The versatility of liquid ­chromatography-tandem mass spectrometry (LC-MS/MS) has made this technique one of the most advantageous in the field. However, the diverse chemical properties of potential analytes pose numerous challenges as clinical laboratory test menus expand.

This article will explore some of these challenges, highlight some best practices for addressing them, and discuss available technology that may represent an opportunity to eliminate some of these challenges today.

How to Optimize Consumables

Many parameters must be optimized when developing LC-MS/MS analytical methods for analysis in biological matrices, including extraction, chromatography, and instrument-specific parameters. Analytical columns are one such area. Columns are an expensive consumable used in routine LC-MS/MS analysis, and laboratories should take care to assure maximum performance of an analytical column throughout an assay.

Laboratories also need to use LC-MS/MS-grade solvents and high-quality deionized water with ultraviolet treatment. Many laboratories are swayed toward lower-priced, lower-quality solvents. However, low-quality solvents come with long-term costs like unanticipated downtime and resource-intensive troubleshooting.

Additionally, inline frits, filters, and any pre-columns used should be changed often between analyses to prevent contaminants from accumulating on the analytical column. Impurities on the analytical column can cause high system backpressure, background noise associated with the flow and gradient, shifting retention times, and abnormal peak shape, adding to the uncertainty of assay results.

Laboratories before analysis also should carefully inspect all vials for particulates and precipitated proteins in extracts. Precipitated proteins can adsorb analyte and contaminate and clog the tubing and injector components of the sample manager.

Paying Attention to the Mobile Phase

Clinical laboratories often use mobile phase additives in LC-MS/MS to optimize the separation and ionization of different compounds of interest. While these additives are frequently necessary, they can contribute to negative outcomes in routine LC-MS/MS analysis if not used properly. Mobile phase should be prepared using only high-quality additives and at the lowest concentration possible in order to achieve the desired result.

When changing over solvents of different composition, lines should be flushed with at least five volumes of high-purity water as an intermediate solvent to prevent salt precipitation. Salt precipitation in the LC-MS/MS system can cause pump failures, high background noise contamination, and reduced sensitivity due to accumulation on source components.

Use of additives and aqueous buffers in LC-MS/MS mobile phase is also conducive to microbial growth that can contribute to many of the same problems encountered with salt precipitation. Laboratories can mitigate microbial growth by sealing mobile phase reservoirs in between assays, discarding mobile phases in use past a certain timeframe, and rinsing solvent bottles thoroughly between uses or at other defined intervals.

LC-MS/MS Maintenance

One key aspect of monthly maintenance is flushing the LC components. Buildup of contaminants from solvents and extracts often causes common problems such as solvent pump failures, carryover, high system pressure, high background, and interferences. Replacement of expensive parts becomes the solution to most of these issues in the absence of regular flushing.

Laboratories should use a manufacturer-recommended mixture of solvents and aqueous acid and ensure that pumps and needle wash systems are primed and a low flow rate set. Injections of solvent at high volume will wash both the sample path and the flow path. To tackle tough system contamination from sample injections of exceedingly high concentrations, laboratories at this step should inject dimethyl sulfoxide at high volume.

During this time, back-flushing using specific solvents and column temperatures based on the column type might revive an analytical column. System flush should always be routed to waste, not into the MS.

Monthly maintenance also includes washing the mobile phase bottles (reservoirs) and testing the solvent pumps for leaks according to the manufacturer’s recommendations. For example, using detergents is not recommended for mobile phase reservoirs. Laboratories may also want to test pumps so that they can be rebuilt before they fail while being used.

Due to the volume of samples and the myriad of sample types injected, preventive maintenance along the sample path is crucial. Mitigating contaminant buildup is absolutely essential—beginning with regular front-end column maintenance, through column fittings and PEEK tubing, and ending with front-end maintenance of the mass spectrometer. While proactive maintenance saves time, some reactive maintenance will be inevitable.

The diversity and volume of samples most LC-MS/MS services see also necessitates proper equilibration of both the LC and MS components while switching over a system. While mobile phase lines should be flushed between assays, MS parameters can be set to allow sufficient time for the MS source and desolvation temperatures and gas flows to reach set points. Starting an analysis without properly equilibrating the MS can cause changes in response and inaccurate quantitation. Labs also need to allow the column temperature to reach its set point between assays to maintain consistent backpressure and retention times. Matrix effects can vary depending on where an analyte elutes in the gradient. Once all of these set points are reached, the analytical column should be equilibrated with 10-20 volumes of mobile phase before beginning to run system suitability samples.

For typical mass spectrometry laboratories, maximizing the capacity of LC-MS/MS systems will often mean performing a wide array of methods on the same system. In order to accommodate a growing test menu, a laboratory may very easily generate a wide variety of methods that utilize different extraction techniques, different analytical columns, and different mobile phase compositions. Depending on the daily or weekly test volume, the laboratory may need to change out columns and mobile phases numerous times between different analytical runs.

The amount of equilibration between runs also can vary depending on an analytical method’s specific parameters. For instance, an analytical method that utilizes an extraction protocol or specific mobile phase modifiers may be incompatible with another method, requiring more time—and sometimes maintenance—before the subsequent method is ready for use.

System Suitability Testing

Due to the large number of assays running on a given system, system suitability should be performed for each new assay set up on a system. While not specifically outlined in current clinical regulatory guidance, system suitability testing has become a valuable mechanism for ensuring proper system configuration before labs begin analyzing prepared samples. The CLSI C62 guidance document also addresses this issue.

The best approach to assessing system suitability varies greatly. It often entails looking for visual cues that something is not quite right in a system and in the resulting chromatograms. Some cues come before injecting any system suitability samples. Equilibration of a system prior to injections is paramount. Attaching fittings to prevent voids and leaks, checking mobile phase volume and quality, and performing the recommended preventive column maintenance before equilibration are the first steps to proper system setup. If a system is running a single test with no changes across a day, system suitability could be performed daily or as needed. However, system suitability should be assessed anytime a change is made to the setup, including column installation, mobile phase change, or parts replacement.

Before assessing system suitability samples, a user needs to identify key chromatographic criteria: retention time, peak shape, contamination, signal-to-noise (S/N), and peak area/height. Depending on the number of injections, retention time should be monitored within the injections or to compared historical data. Changes in retention time can be related to numerous issues stemming mainly from the LC—such as peak shape, which should be Gaussian—with limited tailing/fronting, co-eluting peaks, and, if utilizing a smoothing algorithm, enough data points to elicit symmetry across the peak width.

With chromatography generated prior to the MS, troubleshooting should be directed to the LC and acquisition methods. By injecting system suitability samples without analyte present (only internal standard), users can assess the presence of interferences, contamination from analytes of interest, and baseline and background issues. It also helps to observe consistency from injection to injection, ensuring that each injection achieves the same volume.

As a reminder, the intensity of the background present in the source can suppress analytes of interest and cause poor S/N. The quality of solvents used on the LC system usually causes high background. S/N at the lower limit of quantification is commonly monitored as >10, but depending on the intensity of the background present, problems may arise much sooner than S/N 10. Large drops in signal intensity may point to the cleanliness of the source components as well as solvent/mobile phase quality.

Regular front-end maintenance helps reduce large fluctuations in signal, but the number of injections, extraction types, and matrices contribute to the frequency at which the source and probe require maintenance.

In addition, when switching mobile phases, flushing the lines and pumps with water in between prevents salt from depositing on the surface of the source. With the variety of mobile phase concentrations and pH, the water flush is crucial to reduce the frequency of source cleaning.

Caution on Panels

Analysis using panels helps simplify by minimizing the number of different extraction protocols and changing of columns and mobile phases leading to overall more efficient laboratory workflows. However, this causes instrument inefficiency via wasted analytical time, as most samples will likely contain only a small subset of all included panel analytes eluting during the chromatographic window.

Another negative aspect about using panels is the potential compromise in quality since it’s not always feasible to optimize numerous parameters for a diverse group of compounds. Many fast methods reduce the wasted analysis time, but the changeover and maintenance time between diverse analytical setups remain. Many labs deal with these problems by dedicating instruments to specific analytical methods, which in turn increases operational costs and capital equipment expenditures, and potentially poses space limitations.

Using Laser Diode Thermal Desorption

The laser diode thermal desorption (LDTD) ion source, patented by the Canadian company Phytronix Technologies, may be a solution to these operational problems.

LDTD requires spotting up to 10 microliters of sample extract to individual wells on a 96 or 386 LazWell plate. A laser heats the backside of the well using a thermal ramp pattern specified in the instrument analytical method. The analyte is desorbed and carried through the transfer tube by heated carrier gas. Atmospheric pressure chemical ionization then ionizes the compound which the mass spectrometer subsequently analyses. With no LC component, a lab doesn’t need to spend time switching the column and mobile phases between analytical methods. The impact of one analytical method on another is very minimal since there is no mobile phase and no injection.

An additional benefit of not having an injection is the lack of analytical carry over as a source of contamination. Samples are isolated to individual wells, eliminating needle contact with individual samples. The biggest benefit of using the LDTD is increased throughput. The laser pattern typically takes approximately 6 seconds, with about 10 seconds needed to move from one well to another.

One limitation to such a fast injection cycle is that labs need to limit analytical panels to smaller numbers of analytes and their internal standards. However, the LDTD can analyze large analytical panels if a lab spots samples multiple times based on the maximum number of compounds that can be adequately detected in a single injection. With little difference between methods on the LDTD, there is no reason for dedicated instrumentation and all testing can be run on all equivalent instruments.

When labs use LDTD along with ion mobility or differential mobility, they can circumvent anticipated matrix interferences and loss of specificity. For example, with differential mobility, analytes are subjected to a separation voltage that destabilizes the ion flight path. The application of an analyte-specific compensation voltage offsets this destabilization allowing for ion transmission into the mass spectrometer.

As seen in Figure 1, mycophenolic acid and its glucuronide can be resolved easily despite no chromatographic separation prior to entry into the source. To the unfamiliar eye, what appears to be chromatographic separation is actually separation based on gas phase mobility occurring in the order of milliseconds.

LDTD does, however, have its drawbacks that laboratories should consider carefully. These include the potential need for more extensive sample pretreatment, the lack of chromatographic separation of clinically important isomers and isobars, and possible isobaric interference.

Be Ready to Grow

Labs usually justify an LC-MS/MS system financially based on developing one or two relatively high-volume tests. As a typical laboratory grows, its test menu diversifies, physical space limitations become apparent, and it faces pressures to reduce capital expenditures. All of these factors can inhibit laboratories from fully realizing the potential of mass spectrometry.

Rethinking the analytical process and conducting a capacity assessment similar to how system suitability is done (i.e. seeing the parts as well as the whole) provides opportunities for analytical scalability previously not achievable. A healthy and coordinated effort between operational, technical, and research resources, as demonstrated by the authors of this article, provides a strong foundation to allow every laboratory to meet the challenges in the ever-changing landscape of laboratory testing.

Heidi Schimmelbusch, BS, is an Analyst IV at NMS Labs in Horsham, Pennsylvania. Email: Heidi.Schimmelbusch@nmslabs.com

Jonathan DeCenzi, MS, is an Analyst IV at NMS Labs in Horsham, Pennsylvania. Email: Jonathan.DeCenzi@nmslabs.com

Joseph Homan, MS, is a Senior Scientist III at NMS Labs in Horsham, Pennsylvania. Email: Joseph.Homan@nmslabs.com