extraction of β-blockers from small volume biological fluid samples

4
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 neo TM 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 MicroPlate TM The new TELOS MicroPlate TM 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 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. Kinesis Ltd kinesis.co.uk Kinesis Inc kinesis-usa.com Kinesis GmbH (formerly Abimed) kinesisgmbh.de Kinesis Australia Pty Ltd kinesis-australia.com.au Extraction of β-blockers from Small Volume Biological Fluid Samples using a new Versatile SPE 96-well Plate Format Introduction

Upload: kinesis

Post on 20-Mar-2016

218 views

Category:

Documents


1 download

DESCRIPTION

 

TRANSCRIPT

Page 1: Extraction of β-blockers from Small Volume Biological Fluid Samples

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.

Kinesis Ltd kinesis.co.uk Kinesis Inc kinesis-usa.com Kinesis GmbH (formerly Abimed) kinesisgmbh.de Kinesis Australia Pty Ltd kinesis-australia.com.au

Extraction of β-blockers from Small Volume Biological Fluid Samples using a new Versatile SPE 96-well Plate Format

Introduction

Page 2: Extraction of β-blockers from Small Volume Biological Fluid Samples

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:

Page 3: Extraction of β-blockers from Small Volume Biological Fluid Samples

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

Page 4: Extraction of β-blockers from Small Volume Biological Fluid Samples

Kinesis recognise all trademarks and registered trademarks. E&EO KLTD-1244-1113

UNITED KINGDOMKinesis LtdTel: +44 (0)1480 212122 Fax: +44 (0)1480 212111 E-mail: [email protected] Web: kinesis.co.uk

AUSTRALIA & NEW ZEALANDKinesis Australia Pty LtdTel: +61 (0)7 3829 3996 Fax: +61 (0)7 3829 3997 Email: [email protected] Web: kinesis-australia.com.au

USA & CANADAKinesis IncTel: (518) 289-5817Toll free in USA: (866) 934-6353 Fax: (518) 289-5818 Email: [email protected] Web: kinesis-usa.com

GERMANY, SWITZERLAND & AUSTRIAKinesis GmbH (formerly Abimed) Tel: +49 (0)2173 89 05-0 Fax: +49 (0)2173 89 05-77 Email: [email protected] Web: kinesisgmbh.de

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