Nam Kyung Lee; Kyoung Hwa Jang; Jong Tae Lee; Hee Won Park; Sung Tai Han; Gyo In

doi:10.20307/nps.2019.25.1.49

Journal List > Nat Prod Sci > v.25(1) > 1120590

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Lee, Jang, Lee, Park, Han, and In: Study on the Marker Steroids of New Zealand Deer (Cervus elaphus var. scoticus) Velvet Antler by UPLC-MS/MS and HPLC-PDA Methods–(II)

Original Article

Natural Product Sciences 2019;25(1):49-58.

Published online: 17 December 2019

DOI: https://doi.org/10.20307/nps.2019.25.1.49

Study on the Marker Steroids of New Zealand Deer (Cervus elaphus var. scoticus) Velvet Antler by UPLC-MS/MS and HPLC-PDA Methods–(II)

Nam Kyung Lee^∗, Kyoung Hwa Jang, Jong Tae Lee, Hee Won Park, Sung Tai Han, Gyo In

Laboratory of Fundamental Research, R&D Headquarters, Korea Ginseng Corp., 30, Gajeong-ro, Yuseong-gu, Daejeon 34128, Republic of Korea

^∗Author for correspondence Nam Kyung Lee, Laboratory of Fundamental Research, R&D Headquarters, Korea Ginseng Corp., 30, Gajeong-ro, Yuseong-gu, Daejeon 34128, Republic of Korea Tel: +82-42-870-3010; E-mail: leenamkyung@kgc.co.kr

Received 11 September 2018 Revised 5 November 2018 Accepted 9 November 2018

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Go to :

Keywords: New Zealand deer antler, marker steroid, UPLC-MS/MS, HPLC-PDA, steroid hormone, 7-oxycholesterol

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.

Go to :

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.

undefined

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.

undefined

Table 1.

UPLC-MS/MS detection conditions for the eleven SHs

Compounds (SHs)	Ret. time (min)	Parent ion (m/z)	Adduct	Cone (voltage)	Collision (voltage)	Daughter ions (m/z)
Androstene-3,17-dione	14.8	287.41	[M+H]⁺	36	22	109.04, 97.01
Estrone	15.1	271.37	[M+H]⁺	28	26	133.05
β-Estradiol	15.1	273.38	[M+H]⁺	24	26	107.03
α-Estradiol	15.4	273.37	[M+H]⁺	30	20	135.07
Testosterone	15.8	289.43	[M+H]⁺	36	26	109.04
Dehydroepiandrosterone	16.5	289.43	[M+H]⁺	20	20	213.17
17α-Hydroxyprogesterone	16.7	331.46	[M+H]⁺	36	26	109.04, 96.95
Medroxyprogesterone	19.3	345.49	[M+H]⁺	38	26	123.02, 97.01
Megestrol acetate	19.5	385.51	[M+H]⁺	30	16	325.19
Progesterone	20.3	315.47	[M+H]⁺	22	24	108.98, 96.95
Androsterone	21.4	291.45	[M+H]⁺	22	26	147.10

SH: steroid hormone

Table 2.

Regression equation, correlation coefficients (R²), linear range, the LODs, and the LOQs for eleven SHs and three 7-O-CSs

Compounds	Range (μ g/mL)	Regression equation (n=3)	R²	LOD (ng/mL)	LOQ (ng/mL)
Androstene-3,17-dione (A)	80.1 – 200	y = 825x – 4750	0.999	3.3	10
Estrone	80.1 – 200	y = 722000x – 96700	0.997	0.26	0.78
β-Estradiol	80.1 – 200	y = 6430 x – 33600	0.999	7.13	23.1
α-Estradiol	80.1 – 200	y = 83645x + 5823.7	0.996	0.32	0.96
Testosterone	80.1– 200	y = 753x – 4390	0.999	3.67	11.0
Dehydroepiandrosterone	80.1 – 200	y = 1310x – 2180	0.999	7.0	21.0
17α-Hydroxyprogesterone	80.1 – 200	y = 121921x + 37587	0.999	0.1	0.3
Medroxyprogesterone	80.1 – 200	y = 45393x + 9989.7	0.999	0.38	1.14
Megestrol acetate	80.1 – 200	y = 34299x + 30807.8	0.999	1.1	3.3
Progesterone	80.1 – 200	y = 184045 x + 118121	0.999	0.5	1.50
Androsterone (A)	80.1 – 200	y = 5636.2 x + 1061.8	0.992	3.6	10.8
7-Ketocholesterol (A)	10.0 – 2000	y = 621000x – 28700	0.999	10	30
7α-Hydroxycholesterol (A)	10.0 – 2000	y = 659000x – 29200	0.999	95	285
7β-Hydroxycholesterol (A)	10.0 – 2000	y = 547000x – 31700	0.999	116	348

A: marker steroid of New Zealand deer antler; mixture: mixture of 4 part (tip, upper, middle, and base); LOD: the limit of detection; LOQ: the limit of quantitation; SH: steroid hormone; 7-O-CS: 7-oxycholesterol

Table 3.

Intraday and interday precisions of eleven SHs and three 7-O-CSs (n=3)

Compound	Intraday RSD (%)			Interday RSD (%)
Compound	Day 1	Day 2	Day 3	Interday RSD (%)
Androstene-3,17-dione (A)	1.32	1.64	1.39	1.54
Estrone	1.46	1.95	2.12	1.54
β-Estradiol	1.85	2.08	2.43	2.31
α-Estradiol	1.90	2.37	2.28	2.25
Testosterone	1.53	1.85	1.78	1.77
Dehydroepiandrosterone	1.64	1.94	1.85	1.73
17α-Hydroxyprogesterone	1.76	1.72	1.97	2.08
Medroxyprogesterone	1.69	1.77	2.26	1.95
Megestrol acetate	1.82	2.17	2.17	2.04
Progesterone	1.54	1.42	1.78	1.63
Androsterone (A)	1.57	1.65	1.68	1.45
7-Ketocholesterol (A)	1.63	1.88	1.90	1.67
7α-Hydroxycholesterol (A)	1.38	1.93	1.85	1.63
7β-Hydroxycholesterol (A)	1.54	1.43	1.89	1.78

A: marker steroid of New Zealand deer antler; SH: steroid hormone; 7-O-CS: 7-oxycholesterol; RSD: relative standard deviation

Table 4.

Recovery of eleven SHs and three 7-O-CSs

Compound	Recovery (%)
Compound	low	middle	high
Androstene-3,17-dione (A)	98.9	100.7	102.3
Estrone	97.3	98.7	101.8
β-Estradiol	97.4	98.1	99.2
α-Estradiol	97.5	98.9	103.6
Testosterone	98.7	100.8	103.4
Dehydroepiandrosterone	97.7	100.3	102.3
17α-Hydroxyprogesterone	97.7	99.4	102.9
Medroxyprogesterone	98.9	99.8	99.6
Megestrol acetate	98.4	99.6	101.4
Progesterone	98.9	101.7	103.6
Androsterone (A)	100.5	102.5	104.6
7-Ketocholesterol (A)	99.8	101.6	103.5
7α-Hydroxycholesterol (A)	97.2	99.2	101.5
7β-Hydroxycholesterol (A)	98.7	99.5	100.7

A: marker steroid of New Zealand deer antler; SH: steroid hormone; 7-O-CS: 7-oxycholesterol

Table 5.

The levels of eleven SHs and three 7-O-CSs in NZA

Mean ± SD (μ g/g)	New Zealand deer antler
Mean ± SD (μ g/g)	Tip	Upper	Middle	Base		Mixture
Compound	RM	RM	RM	RM	RM	AE	WE
Androstene-3,17-dione (A)	10.1±0.64	5.72±0.23	1.38±0.43	0.29±0.01	2.94±0.11	14.91±0.63	0
Estrone	17.25±0.59	8.61±0.49	0.72±0.03	0	0.91±0.04	10.05±0.60	0
β-Estradiol	0.33±0.01	0.15±0.01	0	0	0	0	0
α-Estradiol	0.11±0.01	0	0	0	0	0.96±0.04	0
Testosterone	0.27±0.05	0.27±0.01	0.27±0.01	0.27±0.01	0.27±0.01	2.46±0.17	0
Dehydroepiandrosterone	1.22±0.08	0.47±0.02	0.41±0.02	0.12±0.01	0.61±0.04	4.42±0.18	0
17α-Hydroxyprogesterone	0.36±0.02	0.33±0.01	0.32±0.01	0	0.14±0.01	2.97±0.12	0
Medroxyprogesterone	0.25±0.01	0.25±0.01	0	0	0.125±0.01	2.31±0.17	0
Megestrol acetate	0.22±0.01	0.22±0.01	0.22±0.01	0.17±0.01	0.21±0.01	2.08±0.12	0
Progesterone	0.31±0.01	0.27±0.01	0.31±0.01	0.23±0.01	0.31±0.02	2.45±0.11	0
Androsterone (A)	78.57±4.43	7.54±0.35	5.86±0.17	0.52±0.02	8.44±0.36	111.01±3.23	0
7-Ketocholesterol (A)	113.82±5.78	81.42±6.13	72.12±2.76	51.01±2.36	71.33±4.29	1114.47±63.15	0
7α-Hydroxycholesterol (A)	71.61±4.66	44.76±1.14	21.27±1.29	23.57±1.53	35.51±1.85	460.16±16.23	0
7β-Hydroxycholesterol (A)	123.11±5.91	72.38±3.75	48.59±2.85	46.56±1.98	53.36±2.32	805.19±38.21	0

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

TOOLS

Similar articles