Page 14 - Clinical-Medical Solution for Clinical Chemistry

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

Lysis Protocols

Lysis reagents were prepared as following. Ammonium chloride mixture was prepared with ammonium chloride,
NaHCO3 and EDTA in water. HCl solution was diluted HCl at 2 mol/L in water. Each lysis protocols were performed
prior to centrifugation
• Control (1 mL)
• Ammonium Chloride mixture (500 L) + blood (250 L)
• HCl solution (100 L) + blood (1 mL)
• Blood sample (1 mL) with the ultrasound (5 min)
• Blood sample (1 mL) with the ultrasound (10 min)
• Blood sample (1 mL) with the ultrasound (20 min)
• Blood sample (1 mL) with freeze/thaw -20ºC (30 min)
• Blood sample (1 mL) with freeze/thaw -80ºC(30 min)
• Blood sample (0.2 mL) with freeze/thaw -20ºC (30 min)
• Blood sample (0.2 mL) with freeze/thaw -80ºC (30 min)

Figure 2. UV-Visible Spectrophotometer UV-1280.

Pretreatment for UV-visible spectrophotometer
Each lysis blood samples (8 L) are mixed with Drabkin’s reagent (2 mL) which has a role of quantitative, colorimetric
determination of hemoglobin concentration in whole blood with 540 nm band.

Pretreatment for LC/MS

13C, D2-tacrolimus, D3-sirolimus and D4-everolimus were used for ISTD. Zinc sulfate mixture was prepared with
ACN, MeOH, zinc sulfate and ammonium formate. A blood sample (50 L) was mixed with ISTD (25 L) and zinc
sulfate mixture (350 L). After centrifugation, ISP recovery was measured using an online SPE-LC-MS/MS method.
Supernatant obtained in each tested condition was automatically prepared using CLAM-2000. This included addition
of internal standards, protein precipitation, ltration and transfer to the LC autosampler. Acquisition was performed
in MRM mode using a triple quad mass spectrometer (LCMS-8060).

Pump A Pump B
Mobile Phase A Mobile Phase B

Analytical
2 1 Column LCMS
Auto Sampler 3 6 MP A : 90%Buffer + 10%MeOH

MP B : 10%Buffer + 90%MeOH
Elution Load 4 5 * Buffer: Ammonium formate (3 mM, pH 3.6) in water
(Phase B) TRAP Column (Phase A) Valve Position [0] : Red (2-3), [1] : Blue (1-2)
Drain Valve Position 1 : START METHOD (LOAD)
Valve Position 0 : ELUTION

Figure 3. Flow diagram for online SPE

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