Page 56 - CRFTApplication Compendium

Results and Discussion

LC-MS/MS is a highly selective technique for analyzing

drugs from biological matrices. As shown in Figure 1,

samples are loaded to the extraction column via an

autosampler and LC pump combination. After a short

washing time, the central valve is rotated 60° to the injec-

tion position, which allows a second pump to elute the

analytes from the extraction column onto the analytical

column and into the FAIMS-enabled mass spectrometer. In

cases where background or co-eluting interferences appear,

the limiting factor is selectivity.

FAIMS is a unique selectivity enhancing tool for LC-

MS. With FAIMS, gas-phase ions are purified after LC

analysis but before they are mass analyzed. The waveform

shown in Figure 2 separates the gas-phase ions as they are

transferred into the high vacuum region of the MS. The

interference ions in orange are filtered out from the ion

beam, while the analyte ions in blue pass into the MS.

The basic experiment that establishes FAIMS condi-

tions is shown in Figure 3. While infusing a reference

standard of testosterone into the mobile phase, the CV is

scanned over a specific range. The black trace appears for

the transition due to testosterone, and a maximum signal

for testosterone appears at CV –12.5V. To ensure that

mobile phase components with the same transition will

be eliminated, stop the infusion of the reference standard

and repeat the CV scan over the same specified range.

The red trace indicates that chemical background in the

mobile phase emerges from FAIMS not at the CV for

testosterone, but rather in a broad range between CV –15

and –25V. For subsequent LC-FAIMS-MS/MS assays, the

CV set to –12.5V will exclude mobile phase contributions

to the analysis of testosterone. Other endogenous compo-

nents present in the human serum samples may also be

excluded if their FAIMS behavior is different from that of

testosterone.

Regression analysis based on a linear model with

1/[concentration]

2

weighting was used. The average

accuracy, as deviation from theoretical is less than 5% at

all concentrations. The precision of the standards, as

relative standard deviation (%RSD), is less than 17% at

the lower limit of quantitation and less than 11% at all

other concentrations.

Despite the excellent performance of LC at cleaning

up samples, many interferences are still present from the

matrix. Other extraction techniques might remove these

Pump

Waste

Autosampler

Analytical Column

FAIMS-enabled

MS/MS

FAIMS-enabled

MS/MS

Extraction

Column

Extraction

Column

Figure 1: Schematic diagram of LC-FAIMS-MS/MS system

Load sample to extraction column

Inject sample to analytical

column for FAIMS-MS/MS

analysis

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