The eVol – Investigating a Liquid Handling Tool to Improve Consistency and Reliability for Calibrations

We’re taking you to Austin, Texas to meet the amazing team at Santé Laboratories for this investigation! This project, led by the analytical team at Sante Laboratories, put the eVol in use and compared it to standard pipetting techniques in the generation of analytical calibration curves.

Wait….what?

You might be thinking to yourself….What’s an eVol?

An eVol is a digitally controlled dispensing system that’s used for liquid handling.  The eVol covers a liquid volume range from 200 nL to 1000 uL.  You can use this system for a variety of applications including serial dilutions, addition of standards, standard preparation, and delivery of derivatization agents. While the eVol might be a new technology to the cannabis and hemp market, the eVol has been use for a long time by analytical laboratories providing testing services in other industries.  You can find out more information on Trajan’s website.

Now back to the purpose of this study.

There are many factors that can contribute to errors in liquid handling like variability in glassware, volatility of compounds and evaporation, and incomplete fluid transfer.  These errors start to have a larger impact on uncertainties as transfer liquid volumes decrease.  The team at Santé Laboratories also emphasized that, “additional effort to improve precision during preparation of calibration and quality control samples better allows the high throughput analytical laboratory to improve accuracy and repeatability in unknown concentration determinations. Producing a more robust and accurate curve and quality control samples also allows the laboratory to monitor the lifetime and health of their calibration curves, minimizing recalibrations and reducing usage of expensive reference materials (CRM).”

To evaluate the hypothesis that improved liquid handling produces a more reliable and repeatable calibration, Santé Laboratories prepared two sets of external standards; one using calibrated low retention air-cushion pipettes and one using the eVol.  The curve screened Restek’s 11 cannabinoid standards with a serial dilution procedure.  The table below compares the R2 values, multiple minus adjusted R2 values, and variance coefficients.

 

Table 1. R2 Values (Multiple and Adjusted) and Variance Coefficients

Table 2. Multiple Minus Adjusted R2

 

The Santé team commented, “Immediately, comparing the multiple and adjusted R2 values and variance coefficients, we observed lower variance coefficients (between 34 and 66%, 56% on average) and adjusted R2 values closer to unity using the eVol analytical syringe versus air cushion pipettes with low retention tips. Furthermore, when looking at the multiple vs adjusted R2 for the pipette, we see that the adjusted R2 decreases across all cannabinoids, as the adjusted R2 penalizes variance in the individual replicates. Comparing the differences with the eVol, we see a much smaller change.”

But their assessment did not stop there—to really assess the calibration procedure, the team reviewed the residual plots—the differences between the measured and predicted response values in a regression curve. For linear regression, residuals ideally distribute normally across the x-axis. Observing the differences in the residual plots for CBD, CBDa, d9-THC, and THCa, we observed significantly more linear residuals with the eVol than standard pipetting, an important feature as nonlinear residuals lead to repeatable biases in various sections of the calibration curve. In the case of this analysis, the residuals indicate that samples which fall between calibrators 4 and 7 with the pipette would be positively biased from the calibration curve, before taking into account any other random or systematic errors in the analysis.

Figure 1. Residual Plots for CBD, CBDa, THC, and THCa.

Santé Laboratories had the following conclusion, “Precision liquid handling improves calibration procedures, particularly when utilizing small volumes of concentrated materials. This is extremely important for cannabinoids analyses, as commercially available CRMs are concentration and quantity limited due to the controlled nature of the molecules. This requires laboratories in many cases to utilize serial dilutions and low volume inserts or ultra-recovery vials to prepare sufficiently concentrated calibration curves. The eVol analytical syringe, when used for serial dilutions, shows modest improvements in several markers of calibration health. It also enables the user to move from a serial dilution to a straight dilution procedure, allowing for the use of fixed concentration internal standards.

 

The eVol has the additional benefit of being able to pierce septa, allowing use with closed autosampler vials to minimize losses of volatile materials during preparation. This is a particularly useful feature in gas chromatography, especially for laboratories still utilizing full evaporation technique, where analytes are extremely volatile and sample sizes are extremely small—work on this is under way as well.”

 

A special thanks to Tyler West, Andrea Clemente, and Brian R. Sloat at Santé Laboratories (www.santelabs.com).

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