Assessing Accuracy of an Analytical Method In Silico: Application to “Accurate Constant via Transient Incomplete Separation” (ACTIS)

Abstract

Analytical methods may not have reference standards required for testing their accuracy. We postulate that accuracy of an analytical method can be assessed in the absence of reference standards in silico if the method is built upon deterministic processes. A deterministic process can be precisely computer-simulated thus allowing virtual experiments with virtual reference standards. Here, we apply this in silico approach to study "Accurate Constant via Transient Incomplete Separation" (ACTIS), a method for finding the equilibrium dissociation constant (K d) of protein-small molecule complexes. ACTIS is based on a deterministic process: molecular diffusion of the interacting protein-small molecule pair in a laminar pipe flow. We used COMSOL software to construct a virtual ACTIS setup with a fluidic system mimicking that of a physical ACTIS instrument. Virtual ACTIS experiments performed with virtual samples-mixtures of a protein and a small molecule with defined rate constants and, thus, K d of their interaction-allowed us to assess ACTIS accuracy by comparing the determined K d value to the input K d value. Further, the influence of multiple system parameters on ACTIS accuracy was investigated. Within multi-fold ranges of parameters, the values of K d did not deviate from the input K d values by more than a factor of 1.25 strongly suggesting that ACTIS is intrinsically accurate and that its accuracy is robust. Accordingly, further development of ACTIS can focus on achieving high reproducibility and precision. We foresee that in silico accuracy assessment, demonstrated here with ACTIS, will be applicable to other analytical methods built upon deterministic processes. File list (5) download file view on ChemRxiv manuscript.pdf (741.60 KiB) download file view on ChemRxiv supportinginformation.pdf (2.21 MiB) download file view on ChemRxiv thumbnail.png (161.31 KiB) download file view on ChemRxiv models.zip (1.29 MiB)

Template Instrumentation for "Accurate Constant via Transient Incomplete Separation" (ACTIS)

“…The deterministic nature of ACTIS allowed us to create a virtual ACTIS instrument in silico and study the accuracy of ACTIS by computer modeling. 7 This study proved that the accuracy of ACTIS was not affected by large variations in the geometry of the fluidic path and/or the parameters characterizing the flow. The in-silico study led to several important conclusions.…”

“…6 We recently introduced Accurate Constant via Transient Incomplete Separation (ACTIS), a method for finding K d of PL, which was hypothesized to be free of inherent sources of inaccuracy. 7 ACTIS is based on a long-known phenomenon of transient incomplete separation (TIS) of the L from PL in a roundcross-section capillary due to their differential transverse diffusion in a laminar flow with a parabolic velocity profile. 8,9 In ACTIS, a short plug of an equilibrium mixture of P and L in a buffer solution is injected into a capillary prefilled with the pure buffer solution.…”

“…As a result, the instruments likely differed in their ratios of the capillary length to flow velocity; however, ACTIS is robust to variations in this parameter. 7 The instruments also likely differed in focusing performance of the commercial detectors' optics due to the specifics of ball lenses. 12 These differences cumulatively were the reason for significant differences in the relative heights and positions of the non-diffusive peak (the lefthand side peak) between the two instruments (Figure 4, left panels).…”

Template Instrumentation for "Accurate Constant via Transient Incomplete Separation" (ACTIS)

“…The deterministic nature of ACTIS allowed us to assess its accuracy earlier by using a virtual ACTIS instrument built in COMSOL. 7 Our in-silico study revealed that ACTIS accuracy was not influenced significantly by large variations in the parameters of the fluidic setup: the radius of the separation capillary, the radius and length of the injection loop, and the ramp time of the main pump. 7 This invariance of accuracy is important because it gives flexibility in instrument design, and guarantees that the accuracy will not be affected by deviations in the performance parameters of the key instrument components from their nominal values provided by the manufacturer.…”

“…The initial understanding was that a physical ACTIS instrument had to support ideal conditions such as (i) a cylindrical shape of the initial sample plug, (ii) a parabolic flow-velocity profile, and (iii) the absence of ramp time in the pressure pulse. 7 Maintaining such ideal conditions would require a complex ACTIS instrument and would not leave flexibility with fluidic path design. The invariance of accuracy demonstrated in silico suggests that an ACTIS instrument can be much simpler than it was thought initially.…”