High-throughput Microscale Bioanalysis is not a Paradox

Waters Staff

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Reading Time: 4 minutes

microflow at the turn of a keyenhanced MS at a turn of a key

 

Question: When someone says microscale chromatography, what comes to mind?

Increased sensitivity? Reduced sample volume? Enhanced MS sampling efficiency?

Yes, yes, and yes!

You might also say, “finicky,” “slow,” or “only one person in our group knows how to use it!” Maybe even, “there’s no way I can successfully transfer that method, so why bother?”

What if I said, “Reproducible, for routine throughput, and easy to use?”

Believe it or not, it’s true.

Dr. Yun Alelyunas and the research team at Waters have reported (pdf) on a microscale workflow that provides high-sensitivity quantitative results for small molecules and a peptide using a rapid, 3-minute gradient on the ionKey/MS microfluidics platform with an ACQUITY UPLC M-Class System and the Xevo G2-XS QTof Mass Spectrometer. This is comparable to the 2- to 4-minute total gradient/cycle time of traditional throughput workflows on wide-bore columns at analytical scale.

Did Dr. Alelyunas use pixie dust or a magic wand?

Nope. Neither!

Dr. Alelyunas was resourceful and increased the flow rate out to a “sweet spot” where she had optimal speed and enhanced sensitivity at 7 µL/min. As shown in the plot below, there is at least a 5-fold increase in sensitivity using the 150-µm I.D. column at the higher flow rate.  As a result, the effective gradient time is shorter.

microflow-flexibility-high-throughput-slide no title
Flexibility for high throughput with microflow LC.

In drug discovery and development, bioanalytical assays are an integral part of the process. There’s a constant trade-off between collecting the highest quality bioanalytical data (sensitivity, accuracy, linearity), and getting the analysis done in the shortest time (throughput). The unyielding requests to obtain more data – or higher sensitivity data from smaller sample volumes – add to the burden of bioanalytical method development.

The need for lower flow rates and the concomitant improvement in sensitivity is driven by the following:

  1. The increased use of micro-dosing strategy in preclinical studies places high demand on instrument sensitivity
  2. Complex matrices and formulations or the limitation on sample availability from pediatric studies, cerebrospinal fluid, dried blood spot, or biomarker studies
  3. The large number of tests and/or assays required on that reduced sample
  4. The desire to increase sampling of preclinical animals while reducing animal usage

Historically, bioanalysts haven’t had the benefit of achieving all critical bioanalytical measurements at the same moment. I can have high-throughput analysis, but not peak resolution and sensitivity. I can have the highest sensitivity, but I must resort to nanoflow, which has been compromised by its complexity and slow turnaround.

Microflow-LC/MS platforms mark the beginning of a shift in bioanalysis. The bioanalyst can now collect sensitive data from less injected sample (1.0 µL) on 150-µm I.D. columns, using less organic solvents (99% solvent saving realized in this study) and total run time (3 to 4 min) comparable to traditional reversed-phase HPLC high-throughput methods employing 2.1-mm I.D. columns. Furthermore, the analytical characteristics of reproducibility, linearity, precision, and limit of quantitation are on par with the traditional, gold standard wide-bore chromatographic methods. As a result, the ability to obtain high sensitivity and to consume minimal sample using integrated microflow LC expands the utility of LC-MS in multiple areas of drug research and development.

Let’s not leave out our metabolite ID comrades. Achieving that extra sensitivity on a low-level metabolite is easy with this microflow approach. Our biotransformation colleagues can see how clever it is slowing down the gradient and immediately getting better quality MS and tandem MS data. Typically, they would need to consider injecting more sample or employing a pre-concentration step such as solid-phase extraction (SPE).

Thanks to Dr. Alelyunas and the research team at Waters, an analyst or metabolite identification scientist can now harness the power of microflow LC, and see that it CAN be used for routine applications conserving sample and solvent at the same time – while being efficient.

 

You can find additional information on microflow-LC/MS here: