AI-64310-CRFT

Full Optimization of LC/MS Methods to Increase Robustness of Complicated Matrix Containing Samples Using Active Flow Management Chromatography

Overview

Purpose:

This poster demonstrates the proof of concept of using active flow

management (AFM) or curtain flow (CF) chromatography for LC/MS research and

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Methods

Columns were connected in t

2.1 mm i.d. column, a 1 mL/

orens c me o s o ncrease sens v y an ro us ness espec a y w en comp ex

matrices are used.

Methods:

To investigate improvements in sensitivity and reproducibility, a model

50 x 4.6 mm i.d. AFM column was compared with a conventional 50 x 4.6 mm i.d.

column and a conventional 50 x 2.1 mm i.d. column using a series of standard steroids.

FIGURE 2 Illustrations of c

volume to maintain concentra

included a 70% post-column

introduction to the MS source

Accucore™ C18 2.6 µm stati

Comparisons were made of peak heights and areas, S/N, %RSD, and %Diff (precision

and accuracy) between column dimensions. Mobile phase flow rates were adjusted to

supply an equal flow to the MS source. Detection limits were also determined using

alprazolam (2 fg to 2 pg alprazolam on column) in crashed plasma. Column robustness

was investigated through repeated injections (20 µL) of a 5 fg/µL alprazolam solution in

the connective tubing was

velocities and split flow rati

connected directly to inject

column connected directly

column C) Curtain flow 50

(1:1 v) synthetic urine. All analyses were performed on the Thermo Scientific™ TSQ

Quantiva™ triple quadrupole mass spectrometer.

Results:

Comparison between a conventional 50 x 4.6 mm i.d. column with a post-

column split (PCS), a 50 x 2.1 mm i.d. column, and a CF 50 x 4.6 mm i.d. column

showed increases in peak areas, heights, and S/N with the CF column over both the

Pump

Injector

injector to peripheral inlet.

from injector to MS source.

conventional 4.6 mm i.d. (on average, 2.5 times increase) and conventional 2.1 mm i.d.

(on average, 3.5 times increase), while increasing both precision and accuracy of the

analysis. Analysis of alprazolam in 30% crashed plasma showed increased sensitivity,

reducing the LOQ of 6 fg on column and an LOD below 2 fg on column. Further

robustness studies showed excellent peak shape retention following more than 220

injections of 100 fg on column of alprazolam in synthetic urine.

Introduction

Small-bore (2 mm i.d.) columns are a very popular choice for LC/MS separations,

The LC system was a Therm

flow column installation, the fl

autosampler. Mobile phase c

A: 0.1% formic acid (aq), B:

primarily due to their optimum flow rate facilitating both electrospray and atmospheric

pressure chemical ionization methods. One disadvantage of small-bore HPLC columns

is their increased wall effects that decrease chromatographic efficiency. A study by

Gritti and Guiochon

showed that the loss in optimal performance in a 2.1 mm i.d.

column format compared to a 4.6 mm i.d. format (based on the reduced plate height,

)

isocratically. Gradient elution

2 mM ammonium acetate (N

linearity) and 2 mM NH

OAc (

rates were measured volume

source (with the exception of

for superficially porous particles, with particle size diameters of 1.7 to 2.7 µm was

between 13% and 42%, depending on particle size, column length, and manufacturer.

The use of narrow-bore columns may also limit sensitivity in LC/MS research and

forensic methods due to the reduced mass loading capability of the column, which is

essentially proportional to the square of the column radius. One approach to remove

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Results

selected-reaction monitoring (

ionization (HESI) mode

e n uence o wa e ec s on co umn e c ency w c s presen e ere uses cur a n

flow chromatography. In curtain flow columns, the mobile phase is managed at both the

inlet and outlet of the column to create a virtual column inside the analytical column

(Figure 1).

The virtual column has a narrower internal diameter; the dimensions are

related to the volumetric ratio of flow exiting the column through the central zone

relative to the flow exiting the peripheral zones Using an active flow management

Column Performance

Column performance was me

nortestosterone, testosterone

100 pg/µL with six replicate in

chromatograms of the test co

chromatography column with an overall larger inner diameter (4.6 mm) adds an

additional benefit of increased loading capacity (facilitating larger injection volumes)

and increasing robustness to matrix containing samples.

FIGURE 1 Illustration of curtain flow column Mobile phase is split prior to the

parameters of peak area, hei

compared. Figure 4 shows pe

height comparison (C), and

FIGURE 3. TIC Chromatogr

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sample injector. Sample is injected to the central flow region. Mobile phase flow

is managed at both the inlet and outlet ends of the column. Virtual column

diameter is related to the ratio of flow between the central flow region and the

peripheral flow region.

es os erone an ep es os

PCS, B) Conventional 2

1 m

Intensity was normalized to

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Central flow

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to source

from injector

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