6 research outputs found
Green Sample Preparation for Liquid Chromatography and Capillary Electrophoresis of Anionic and Cationic Analytes
A sample preparation
device for the simultaneous enrichment and
separation of cationic and anionic analytes was designed and implemented
in an eight-channel configuration. The device is based on the use
of an electric field to transfer the analytes from a large volume
of sample into small volumes of electrolyte that was suspended into
two glass micropipettes using a conductive hydrogel. This simple,
economical, fast, and green (no organic solvent required) sample preparation
scheme was evaluated using cationic and anionic herbicides as test
analytes in water. The analytical figures of merit and ecological
aspects were evaluated against the state-of-the-art sample preparation,
solid-phase extraction. A drastic reduction in both sample preparation
time (94% faster) and resources (99% less consumables used) was observed.
Finally, the technique in combination with high-performance liquid
chromatography and capillary electrophoresis was applied to analysis
of quaternary ammonium and phenoxypropionic acid herbicides in fortified
river water as well as drinking water (at levels relevant to Australian
guidelines). The presented sustainable sample preparation approach
could easily be applied to other charged analytes or adopted by other
laboratories
Electrokinetic Removal of Dodecyl Sulfate Micelles from Digested Protein Samples Prior to Electrospray-Ionization Mass Spectrometry
In
proteomics, dodecyl sulfate (DS<sup>–</sup>) as sodium
salt is commonly used in protein solubilization prior to tryptic digestion,
but the presence of the DS<sup>–</sup> hampers the electrospray
ionization mass spectrometric (ESI-MS) analysis. The development of
DS<sup>–</sup> depletion techniques is therefore important
especially when dealing with small samples where there could be poor
sensitivity due to sample loss or dilution during sample preparation.
Here, we present a simple and fast electrokinetic removal method of
DS<sup>–</sup> from small volumes of peptide and digested protein
samples prior to ESI-MS. The selective removal was accomplished using
an acidic extraction solution (ES) containing acetonitrile (ACN) inside
a fused-silica capillary that was dipped into the sample. The use
of acidic ES suppressed the electroosmotic flow; allowing the electrokinetic
movement of DS<sup>–</sup> monomers and micelles into the capillary.
The high amount of ACN present at the tip of the capillary served
to collapse the micelles migrating into the capillary, thereby releasing
the peptides that were bound to these micelles, facilitating peptide
retention in the sample and efficient DS<sup>–</sup> removal.
Increased % MS signal intensity (SI) restoration of the peptide was
observed, while DS<sup>–</sup> removal was unaffected when
the amount of ACN in the ES was increased. This is because of the
micelle to solvent stacking mechanism (effective electrophoretic mobility
reversal) working at high concentration of ACN for the improved recovery
of the peptides. % MS SI restoration for the Z-Gly-Gly-Val and bradykinin
peptides were 75–83% while % MS SI reduction of DS<sup>–</sup> was up to 99% under optimal conditions, that is, 40% ACN in the
ES. Higher % peptide recoveries from digested protein samples were
obtained using the proposed method compared to the conventional cold
acetone precipitation method
Rapid Method Development in Hydrophilic Interaction Liquid Chromatography for Pharmaceutical Analysis Using a Combination of Quantitative Structure–Retention Relationships and Design of Experiments
A design-of-experiment
(DoE) model was developed, able to describe
the retention times of a mixture of pharmaceutical compounds in hydrophilic
interaction liquid chromatography (HILIC) under all possible combinations
of acetonitrile content, salt concentration, and mobile-phase pH with <i>R</i><sup>2</sup> > 0.95. Further, a quantitative structure–retention
relationship (QSRR) model was developed to predict retention times
for new analytes, based only on their chemical structures, with a
root-mean-square error of prediction (RMSEP) as low as 0.81%. A compound
classification based on the concept of similarity was applied prior
to QSRR modeling. Finally, we utilized a combined QSRR-DoE approach
to propose an optimal design space in a quality-by-design (QbD) workflow
to facilitate the HILIC method development. The mathematical QSRR-DoE
model was shown to be highly predictive when applied to an independent
test set of unseen compounds in unseen conditions with a RMSEP value
of 5.83%. The QSRR-DoE computed retention time of pharmaceutical test
analytes and subsequently calculated separation selectivity was used
to optimize the chromatographic conditions for efficient separation
of targets. A Monte Carlo simulation was performed to evaluate the
risk of uncertainty in the model’s prediction, and to define
the design space where the desired quality criterion was met. Experimental
realization of peak selectivity between targets under the selected
optimal working conditions confirmed the theoretical predictions.
These results demonstrate how discovery of optimal conditions for
the separation of new analytes can be accelerated by the use of appropriate
theoretical tools
Benchmarking of Computational Methods for Creation of Retention Models in Quantitative Structure–Retention Relationships Studies
Quantitative
structure–retention relationship (QSRR) models
are powerful techniques for the prediction of retention times of analytes,
where chromatographic retention parameters are correlated with molecular
descriptors encoding chemical structures of analytes. Many QSRR models
contain geometrical descriptors derived from the three-dimensional
(3D) spatial coordinates of computationally predicted structures for
the analytes. Therefore, it is sensible to calculate these structures
correctly, as any error is likely to carry over to the resulting QSRR
models. This study compares molecular modeling, semiempirical, and
density functional methods (both B3LYP and M06) for structure optimization.
Each of the calculations was performed in a vacuum, then repeated
with solvent corrections for both acetonitrile and water. We also
compared Natural Bond Orbital analysis with the Mulliken charge calculation
method. The comparison of the examined computational methods for structure
calculation shows that, possibly due to the error inherent in descriptor
creation methods, a quick and inexpensive molecular modeling method
of structure determination gives similar results to experiments where
structures are optimized using an expensive and time-consuming level
of computational theory. Also, for structures with low flexibility,
vacuum or gas phase calculations are found to be as effective as those
calculations with solvent corrections added
Retention Index Prediction Using Quantitative Structure–Retention Relationships for Improving Structure Identification in Nontargeted Metabolomics
Structure
identification in nontargeted metabolomics based on liquid-chromatography
coupled to mass spectrometry (LC-MS) remains a significant challenge.
Quantitative structure–retention relationship (QSRR) modeling
is a technique capable of accelerating the structure identification
of metabolites by predicting their retention, allowing false positives
to be eliminated during the interpretation of metabolomics data. In
this work, 191 compounds were grouped according to molecular weight
and a QSRR study was carried out on the 34 resulting groups to eliminate
false positives. Partial least squares (PLS) regression combined with
a Genetic algorithm (GA) was applied to construct the linear QSRR
models based on a variety of VolSurf+ molecular descriptors. A novel
dual-filtering approach, which combines Tanimoto similarity (TS) searching
as the primary filter and retention index (RI) similarity clustering
as the secondary filter, was utilized to select compounds in training
sets to derive the QSRR models yielding <i>R</i><sup>2</sup> of 0.8512 and an average root mean square error in prediction (RMSEP)
of 8.45%. With a retention index filter expressed as ±2 standard
deviations (SD) of the error, representative compounds were predicted
with >91% accuracy, and for 53% of the groups (18/34), at least
one false positive compound could be eliminated. The proposed strategy
can thus narrow down the number of false positives to be assessed
in nontargeted metabolomics
In Silico Screening of Two-Dimensional Separation Selectivity for Ion Chromatography × Capillary Electrophoresis Separation of Low-Molecular-Mass Organic Acids
A prerequisite
for ordered two-dimensional (2D) separations and
full utilization of the enhanced 2D peak capacity is selective exploitation
of the sample attributes, described as sample dimensionality. In order
to take sample dimensionality into account prior to optimization of
a 2D separation, a new concept based on construction of 2D separation
selectivity maps is proposed and demonstrated for ion chromatography
× capillary electrophoresis (IC×CE) separation of low-molecular-mass
organic acids as test analytes. For this purpose, 1D separation selectivity
maps were constructed based on calculation of pairwise separation
factors and identification of critical pairs for four IC stationary
phases and 28 levels of background electrolyte pH in CE. The derived
IC and CE maps were then superimposed and the effectiveness of the
respective 2D separations assessed using an in silico approach, followed
by testing examples of one successful and one unsuccessful 2D combination
experimentally. The results confirmed the efficacy of the predictions,
which require a minimal number of experiments compared to the traditional
one-at-a-time approach. Following the same principles, the proposed
framework can also be adapted for optimization of separation selectivity
in various 2D combinations and for other applications