Application Notes
Combinatorial Sample Analysis by HPLC/ELSD
Examples of the use of reversed phase HPLC with ELSD detection are given for combinatorial drug analysis. Separations of an aspartate antagonist in synthesis solution and mixture of catecholamines are shown.
Combinatorial Sample Analysis, Alltech Application Note 0050LE, July 7, 2000.
Evaporative light scattering detection (ELSD) is presented as an alternative to MS and UV detection from HPLC separations of combinatorial samples, pharmaceutical compounds produced for the first time for which there are no standards. Because the response of the ELSD is based entirely on the mass of the sample, internal standards can be used instead of standards of the target analytes.
An n-methyl-d-aspartate antagonist in a drug synthesis solution was separated from its impurities by gradient elution in around 20 minutes by reversed phase HPLC on a Zorbax SB-Phenyl, 250 x 4.6mm column (Agilent Technologies Part No. 880975-912) using water/acetonitrile and detected by evaporative light scattering using an Alltech Model 2000 (Evaporative Light Scattering Detector.) The same separation with UV detection gave misleading results due to weak chromophores for the antagonist and strong ones for the impurities.
A mixture of catecholamines (noradrenaline, adrenaline, 3-hydroxytyramine, 3,4-dihydroxyphenylalanine, phenylalanine and homovanillic acid) was separated by gradient elution in around 7 minutes by reversed phase HPLC on a Platinum EPS C18, 3µm, 53 x 7mm Rocket column (50573) using water and TFA/acetonitrile and detected by the ELSD. The same separation with UV detection suffered from a drifting baseline.
In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
Key Analyte(s)
3,4-dihydroxyphenylalanine3,4-dihydroxyphenylalanine (L-phenylalanine)is the primary component of the artificial sweetener aspartame. It is most frequently measured in food products using HPLC techniques.
3-hydroxytyramine (Dopamine)3-hydroxytyramine (Dopamine) is a neurotransmitter in the brain also used to treat Parkinson\'s disease. It is most often measured using HPLC techniques.
adrenaline (epinephrine)Adrenaline (epinephrine) is a catecholamine hormone produced by the body and used therapeutically to reduce swelling due to allergies. I tis msot often measured using HPLC.
homovanillic acid
n-methyl-d-aspartate antagonist
noradrenaline
phenylalanine
Technique
HPLCHigh Performance Liquid Chromatography (HPLC)is a technique for separating components in a sample on the basis of interactions between the component, a liquid mobile phase and a solid stationary phase.
Source
Alltech Associates, Inc.Alltech Associates, Inc., one of the leading suppliers of HPLC columns, manufactures and distributes HPLC, GC and IC columns, accessories and consumables and maintains a large searchable database of application notes and chromatograms on its website.
