Page 27 - Clinical-Medical Solution for Clinical Chemistry

Application Clinical Research / LCMS-8060

News A Fast LC/MS/MS Method for Quantitative Analysis
of Five β-Lactam Antibiotics in Human Plasma

Zhi Wei Edwin Ting , Kelvin Loh Shun Cheng*, Daryl Kim Hor Hee , Lawrence Soon-U Lee , Jie Xing 1
2
2
1
1
AD-0135 & Zhaoqi Zhan 2
Shimadzu (Asia Pacific) Pte Ltd, Singapore; Clinical Analysis Centre, Department of Medicine
1
Research Laboratories, National University of Singapore; * ITP student from NTU, Singapore
 Introduction
The β-lactam type antibiotics are used in the treatment of prepared: 20, 40, 80, 200, 400, 2000 and 4000 ng/mL in
various bacterial infections in human over decades. One of plasma. The concentrations of internal standards were 200
the consequences of continuous usage of antibiotics is the ng/mL or 800 ng/mL in these calibrants. A LCMS-8060, a
progressive development of drug resistance of bacteria in triple quadrupole LC/MS/MS system with heated ESI was
human [1]. Therapeutic Drug Monitoring (TDM) aims at employed in this work. The analytical conditions and
obtaining pharmacokinetic pattern of an antibiotic in patient instrumental parameters are compiled into Table 1.
to develop personalized medicine treatment. Conventional
TDM methods such as immunoassays are well-established. Table 1: Analytical conditions and parameters on LCMS-8060
However, one of the drawbacks of immunoassays is lack of Kinetex 1.7µ C18 100A
specificity due to cross-reactivity with metabolites, which Column (100 mmL x 2.10mm I.D.)
may give false positives result [2,3]. Recently, LC/MS/MS A: Water with 0.1% FA
has been used for fast and direct measurement of β-lactam Mobile Phase B: Acetonitrile with 0.1% FA
antibiotics such as amoxicillin [4] and piperacillin, etc. [5,6] Gradient elution (5.5 minutes)
in human plasma. In this application news, a fast LC/MS/MS Elution Program B: 5% (0 to 0.2 min)  90% (3.5 to 4.0
method with a simple sample pre-treatment procedure for min)  5% (4.1 to 5.5 min)
quantitative analysis of five β-lactam antibiotics meropenem Flow Rate 0.5 mL/min
(MER), tazobactam (TAZ), piperacillin (PIP), cefepime Oven Temp. 40ºC
(CEF) and ceftazidime (CFT) is described. A small injection Injection 2 µL
volume of sample of this MRM-based method is required Interface ESI (heated)
only, which minimizes the contamination of sample matrix, MS Mode MRM, Positive
as such, reducing the cleaning and maintenance time of the Block Temp. 400ºC
interface of LC/MS/MS in clinical research work. DL Temp. 250ºC
Interface Temp. 300ºC
CID Gas Ar, 270 kPa
Nebulizing Gas N 2 , 3.0 L/min
Drying Gas N 2 , 5.0 L/min
Heating Gas Zero Air, 15L/min

125 µL of Pool Human Plasma
Figure 1: Structure of meropenem (MER) with a β-lactam ring.
(1) Spiked 5 µL of I.S. (2) Add 365 µL of ACN/MeOH (1:1)
 Experimental Shake & Vortex for 10 mins

Sample preparation and analytical conditions
Five antibiotics used in this study are meropenem (MER), Centrifuge for 10 mins at 13,000 rpm
tazobactam (TAZ), piperacillin (PIP), cefepime (CEF) and
ceftazidime (CFT). The compounds and four stable isotope- ~480 µL of Supernatant
labelled meropenem-d6, piperacillin-d5, cefepime-cd3 and
ceftazidime-d6 as internal standards were purchased from
certified suppliers. Pool human plasma was obtained from i- 0.2 µm Nylon Filter
DNA Biotechnology Pte Ltd and used as matrix. The
sample pre-treatment and spiked sample preparation ~400 µL of Filtered Spiked Plasma
procedure are illustrated in Figure 1. A simple protein crash
method was applied by adding ACN:MeOH (1:1) to plasma 2 µL injected to LCMS-8060
in a ratio of 3:1, followed by vortex and centrifuge. A
calibration series of spiked standard samples were Figure 2: Procedure of protein crash and spiked-sample preparation