Table 1 High performance liquid chromatography gradient and mass spectrometry operating condition

HPLC gradient

Time (min)

Mobile A (%)

Mobile B (%)

Flow rate (mL/min)

 0

100

0

0.4

 1

75

25

0.4

 2

50

50

0.4

 6

0

100

0.4

 7.5

0

100

0.4

 8

100

0

0.4

 10

100

0

0.4

MS operating condition

 Mode

ESI positive mode

 Scan type

Full scan or multi reaction monitoring

Compounds

MW

[M + H]⁺

Ion transition

Collision energy

Retention time (min)

 Cholesterol

386.65

369.35

369.35 → 243.15

35

6.67

 Pregnenolone

316.48

299.26

299.26 → 281.23

35

4.49

 Finastride (IS)

372.54

373.28

373.28 → 305.22

35

4.14