extraction of β-blockers from small volume biological fluid samples
DESCRIPTION
ÂTRANSCRIPT
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 100µl, it is important elution volumes and the sample prep format are fully compatible with the original sample volumes being processed.
This poster summarises a solid phase extraction method for two drugs from human plasma, Metoprolol and Propranolol using TELOS neoTM PRP, a water-wettable polymer-based SPE sorbent. Analyte concentrations range from 0.25 to 25ng/ml. The data highlights the minimum elution volumes that can be achieved from the 5mg sorbent mass.
TELOS MicroPlateTM
The new TELOS MicroPlateTM is optimised for the sample processing and extraction of small volume biological fluids. The internal diameter, frit design and sorbent allow biological fluids (plasma or urine) to be processed.
The TELOS MicroPlate is a modular design, allowing for flexibility in sample numbers. Full or partially populated plates can be processed using vacuum or positive pressure. In addition, the well outlet design ensures good collection plate penetration, removing any possibility of well to well cross contamination.
About TELOS neo PRPTELOS neo PRP (Polar-modified Reversed Phase) is a water-wettable, non-polar SPE sorbent utilising a proprietary polymeric backbone. The surface chemistry has been optimised to provide the necessary balance of non-polar interactions for retaining compounds of varying polarity, from polar metabolites to higher molecular weight analytes. TELOS neo PRP is part of the TELOS neo family of solid phase extraction sorbents.
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Extraction of β-blockers from Small Volume Biological Fluid Samples using a new Versatile SPE 96-well Plate Format
Introduction
ApplicationWe used β-blockers, Metoprolol and Propranolol (see Figure 1 for structures), for the investigation work. The two analytes were spiked at the desired concentrations into human plasma and extracted using a TELOS neo PRP 5mg MicroPlate.
Figure 1: Structures of Metoprolol and Propranolol
Extraction MethodologySAMPLE PREPARATIONHuman plasma samples were spiked with the two analytes in the concentration range 0.25 to 25ng/ml.
SPE METHODThe following TELOS MicroPlate was used:
TELOS neo PRP MicroPlate, 5mg (part number 600-005M-096MP)
Metoprolol
Propranolol
STEP SOLVENT AND VOLUMES
Conditioning Methanol (200µl)
Equilibration Deionised Water (200µl)
Sample Application
Human Plasma (100µl)
Interference Elution
Deionised Water/Methanol, 95/5, v/v (200µl)
Analyte ElutionAcetonitrile/IPA, 40/60, v/v containing 2% formic acid (50µl)
MS CONDITIONSANALYTE DP CE IS TEMP GSI GS2 CUR
Metoprolol 70V 27V 5500V 500°C 40Pa 40Pa 12Pa
Propranolol 70V 27V 5500V 430°C 60Pa 60Pa 12Pa
ANALYTE Q1 Q3
Metoprolol 268.3 191.0
Propranolol 260.1 157.1
HPLC Column: C18 5µm 15cm x 2.1mmid
Mobile Phase:Acetonitrile/H20/TFA, 22/78/0.1%, v/v/v
Temperature: 30°C
Flow rate: 300µl/min
Injection: 10µl
Detection:MS (ABI4000+) – see below for conditions
LC Instrument: Shimadzu LC-20A
Analytical MethodologyDetails of the analytical conditions are as follows:
Figure 4: Chromatogram of blank sample
Figure 3: Chromatogram of spiked plasma sample with 5ppb Metoprolol and Propranolol
ANALYTE CONCENTRATION RESPONSE
Metoprolol
0.25ng/ml 7.98E+03
0.75ng/ml 2.39E+04
2ng/ml 5.87E+04
5ng/ml 1.59E+05
20ng/ml 6.16E+05
Propranolol
0.5ng/ml 3.25E+03
2ng/ml 1.31E+04
5ng/ml 3.11E+04
10ng/ml 6.53E+04
25ng/ml 1.57E+05
Table 5: Responses for Metoprolol and Propranolol standards
ANALYTE LOD
Metoprolol 0.02ng/ml
Propranolol 0.1ng/ml
Table 4: LOD of Metoprolol and Propranolol
Figure 5: Linearity check for Metoprolol
Standard Curves and Limits of Detection
Figure 2: Chromatogram of 5ppb Metoprolol and Propranolol
STANDARD CURVE
LIMITS OF DETECTION
Figure 6: Linearity check for Propranolol
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ANALYTE CONCENTRATION SAMPLE - % RECOVERY MEAN (%) RSD (%)
1 2 3 4 5
Metoprolol
0.5ng/ml 88.3 81.6 87.8 85.5 90.6 86.8 3.93
2.5ng/ml 91.8 93.5 89.7 97.1 90.8 92.6 3.12
10ng/ml 95.8 93.6 96.1 92.5 91.3 93.9 2.21
Propranolol
1ng/ml 92.1 83.9 89.7 88.5 86.5 88.1 3.54
5ng/ml 88.3 93.2 89.5 90.3 89.6 90.2 2.03
20ng/ml 91.7 93.5 95.8 99.1 97.8 95.6 3.17
• TheTELOSMicroPlateformatissuitablefortheextractionofsmallvolumebiologicalfluids.
• Respective recoveries for Metoprolol and Propranolol ranged from 86.8 to 93.9 and 88.1 to 95.6 through the concentrations used (0.5 – 20ng/ml), with good RSDs.
• LODs for Metoprolol and Propranolol were 0.02 and 0.1ng/ml respectively.
• The 5mg sorbent mass allows an elution volume of 50µl to be used, providing a volume suitable for subsequent analysis and minimal analyte dilution.
• Good linearity was observed through the concentrations used.
Table 3: Recoveries of Metoprolol and Propranolol using 50µl elution
Determination of Minimum Elution VolumeUsing the above extraction procedure, the minimum volume of Acetonitrile/IPA (containing 2% formic acid) elution solvent was determined for the analytes for the 5mg sorbent mass.
Conclusions