Page 13 - Clinical-Medical Solution for Clinical Chemistry

Evaluation of Blood Lysis Procedures prior to Automated
Sample Preparation for Immunosuppressant Assay by LC-MS/MS

Introduction

1)
CLAM-2000 (Shimadzu Corp., Japan) fully automates in erythrocytes . Thus, it is mandatory to release
blood or other samples pre-treatment prior to LC-MS erythrocytes prior to any centrifugation of the blood.
analysis (Fig.1). The whole blood has cell debris, brin clots Current practice is to freeze and thaw samples, which is
and so on. It is desirable to remove such particulates for not convenient for emergency analysis. To address this,
accurate sample dispensing by centrifugation. However, several protocols of hemolysis were tested. Evaluation of
this poses a challenge to the measurement of the protocol was based on lysis ef ciency,
immunosuppressants (ISP) in whole blood samples since immunosuppressant recovery and time consumption.
large proportion of ISP are bound to cytoplasmic proteins

Figure 1. Fully automated sample preparation module CLAM-2000 and
triple quadrupole mass spectrometer LCMS-8060.

Methods and Pretreatment

Individual blood sample from healthy volunteer was spiked aliquots of each sample were subjected to all lysis
with tacrolimus (MW: 804.0), sirolimus (MW: 914.2), protocols. Lysis ef ciency was estimated by measuring
everolimus (MW: 958.2), and cyclosporine A (MW: 1202.6) hemoglobin absorbance in supernatant using a UV-Visible
and incubated for 30 minutes at room temperature. Then spectrophotometer (UV-1280. Fig.2).

2