Chromatography
Key UHPLC Characteristics Required for High-throughput LC-MS
Dr Gesa J. Schad, Shimadzu Europa GmbH, Duisburg, Germany Dr Kyoko Watanabe, Dr Davide Vecchietti, Yuki Ito, Hidetoshi Terada, Dr Takato Uchikata, Yusuke Osaka, Shimadzu Corporation, Kyoto, Japan Dr Curtis Campbell, Shimadzu Scientifi c Instruments, Columbia, Maryland, USA
With high sensitivity and selectivity, LC-MS is a workhorse technique for quantitative assays. Mass spectrometry (MS) detection allows simultaneous identifi cation and quantitative analysis of multiple compounds, even if the chromatographic separation is imperfect. This ability dramatically increases the throughput for multiple component assays. In an ultra-fast UHPLC-MS method, the analytical cycle time is the most critical consideration. New generation UHPLC columns packed with sub-2 ¬µm or superfi cially porous particles for high effi ciency, allow for shorter column length and faster analysis. However, ultra-high throughput is not achieved simply by using the shortest columns. First and foremost, a rapid injection speed and high sample capacity are required. Secondly, the system must reduce other time-consuming factors, such as the rinsing time. If the target compounds have a strong tendency to adsorb to a system’s inner surface, the LC instrument must be rinsed carefully after every injection to avoid carryover that causes quantitative errors. This rinsing phase can considerably increase the analytical cycle time. And in addition, ‘non-data-acquisition time’, for column rinsing and system equilibration, has to be considered.
This article introduces the key front-end UHPLC competencies required for high- throughput LC-MS assays.
1. Injection Speed and Sample Capacity
Injection speed plays a pivotal role in achieving high sample throughput, as the duration of an injection sequence (= time required for the autosampler to inject a sample - not including post-injection rinsing) adds to the analytical cycle time. If it takes 30 seconds to inject a sample in a 30 second gradient run, half of the analytical cycle time is ‘non-data- acquisition time’. This is not conducive to a high-throughput objective. New generation UHPLC equipment, such as the Nexera series Sil-40 autosampler offer high-speed injections, with an injection cycle time of only 6.7 seconds to not impede a rapid analysis.
An example application that highlights the effectiveness of sample analysis using an ultra-fast injection sequence, is the high-speed analysis of drugs in blood plasma by LC-MS/MS [1]. The plasma samples, spiked with verapamil and its isotopically labelled analogue, were analysed in an 18 second isocratic method using a 5 x 2.1 mm UHPLC column. Figure 1 shows a representative chromatogram of the assay.
Sample capacity also plays a critical role in enabling high-throughput analysis, as it allows an extended time of unattended operation. The ease of adding samples to the running system is another important characteristic for high-throughput devices. An open-access design allows to add samples to an analytical sequence without interrupting an ongoing analysis.
2. Carryover and Rinsing Capability
With their ultra-high sensitivity, LC-MS/MS systems can detect trace levels of analyte that remain in the system and elute in a subsequent run. This so-called ‘carryover’ negatively impacts an analysis and can result in quantitative errors. It is also a serious issue with regards to throughput, as it requires additional rinsing phases within an analytical sequence, leading to longer cycle times. Autosampler and separation column are the most likely sources of carryover. Column-based carryover is best dealt with during method development - making sure that the mobile phase/gradient conditions are suffi cient to remove any trace amounts of the analytes. As for samples remaining in the autosampler, there are two ways to minimise the risk of system carry-over - the sampler design, and effective countermeasures.
In a ‘needle-in-fl ow-path’ design, the sample is aspirated into the needle and the total volume is swept directly onto the system. The sample loop is therefore part of the fl ow path, hence continually washed with the method gradient, aiding in reducing chemical adsorption. This design eliminates the intermediate step of loading a separate sample loop and the required valve openings and closings, which can trap the sample and lead to carryover. The total injection method is fast and clean - a pre-requisite for high- throughput LC-MS [2].
However, not all molecules allow to skip post-sample injection cleanup, they require additional methods to remove any remaining compound from the system. Typical areas of concern are the injection port and the autosampler needle. Rinsing the needle is one of the most effective solutions to fl ush out any remaining analyte on its surface. Most of the newer autosamplers have rinsing functions that allow dipping the needle into a washing solution. But it’s important to note that not only the outer but also the internal surface of the needle is exposed to a risk of adsorption. Additionally, the inside wall of the injection port can also be contaminated if the needle is not perfectly clean. Compounds may accumulate around these parts and become a source of carryover.
Overcoming this issue requires a well-designed washing program. Various rinsing methods and multiple rinsing solvents to wash the inside and outside of the needle, the sample loop, and the inside and surface of the injection port are helpful to address a wide range of chemical properties of the target compounds. One can design a wash routine to eliminate any carryover - strong organic wash, acidic or basic wash, ionic wash - whichever is needed for the class of compound in question. Figure 2 highlights the reduction of carryover with an additional internal rinse of the needle and injection port, compared to only an external rinse.
Figure 1. Linearity over the bioanalytically relevant concentration range (0.4 – 100 g/L). Black: Verapamil chromatogram. Pink: Verapamil-D6 chromatogram.
Of course, any wash/rinse routine will add time to the total autosampler sequence. And this may have an impact on the analytical sequence time.
INTERNATIONAL LABMATE - NOVEMBER 2022
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