Page 56 - Clinical-Medical Solution for Clinical Chemistry

A Novel Platform of On-line Sample Pre-treatment and LC/MS/MS
Analysis for Screening and Quantitation of Illicit Drugs in Urine

Introduction

In recent years, LC/MS/MS methods are adopted in ltering by vacuum ltration, and sample derivatisation
analyses of illicit and prescription drugs in toxicological with heating. Internal standard and reagent for
samples such as urine and serum. Sample pre-treatment is derivatization or other purposes can be added to a sample
always a critical step in the whole analysis procedure and before or after protein crash. We describe development of
on-line sample pre-treatment is desired not only for an automated sample pre-treatment using a Shimadzu
improving analysis throughput, but also minimizing human CLAM-2000 module coupled with Shimadzu LCMS-8040
errors. The CLAM-2000 module is designed for on-line TQ system. It involves IS addition, protein precipitation,
sample pre-treatment in high throughput LC/MS/MS ltration and transferring the nal solution to LC/MS/MS
analysis of drugs and metabolites in biological samples for analysis. This new platform was applied and evaluated
such as plasma/serum and urine. Many sample preparation for quantitation of 18 illicit drugs with 14 isotope-labelled
process can be performed automatically such as dispensing internal standards (IS).
solvents, sample-reagent mixing by vortexing, sample

Sample Reagent Shaking Filtration Sample
pipetting Pipetting Transfer Samples are transferred to
LC/MS/MS

Figure 1: Procedure of protein crash and spiked-sample preparation

Experimental

Sample preparation and analytical conditions

A total of 18 illicit drugs and 14 isotope-labeled internal mixed internal standard (IS) stock solution was added to
standards (except for phencyclidine, methaqualone, the sample, followed by addition of 40 µL of organic
methadone and propoxyphene) were used for setting up solvent (MeOH : ACN = 1 : 1 in volume). The sample
the MRM quantitation method. The urine samples, mixture was vortexed and ltered into a collection vial
internal standards mixed solution and organic solvents before injecting to LCMS-8040. A Phenomenex Biphenyl
were pre-loaded onto the CLAM-2000. An automated column (100 x 2.1 mm I.D., 2.6µm) was used for the
batch-run program allows sample pre-treatment and analysis of 18 analytes and 14 IS with a gradient elution
analysis to perform concurrently on the CLAM-LC-MS/MS program of 11 minutes. A calibration series of spiked
platform. Table 1 shows the analytical conditions on standard samples in urines were prepared in
LCMS-8040. Figure 2 illustrates the automated work ow concentrations of 20, 50 and 200 ng/mL. The
on the CLAM-2000 module. An aliquot of 20 uL of urine concentration of each IS was 100 ng/mL. A LCMS-8040
sample was dispensed into a ltration vial. Then, 20 µL of with ESI was employed in this work.

2