Page 17 - Shimadzu Journal vol.5 Issue1

Selection 10 Clinical research
Quantitative Analysis of β-Lactam Antibiotics in Human Plasma by High Sensitivity LC/MS/MS
Method
The β-lactam type antibiotics are used in the treatment of various bacterial infections in human over decades. One of the
consequences of continuous uses of antibiotics is the progressive development of drug resistance of bacteria in human [1]. Therapeutic
Drug Monitoring (TDM) aims at obtaining pharmacokinetic pattern of an antibiotic in patient to develop personalized medicine
treatment. Conventional TDM methods such as immunoassays are well-established. However, one of the drawbacks of immunoassays
is lack of specificity due to cross-reactivity with metabolites, which may give false positives [2,3]. Recently, LC/MS/MS has been used for
fast and direct measurement of β-lactam antibiotics such as amoxicillin [4] and piperacillin, etc. [5,6] in human plasma. In this
application news, a fast LC/MS/MS method with a simple sample pre-treatment procedure for quantitative analysis of five β-lactam
antibiotics, meropenem (MER), tazobactam (TAZ), piperacillin (PIP), cefepime (CEF) and ceftazidime (CFT) is described. A small injection
volume of sample of this MRM-based method is required only, which minimizes the contamination of sample matrix, as such, reducing
the cleaning and maintenance time of the interface of LC/MS/MS in clinical research work

Selection 11 Clinical research
Solid Phase Extraction and SFC-MS/MS Method for Analysis of Aflatoxins M1, M2, B1, B2, G1 and G2
in Milk Powders
Aflatoxins B1, B2, G1 and G2 are secondary metabolites that are produced by Aspergillus fungi. If aflatoxins B1 and B2 are present
in feeds consumed by lactating animals, they can be converted to hydroxylated metabolite aflatoxins M1 and M2 in the animals
respectively, which are found eventually in the milk [1]. Aflatoxins B1, B2, G1, G2 are carcinogenic and aflatoxin M1 is potentially
carcinogenic. The levels of these aflatoxins in dairy products like milk powders are strictly regulated around the world. For example,
European Union regulation limits aflatoxin M1 in milk below 0.050 µg/kg [2]. Many LC/MS/MS methods were reported for analysis
of aflatoxins B1, B2, G1, G2 and M1, but few including aflatoxin M2 [4]. In addition, sample preparation method is varied for
different matrixes and is often a critical factor to the analysis sensitivity and accuracy. The aim of this study is to develop a novel
method - supercritical fluid chromatography SFC-MS/MS for high sensitivity analysis of aflatoxins B1, B2, G1, G2, M1 and M2 in
milk powders. The supercritical fluid (SF) CO2 mobile phase has advantages of cost-effective and environmental friendly as
compared to organic solvents. A special sample preparation procedure was explored, which involved extraction with adding Q-Sep
extract salt and purification with Supel™ Tox Alfazea SPE cartridge. Conventional SPE uses the concept of binding target
compounds to the stationary phase first and eluted out by organic eluent subsequently. However, Supel™ Tox Alfazea SPE
cartridge acts as a filter where the sample matrix is trapped while the target aflatoxins are eluted out.

Selection 12 Clinical research
A High Sensitivity LC/MS/MS Method for Quantitative Analysis of Eight Antifungal Drugs in Human
Serum
Triazoles and echinocandins are commonly used for the treatment of invasive fungal infections via systemic antifungal
chemotherapy. However, these drugs exhibit substantial pharmacokinetic variability in patients such as bioavailability and
drug-drug interactions [1,2]. Clinicians often find it challenging to select proper drug doses and evaluate the potential toxicity
effects. Therapeutic drug monitoring (TDM) of antifungals is essential to maximise the efficacy and minimise drug overdose risk in
patients, hence individualising the treatment [3]. In this study, we aim at developing a fast and reliable LC/MS/MS method with
high sensitivity and simple sample pre-treatment. The method is established for simultaneous determination of two classes of
antimycotic compounds, five triazoles and three echinocandins in human serum. The method performance is evaluated with spiked
serum samples thoroughly before further implementation and validation with clinical samples.

Selection 13 Clinical research
Quantitative clinical toxicological screening comparing Library ID from production scan MS/MS to
MRM spectrum-mode ID.
Forensic toxicological sample measurement is commonly performed in a targeted analysis on selected panels of compounds. When
using triple quadrupole platforms for analysis, typically two MRMs are used for compound measurement with a quantifier ion
transition and reference ion transition. To help reduce false positive and false negative reporting two alternative approaches have
been considered; MRM triggered product ion spectrum and MRM Spectrum mode. MRM Spectrum mode acquires a high number
of fragment ion transitions for each target compound generating a fragmentation spectra that could be used in routine library
searching and compound verification using reference library match scores. In this work, we compare different approaches in target
quantitation and identification applied to clinical and forensic toxicology.

Selection 14 Clinical research
Toxicological screening for over 1,000 compounds in an MRM based acquisition for Library ID in
whole blood samples
A library of product ion spectra for 1,222 compounds has been developed for clinical and forensic toxicology screening to help
reduce false positive and false negative reporting. The library enables multi-targeted methods to be developed for routine
screening, library identification and quantitation. The scope of the library considers two approaches; a MRM triggered full scan
product ion spectra and MRM Spectrum mode. MRM Spectrum mode acquires a high number of fragment ion transitions for each
target compound generating a fragmentation spectra which can be used in routine library searching and compound verification
using reference library match scores. In this work, MRM Spectrum mode has been applied to analysis of patient samples to
quantify and identify targets in whole blood samples extracted using a QuEChERS method.