Sex hormones refer to steroid hormones synthesized by tissues such as the gonads, placenta, and adrenal cortex of animals, including estriol, estrone, diethylstilbestrol, estradiol, testosterone, methyl testosterone, and progesterone. Sex hormones have the physiological function of controlling the attached sex organs and secondary sexual characteristics. Adding them to cosmetics can promote hair growth, breast enhancement, whitening, wrinkle removal and skin elasticity. Generally speaking, the estrogen secreted by the female body can fully meet the physiological needs of the female skin. However, some illegal cosmetic manufacturers have seen business opportunities and added estrogen in cosmetics. The long-term use of these estrogen-containing cosmetics will cause a series of skin problems and even the risk of cancer. For example, estrogen cosmetics have been reported to cause cell cancer, endometrial cancer, breast cancer, testicular cancer and precocious puberty in children. There is a huge potential threat to the health of the people.

In China’s current “Cosmetic Safety Technical Specifications” (2015 edition), sex hormones (estrogens, substances with androgenic effects, progesterone) are listed as prohibited substances, and are prohibited from being added to cosmetics as cosmetic raw materials. A rapid and sensitive detection method for the determination of seven sex hormones in cosmetics has important practical significance for regulating the cosmetics market and protecting people’s health.

Due to the differences in cosmetic bases, 7 sex hormones such as estriol are not easily separated from the bases, which affects their qualitative and quantitative determination. Welch materials specially launched this application scheme.


Cosmetics Safety Technical Specifications 2015 Edition.

Scope of application

Determination of 7 sex hormones including estriol in cosmetics.

Configuration of standard solutions

Estrogen standard solution: Weigh 0.2 g each of estrone, estradiol, estriol, and diethylstilbestrol, dissolve in methanol, transfer to a 100 mL volumetric flask, and dilute to the mark with methanol.

Androgen standard solution: weigh 0.6 g of testosterone and methyl testosterone, dissolve in methanol and transfer to a 100 mL volumetric flask, and dilute to the mark with methanol. Take 10mL of this solution and put it in a 100mL volumetric flask, and dilute to the mark with methanol.

Progesterone standard solution: Weigh 0.6g of progesterone and transfer it to a 100mL volumetric flask with methanol, and dilute it to the mark with methanol. Take 10mL of this solution and place it in a 100mL volumetric flask, and dilute to the mark with methanol.

Mixed standard stock solution: Pipette 50.00 mL of estrogen standard solution, 5.00 mL of androgen standard solution, and 5.00 mL of progesterone standard solution into a 100 mL volumetric flask, dilute to the mark with methanol, and make 1 mL containing 4 kinds of estrogens respectively. 1.00 mg, a mixed standard stock solution of 30.0 μg each of 2 androgens and 30.0 μg of 1 progestogen.

Dilute 1 mL of mixed standard stock solution 0 μL, 100 μL, 200 μL and 500 μL with methanol to prepare mixed standard series solutions.

Chromatographic conditions

Chromatographic column: Welch Ultisil® LP-C18 (4.6×300 mm, 5 μm).

Mobile phase: methanol: water = 82: 18

Column temperature: 45℃

UV detector: 254nm

Flow rate: 0.6mL/min

Injection volume: 10μL


1.Standard solution

NameRetention timePeak areaPeak area%Peak heightTailing factorDegree of separationPlates
Methyl testosterone12.59240.433.28521.361.024.9527480.28

2.Lotion sample

NameRetention time Peak area Peak area% Peak height Tailing factor Degree of separation Plates
Estriol 6.5314.771001.480.988920.68

3.Sample spike

Name Retention time Peak area Peak area% Peak height Tailing factor Plates Degree of separation Plates
Estriol 6.53138.331.4818.740.8819078.19
Diethylstilbestrol 8.037877.4784.52987.831.077.4723410.63
Estradiol 8.95130.281.4015.351.092.6125517.23
Estrone 9.36127.181.3615.121.011.6927806.24
Testosterone 11.18295.323.1729.121.012.1927796.79
Methyl testosterone 12.61219.502.3619.521.015.0528321.95
Progesterone 15.07486.075.2237.121.007.5829979.22

Recovery rate

/EstriolDiethylstilbestrolEstradiolEstroneTestosteroneMethyl testosteroneProgesterone
Peak area138.337877.47130.28127.18295.32219.50486.07
Recovery rate88.8%88.1%88.5%90.6%90.3%91.3%89.8%


Under these chromatographic conditions, Welch Materials acid-resistant Ultisil® LP-C18 (pH tolerance range of 0.5-8) can have good peak shape, and because this column uses an uncapped, side chain sterically protected bond It can avoid the instability of the column due to the hydrolysis of the end-capped small groups under acidic conditions; at the same time, it also shields the hydroxyl group to a certain extent, so that the chromatographic column with polar functional groups such as estriol has a symmetrical peak shape. Good peak capacity can effectively separate various impurities and meet the daily testing requirements of 7 sex hormones such as estriol in cosmetics.

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