Factors that affect the reproducibility of your assay

As scientists, we know our experiments must be reproducible. Without it, there is always doubt as to whether our data interpretations and conclusions are truly sound. With LC-MS/MS quantitative methods, high precision and reproducibility are key to generating trustworthy and reliable answers. In fact, most regulatory agencies demand a high level of precision when it comes to LC-MS/MS assays that are used in the development of drugs or the detection of endogenous species for clinical purposes. LC-MS/MS methods should not only demonstrate a high degree of precision and reproducibility for repeat injections of the same sample, different samples of the same type, and different instances of the assay within a laboratory but in many cases, the method must exhibit high reproducibility across different laboratories.

There are many aspects of an assay that can affect reproducibility. From an LC-MS/MS solutions standpoint, SCIEX has thought long and hard about how to design instruments and software that maximize analytical precision and reproducibility. It’s no surprise that the stability and robustness of the hardware can play a big part. And when it comes to software, a little intelligence goes a long way.

For SCIEX instruments, stability and ruggedness have always been a central principle of design. The stability of the ion source is actually a key component to generating high precision and reproducibility for LC-MS/MS assays. Sources must be able to maintain consistent drop formation, spray conditions, and ionization for hundreds to thousands of injections spanning multiple days without any significant change in response. The SCIEX TurboV™ ion source, originally developed over 15 years ago, is still considered the gold standard for mass spec ionization because of the high consistency, sensitivity, and reliability it provides. The IonDrive, OptiFlow, and DuoSpray Turbo V ion sources all evolved from the Turbo V design and provide additional flexibility in flow rates and ionization modes. The plug-and-play aspect of these sources implicitly dictate a requirement for a high degree of precision. They must work the same every time, all the time, with virtually no optimization. That requirement alone imparts a degree of reproducibility built into the design.

The Curtain Gas™ is another component on SCIEX mass spectrometers that is key to high precision and reproducibility. It provides underlying robustness to the system where clustering problems and clogging problems with the orifice are minimized. Charged analytes get through but not much else. The stability that results also manifests as improved reproducibility and precision.

Besides stability, another important aspect of mass spectrometry design that is intricately linked to the precision and reproducibility of LC-MS/MS assays is the speed of acquisition. For the highest precision, each peak must be sampled enough times across its elution profile. We want the peak to be smoothly ‘drawn’ as the analyte elutes. Inadequate sampling leads to poor peak shape and less reproducibility for data integration. With triple quadrupoles, the speed comes down to the dwell time and cycle time. With many concurrent transitions to monitor, one or the other must be sacrificed and quantitative statistics can suffer. The Scheduled MRM™ Pro Algorithm can significantly reduce the number of concurrent transitions that need to be monitored. It enables thousands of MRM transitions to be scheduled in time so that the instrument only collects data when the analyte is present. This provides a huge benefit, especially for complex samples where many MRM transitions are required. Rather than collecting data for all the transitions all the time, the algorithm allows the acquisition to be focussed on only those MRM transitions that may be present at that exact moment in time during the LC run. More time can be spent on each component and more data points can be collected for each peak. This equates to better quant statistics overall, including precision and reproducibility.

Speed is also a big factor for high resolution instruments that are used for quantitation. Scan speed needs to be slow enough to provide good sensitivity. But if the scan speed is too slow, the chromatographic peak shape will not be ‘drawn’ smoothly with enough data points, and precision and reproducibility will suffer. With the SCIEX QTOF X500 and TripleTOF^® instruments and the MRM^HRworkflow, MS/MS spectra are collected at high-resolution and fast scan speeds without sacrificing sensitivity. Fragment ions are extracted post-acquisition to generate MRM-like peaks for integration and quantification. The Scheduled MRM Pro Algorithm can also be used here to reduce the number of concurrent acquisitions, thereby improving reproducibility and precision even further. 

Another important software feature that can improve precision is the ability to sum more than one MRM transition or fragment ion together. In many cases, more than one MRM transition is monitored for each analyte because this can increase confidence. And in the case of MRM^HR, the entire MS/MS spectrum is collected so all of the fragment ions are present anyway. By quantifying on the summation of the fragment ions or MRM transitions, reproducibility and precision can be improved versus quantifying on individual ions. This can be particularly useful for low abundant analytes where better reproducibility is often achieved through summation.  

LC equipment is another area where reproducibility can be intricately tied to the quality of the instrumentation. With LC, maximizing the precision of the gradient, flow control, temperature control, and injection volume matter a lot, as does minimizing carryover. Any variation in the LC gradient or flow rate, or carryover from a previous injection can have an adverse effect on the elution profile of an analyte and its quantitation. For analytical flow assays, SCIEX ExionLC systems feature temperature-controlled column ovens, cooled autosamplers, high precision flow control, high precision injection volume, and ultralow carryover to virtually non-detectable levels (e.g., a maximum of 0.0015% for the ExionLC). For microflow assays, gradient and flow control and injection volume become even more critical. The SCIEX M5 MicroLC uses patented Microfluidic Flow Control™ technology that provides consistent, accurate results without flow splitting all the way down to 1uL/min flow rates. In fact, because microflow can improve sensitivity while saving on sample, reagents and buffers, microflow LC-MS/MS has gained in popularity and SCIEX has developed a complete solution around this flow regime.

At SCIEX we know the power of precision. The higher the precision and reproducibility, the higher the validity we can assign to our scientific conclusions. Reproducible, replicable, and repeatable results are the key to success. Learn more about how SCIEX values precision and reproducibility by exploring our products today.