This is a LC-MS/MS method for determination of steroid hormones in serum by Microplate.
Table 1 Information of the analytes
The standards were dissolved by methanol to get stock solutions at the concentration of 1 mg/mL. Then the stock solutions were diluted to required concentration by methanol.
This experiment employed Cleanert® PEP 96-well plate (5 mg/well) for sample purification.
Column condition: 200 μL of Methanol was added into each well of Cleanert® PEP, followed by 200 μL of Water to condition the packing material.
Sample loading: 100 μL of serum was diluted with 100 μL solvent which contained 1% formic acid in methanol / water (50:50,v/v), then loaded the sample onto the plate.
Washing: 400 μL of methanol/water (35/65, v/v) was used to wash the plate.
Elution: 200 μL of methanol with 1% formic acid was used to elute the plate.
Then, the elution was collected and analyzed by LC-MS/MS. (The elution could be evaporated and reconstituted to obtain lower LOD)
Instrumentation: LC-MS/MS, API 4000+
Column: Venusil® ASB C18, 2.1 × 50 mm, 3 μm, 150 Å;
Mobile phase: 0.1% FA in acetonitrile / 0.1%FA in Water (60:40, v/v)
Flow rate: 0.2 mL/min
Column temperature: 30 °C
Injection volume: 3 μL
Ion source: ESI - Positive
Scan mode: MRM
Table 2 MS/MS transitions and Retention time of target compounds
Results and Discussion
Table 3 Recovery data
The assay described good result of steroid hormones extract from serum by Cleanert® PEP Microplate. The recovery from 85.9% to 110.6% at 30ppb. The usage of the 96-well plate is sufficient for high throughput sample clean-up procedure prior to LC-MS/MS.