This method is a LC-MS/MS method for the determination of steroid hormones in serum.
Table 1 Information of the analytes
The standards were dissolved by methanol to get stock solutions at the concentration of 1 mg/mL. Then stock solutions were diluted to required concentration by methanol.
This experiment employed Cleanert® SLE (200mg / 3mL) for sample purification.
Sample loading: Appropriate volume of methanol was added to 200 μL of serum sample, adjusted the content of methanol to 5%. Shook the sample and loaded onto the cartridge, then drew through the top frit under low vacuum (< -0.04 MPa) and stood for 10 min.
Elute analytes: 600 μL MTBE was used to elute the cartridge, and then the elution was collected at 1~2mL/min, repeated the elute operation after standing 1min, repeated twice. Then the elution was combined together for concentrate.
The elute was evaporated to dryness at 40 °C and reconstitute the residue by 200 μL of Acetonitrile:Water (3:7, v/v), and then analyzed by LC-MS/MS.
LC-MS/MS, API 4000+
Column: Venusil® ASB C18, 2.1 × 50 mm, 3 μm, 150 Å
Mobile phase: Acetonitrile:Water (55:45, v/v) for analysis of progesterone, testosterone and boldenone; Acetonitrile:Water (30:70, v/v) for analysis of cortisone
Flow rate: 0.2 mL/min
Column temperature: 30 °C
Injection volume: 5 μL
Scan mode: MRM
Two ionization modes were employed on the basis of compound structure. Analyzed progesterone, testosterone and boldenone with positive mode and analyzed cortisone with negative mode.
Table 2 MS/MS transitions and Retention time of target compounds
Table 3 MS Conditions
Boldenone, Testosterone and Progesterone
Results and Discussion
Table 4 Recovery data
Spiked two serum samples with the concentration of 5 ng/mL. Results of spiked recovery were showed in Table 4. Actual serum samples were also extracted and analyzed by using the same procedures. Approximate 4ng/mL of cortisone was detected while the other three steroid hormones were free. Background should be subtracted from the response of cortisone in spiked samples to calculate the recovery data.
It is a preliminary method for extracting steroid hormones from serum because the recovery data of progesterone and testosterone is a little unsatisfactory. According to our experience, approximate content of organic solvent, such as methanol, in the serum sample could benefit to increase recoveries, because methanol will help progesterone and testosterone dissolve in aqueous serum solution.