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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

Copyright © 2019 The Korean Society of Pharmacognosy

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.

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

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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
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
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 (%)
Day 1 Day 2 Day 3
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 (%)
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
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

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