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Amitriptyline and related compounds may be present in the bloodstreamat various levels fromthetreatment of depression. It is of interest to determine levels of amitriptyline and metabolites in thebloodstreamwith minimal background fromthe matrix (e.g., blood proteins). As shown below, it ispossible to significantly reduce levels of matrix compounds by the use of a Zorbax SPE C18 EC

cartridge. The analytes of interest are then easily quantitated by reversed-phase HPLC. Recovery ofamitriptyline in this method was very high,see Recovery.

Analysis of Amitriptyline in Serum:Zorbax SPE C18 EC Cartridge

Solid Phase Extraction Method

CARTRIDGE: 100 mg, 1 ml Zorbax SPE

C18 EC (P/N 5184-3597, Box of 100)

CONDITION:-1.0 mL Methanol-1.0 mL Deionized Water

LOAD:-1.0 mL Sample (see sample preparation)

WASH:-1.0 mL Deionized Water

ELUTE:

-1.0 mL 1% ammonia in Methanol INTERNAL STANDARD:-10 L (1 mg/mL) nit robenzene in methanolas internal standard for High Load.

-10 L (0.05 mg/mL) nit robenzene inmethanol as internal standard for LowLoad.

Sample Preparation

1mg/mL amitriptyline for High Load

0.05 mg/mL amitriptyline for Low Load Spike 10 L above solutions-0.5 mL bovine serum-0.1 mL Water

Internal Standard:see SPE Method,above.

Application Brief

Pharmaceutical

Wu Chen

Figure 1.Comparison of amitriptyline-spiked serum before (panel A) and after (panel B) extract ion using the Zorbax SPEC18 EC cartridge. Notice effectiveness of the extract ion. In panel A, the large amount of protein in the sample interferes withoptimal quantit ation. Once the sample has passed through the cartridge, analytes are clearly detected and quantif iable (panelB). Nitrobenzene is included in the samples as an internal standard.

SerumProteins

SerumProteins

Amitriptyline

A

B

Nitrobenzene

Amitriptyline

Amitriptyline

N

CH3

CH3

Nitrobenzene

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Copyright2001 Agilent Technologies

All Rights Reserved. Reproduction, adaptation or

translation without prior written permission is

prohibited, except as allowed under the copyright

laws.

Publication Number 5988-2522EN

For more information on our products and services,

please contact your local Agilent Technologies

office or your local authorized Agilent distributor, or

visit our website at: www.agilent.com/chem

Dr. Wu Chen is a research chemist at Agilent

Technologies, based in Wilmington, Delaware.

HPLC Method

LC Instrument: Agilent 1100Column: Zorbax Bonus-RP, 4.6 mm x 150 mm, 5 m (P/N 883668-904)Eluent: 75% Acetonitrile, 25% K2HPO4(18 mM, pH 6.85)UV: 254 nmFlow: 1.0 mL/min.Temperature: 30CInj. Vol.: 5 L for high load 20 L for low load

Recovery

Amitriptyline ( 1 g/mL) X= 100%, n = 5

Recovery of analyte in these experiments isan indication of goodness of technique, aswell as quality of the SPE packing. For bestresults, solutions should not be drawncompletely through the cartridge until thefinal elute step.