Chromatography
The Use of Short 10 mm Columns for Rapid LC-MS Analyses Matt James1 , Arianne Soliven1 , Colin Pipe, David Dunthorne, Mark Fever, Tony Edge*, Avantor, 1 The Markham Centre, Station Road, Theale, Reading, RG7 4PE, UK. 1Joint fi rst authorship *Corresponding author
anthony.edge@avantorsciences.com Abstract
This brief communication demonstrates the utilisation of a short 10 mm length, narrow bore 2.1 mm internal diameter (ID) column for rapid LC separations coupled with mass spectrometry (MS) detection for high sample throughput (HTP). We demonstrate the capabilities for three applications that differ in sample and separation complexity; a sample separated via isocratic separation conditions and two samples that require differing gradient conditions. For all three cases, high laboratory productivity rates were calculated based on the number of samples that can be analysed in a 24-hour time frame and ranged from 464- 1200 samples.
Introduction
Rapid LC-MS assays are valuable for analytical laboratories that have a large number of samples to analyse and/or increase laboratory productivity [1]. The MS detector’s speed and selectivity for ionised species can facilitate the use of direct injection HTP assays [2,3]. However, direct injection-MS analyses are prone to sample matrix effects, where the presence of competing ionised species can lead to suppression and in some cases the enhancement of the signal response [4]. Hence, the integration of a front-end liquid chromatography column separation provides an additional degree of resolution/peak capacity to separate targeted analytes or isobaric species from the sample’s matrix, before ionisation, desolvation and MS detection [5].
The column length selection is a critical practical parameter for maximising the peak capacity for the separation of complex low molecular weight samples [6]. On the other hand, to develop rapid LC HTP assays, we must reduce the column volume used in the LC front-end separation strategy [7]. Moreover, for gradient elution conditions, reducing the sum of the gradient time and re-equilibration ‘total cycle time’, can be achieved by employing low-volume, highly effi cient columns [8]. In this short communication, we highlight how to exploit short 10 mm columns to achieve rapid LC-MS analysis for three different low molecular weight applications, with differing sample complexities.
Experimental
The short 10 mm length column with a narrow bore 2.1 mm ID, was packed with 2-micron particles (dp
) to provide suffi cient plates and minimise the column volume.
The cartridge style small column format is housed within hardware that facilitates the use of standard 1/16-inch fi ttings and outer-diameter connection tubing. For optimum use, the data acquisition rate must be increased. For quantifi cation purposes, we recommend collecting at least 10-15 data points across each peak by applying fast detector sampling rates/dwell times for the small volume peaks [9]. The column separation and MS detection details are listed in the respective application (Figures
1-3). For all post-column fl uidic connections, 0.12 mm ID PEEK tubing was used. Standards were of high analytical grade and solvents were of HPLC grade and were sourced via VWR International, UK – part of Avantor® were sourced through Wellington Laboratories, US.
, except for PFAS standards, which
With respect to minimising the extra-column dispersion, direct connection to grounded ionisation sources is ideal, otherwise it is best to minimise the length and internal diameter of the connection tubing of the LC-MS workfl ow, particularly between the outlet of the column and the inlet of the ion source. For safe practice, the column must be installed in a manner that ensures it is always electrically grounded.
Discussion
To demonstrate the high throughput capabilities when exploiting the use of the 10 mm short length column, we highlight the application of the HTP-MS column for separations that vary in sample and separation complexity (Figures 1-3). In Figure 1, isocratic separation conditions were employed for the HTP separation of three immunosuppressants. The additional advantage of exploiting high-temperature liquid chromatography was used (compatible with both the sample, stationary phase, and instrumentation). An isocratic peak width of 0.25 min was achieved. The sample complexity does not require the use of gradient elution, hence there is no need to re-equilibrate the column with a fi xed mobile phase composition fl owing through the column from injection to injection. A total cycle time of <1.2 min was achieved in the fi nal assay, representing a lab productivity rate equivalent to analysing approximately 1,200 samples in a 24-hour time frame. This is the maximum sample throughput if operated with stacked injections (no injection cycle time).
Figure 2: a series of nine different low-molecular-weight nonsteroidal anti-infl ammatory drugs (NSAIDs), were separated using the 10 mm length column hyphenated to the MS.
Figure 1: isocratic separation conditions were employed for the HTP separation of three immunosuppressants.
In the second application (Figure 2), a series of nine different low-molecular-weight nonsteroidal anti-infl ammatory drugs (NSAIDs), were separated using the 10 mm length column hyphenated to the MS. The sample complexity was higher compared to the simpler fi rst application and therefore required gradient elution. The gradient separation
INTERNATIONAL LABMATE - NOVEMBER 2023
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