Extraction of β-blockers from Small Volume Biological Fluid Samples using a new Versatile SPE 96-well Plate Format Introduction 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 100¾l, 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 waterwettable 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.
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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 PRP TELOS 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.
Application
Analytical Methodology
We 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.
Details of the analytical conditions are as follows:
Metoprolol
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
Propranolol
Figure 1: Structures of Metoprolol and Propranolol
Extraction Methodology SAMPLE PREPARATION Human plasma samples were spiked with the two analytes in the concentration range 0.25 to 25ng/ml.
SPE METHOD The following TELOS MicroPlate was used: TELOS neo PRP MicroPlate, 5mg (part number 600-005M-096MP) 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 Elution
Acetonitrile/IPA, 40/60, v/v containing 2% formic acid (50μl)
MS CONDITIONS ANALYTE
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
Standard Curves and Limits of Detection STANDARD CURVE ANALYTE
Metoprolol
Figure 2: Chromatogram of 5ppb Metoprolol and Propranolol
Propranolol
CONCENTRATION
RESPONSE
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
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
LIMITS OF DETECTION ANALYTE
LOD
Metoprolol
0.02ng/ml
Propranolol
0.1ng/ml
Table 4: LOD of Metoprolol and Propranolol
Figure 3: Chromatogram of spiked plasma sample with 5ppb Metoprolol and Propranolol
Figure 5: Linearity check for Metoprolol
Figure 4: Chromatogram of blank sample
Figure 6: Linearity check for Propranolol
Determination of Minimum Elution Volume Using 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. ANALYTE
CONCENTRATION
Metoprolol
Propranolol
SAMPLE - % RECOVERY
MEAN (%)
RSD (%)
1
2
3
4
5
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
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
Table 3: Recoveries of Metoprolol and Propranolol using 50µl elution
Conclusions • The TELOS MicroPlate format is suitable for the extraction of small volume biological fluids. • 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.
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