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High Resolution LC-MS for Screening and Quantitative Analysis of Antibiotics in Drinking Water Using an Orbitrap and Online Sample Preparation
Overview
Purpose:
To demonstrate online sample pre-concentration and extraction of water
samples and analysis with high-resolution, accurate mass (HR/AM) detection,
quantitation and confirmation.
Methods:
Inject 1 mL water samples directly onto a trapping column. The trapped
compounds are then backflushed onto an analytical HPLC column and detected using
a Thermo Scientific Orbitrap mass analyzer.
Results:
This poster describes a method to perform screening and quantitation of
antibiotics at ppt and sub-ppt levels in drinking water using online pre-concentration
together with HR/AM confirmations of the compounds.
Introduction
Most current methodologies for the quantitation of antibiotics in drinking water revolve
around analysis using triple stage quadrupole platforms with offline sample
preparation. While this is a proven technique for the analysis of many contaminants in
drinking water, ground water and other environmental water samples, the offline
sample preparation steps are time-consuming and prone to operator error and
reproducibility problems. In addition, the need to transport large sample volumes from
the collection site to the laboratory, typically 1 L samples, is laborious. This poster
illustrates the ability to directly inject the water sample without any offline pre-
concentration steps, while achieving the same sensitivities required for the experiment.
Thus smaller sampling volumes can be used.
The method described here utilizes liquid chromatography-mass spectrometry (LC-MS)
with a Thermo Scientific Exactive Plus Orbitrap™ mass spectrometer using HR/AM.
While the triple stage quadrupole instrument is routinely used in these types of
experiments, we demonstrate the ability to use a benchtop HR/AM instrument to
quantitate and confirm the contaminants of interest. The advantages of HR/AM
instruments includes high resolution to isolate contaminants of interest from interfering
matrix peaks at similar masses as well as the ability to re-interrogate data at a later
date for additional compounds. Furthermore, compared to the triple stage quadrupole
instrument, method development time is greatly reduced as there is no need to
individually optimize each analyte of interest.
Methods
Sample Preparation
Samples were prepared from a stock solution of
antibiotics
in methanol. Calibration
solutions were prepared from the stock solutions, resulting in 8 levels of antibiotics for
positive analysis. Dilutions were made in laboratory water (HPLC-grade) to create eight
different calibration levels. The antibiotic calibration samples were acidified with formic
acid to a concentration of 0.1% formic acid. The concentration range varied for each
compound, but were in the approximate range of 1 ppt to 10 ppb. This ensured
compatibility with the mobile phase for chromatography. No further sample preparation
was conducted. The antibiotics studied for this poster were: carbamazepine,
erythromycin, ketoprofen, norethindrone, roxithromycin, sulfachloropyridazine,
sulfadimethoxine, sulfamerazine, sulfamethazine, sulfamethizole, sulfamethoxazole,
sulfathiazole, trimethoprim and tylosin.
Liquid Chromatography
Liquid chromatography was performed using the Thermo Scientific EQuan MAX
system. The EQuan MAX system consists of two high pressure liquid chromatography
(HPLC) pumps, autosampler and switching valves. The first HPLC pump, a Thermo
Scientific Accela 600 pump, is use to transfer the large volume sample from the
autosampler loop to the loading column (Thermo Scientific Hypersil GOLD aQ column,
20 x 2.1 mm, 12
μ
) at a flow rate of 1.0 mL/min. After 1.2 minutes, a six-port valve is
switched to back-flush the loading column onto the analytical column (Thermo
Scientific Accucore aQ column, 100 x 2.1 mm, 2.6
μ
), and remains inline for 11 minutes.
The analytes are eluted using an 11-minute reversed-phase gradient from the second
HPLC pump, the Thermo Scientific Accela 1250 pump. The mobile phases were water
(A) and methanol (B), both containing 0.1% formic acid and 4mM ammonium formate.
The gradient program for both pumps is shown in Table 1. After 12 minutes of runtime,
the loading column is returned to its original position, taking the analytical column
offline from the loading column, and the system is re-equilibrated for the next injection.
The total run time is 15 minutes. The flow diagram for the EQuan MAX system is
shown in Figure 1.
Mass Spectrometry
The Exactive™ Plus Orbitrap mass spe
Exactive Plus was operated in alternati
mode with positive electrospray ionizati
collected, and subsequently, all of the i
higher-energy C-trap dissociation (HC
30 eV with a 20% stepped CE, and ana
resolution for the full scan experiment
experiment was 35,000. The mass ran
and 80-1000 amu in the AIF experimen
Data Analysis
Data was collected and analyzed using
Spectral confirmation was carried out
Time
(min)
Loading Pump
%A
Flow Rate
(
µ
L/min)
Ti
(
0.0
100
1000
1.3
100
1000
1.5
100
100
12.0
100
100
12.1
100
1000
15.0
100
1000
1
TABLE 1. HPLC gradients for the loadi
FIGURE 1. EQuan MAX system flow