Developments in LC-MS/MS detection levels have seen biological fluid sample volumes reduced to such an extent that conventional SPE formats are not always suitable, or provide considerable analyte dilution and extended evaporation times. When extracting from sample volumes of less than 100 μL, it is important elution volumes and the sample preparation format are fully compatible with the original sample volumes being processed.
This application summarized the solid phase extraction of two drugs from human plasma, Indomethacin and Ibuprofen using Cleanert® PEP, a water-wettable polymer-based SPE sorbent. Analyte concentrations range from 5 to 50 ng/mL. The data highlights the minimum elution volumes that can be achieved from the 5 mg sorbent mass and the versatile nature of the modular plate design.
Figure 1 Chemical structure of Indomethacin and Ibuprofen
Human plasma samples were spiked with the two analytes in the concentrations range 0.25 to 25 ng/mL.
SPE Method using Cleanert PEP MicroPlate, 5 mg.
Conditioning: 200 μL Methanol.
Equilibration: 200 μL Deionised Water
Sample Application: 50 μL Human plasma diluted by 50 μL 2% formic acid aqueous solution
Interference Elution: 200 μL Deionised Water/Methanol (95/5, v/v)
Analyte Elution: 100μL Acetonitrile
The Cleanert PEP MicroPlate was processed using a vacuum manifold.
Instrumentation: LC-MS/MS, API 4000+
HPLC Column: Venusil XBP C18, 150×2.1 mm, 5 μm, 120 Å
Mobile Phase: Acetonitrile/Water(75/25, v/v)
Flow rate: 0.2 mL/min
Injection: 3 μL
Ion source: ESI Negative
Scan mode: MRM
Table 1 MS Conditions
Indomethacin and Ibuprofen
Table 2 Precursor/Product Ions
Retention time /min
Results and Discussion
Using the SPE method developed, both analytes, Indomethacin and Ibuprofen, were extracted from spiked human plasma (50 µL) in the concentration range 5 to 50 ng/ml. The analytes were eluted in only 100 µL of elution solvent. This ensured little or no analyte dilution compared with the original sample volume. In addition, the 100µL elution volume ensured evaporation and reconstitution time was minimised and throughput improved.
Recoveries for the two analytes were >85% for Indomethacin and >90% for Ibuprofen. The RSDs were <5%. Linearity checks across the full concentration range were good. Increasing the number of samples per concentration to develop and report a robust method. Blank plasma was also extracted and analyzed using the same procedures to ensure it was drug free over the required concentrations.
Figure 1 Chromatogram of 10ng/mL Indomethacin and Ibuprofen
Table 3 LOD of Indomethacin and Ibuprofen
Table 4 Responses for Indomethacin and Ibuprofen standard
Table 5 Recoveries of Indomethacin and Ibuprofen spiked in plasma
Sample - % Recovery
Extraction procedure using Cleanert® PEP MicroPlate assure the number of wells to match sample numbers being processed. Only 100 μL of elution solvent ensured elute Indomethacin and Ibuprofen completely which were spiked into 50μL of plasma. The SPE method included an organic solvent/water combination as the interference elution was to remove polar interferences from the sorbent. This ensured a short analytical run time of less than 10 min, supporting a high throughput application.
Cleanert® PEP Micro Plate
96-well Collection plate
2.2 mL Square well
96-well Vacuum Manifold
Adapt to 96-well plate
Cleanert® M96 Positive Pressure Device
Adapt to 96-well plate
Venusil® XBP C18(A)
2.1 × 150 mm, 5 μm, 150 Å
1.5 mL vials
1.5 mL short thread vial, amberglass
1.5 mL vials caps
9 mm screw neck cap
Micro-insert clear glass
300 μL micro-insert
φ13; 0.22 μm
2 mL, needless