CSs (n=3)
Table 4.
Recovery of eleven SHs and three 7-O-CSs
Table 5.
The levels of eleven SHs and three 7-O-CSs in NZA
The data (mean±standard deviation) are based on the triplicate determinations; A: New Zealand deer antler marker steroid; Mixture: mixture of 4 part (tip, upper, middle, and base); RM: raw material; AE: alcohol extract.; WE: water extract; SH: steroid hormone; 7-O-CS: 7-oxycholesterol; NZA: New Zealand deer antler