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RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise "undruggable" using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products.

An approach is described for determining if there is an intrinsic advantage, from a selectivity and resolution perspective, of using two different UHPLC/HPLC reversed-phase columns in tandem for a separation of a given sample compared to a single U/HPLC reversed-phase column that provides the same plate number. Retention data for 16 compounds extracted directly from the hydrophobic subtraction model (HSM) database at HPLCColumns.org are used to simulate and then compare the critical resolution of those compounds obtained using HSM conditions (isocratic elution at 35°C using 50% acetonitrile, 50% aqueous phosphate buffer at pH 2.8 or 7) for each of 662 U/HPLC single columns or 218,791 combinations of tandem columns and assuming a modest plate number of 8000. The critical resolution obtained for 16 additional "n-1" samples created by the systematic removal of one of the original 16 compounds was also compared using single- and tandem-column LC, as was the critical resolution obtained for thousands of synthetic samples generated by randomly varying HSM solute descriptors for each synthetic compound. When all possible single-column or tandem-column results were compared, a significant advantage was observed with tandem-column liquid chromatography (TC-LC), with an average increase in critical resolution of 0.63 (pH 2.8) or 0.75 (pH 7) units observed for the synthetic samples with the smallest number of components (m = 5). As the number of components in a sample increased, the average improvement in critical resolution (∆R

The size-exclusion separation of a water-soluble polyelectrolyte polymer, sodium polystyrene sulfonate (NaPSS), was demonstrated on common reversed-phase (C(18,) C(4), phenyl, and cyano) and hydrophilic interaction chromatography (HILIC) columns. The effect of common solvents – acetonitrile (ACN), tetrahydrofuran (THF), and methanol (MeOH), used as mobile phase modifiers – on the elution of NaPSS and the effect of column temperature (within a relatively narrow range corresponding to typical chromatographic conditions, i.e., 10 °C–60 °C) on the partition coefficient, K(SEC), were also investigated. Non-size-exclusion chromatography (non-SEC) effects can be minimized by the addition of an electrolyte and an organic modifier to the mobile phase, and by increasing the column temperature (e.g., to 50 °C or 60 °C). Strong solvents such as THF and ACN are more successful in the reduction of such effects than is the weaker solvent MeOH. The best performance is seen on medium polarity and polar stationary phases, such as cyanopropyl- and diol-modified silica (HILIC), where the elution of the NaPSS polyelectrolyte is by a near-ideal SEC mechanism. Hydrophobic stationary phases, such as C(18), C(4), and phenyl, require a higher concentration of a strong solvent modifier (THF) in the mobile phase to reduce non-SEC interactions of the solute with the stationary phase.

A chromatographic analytical method for the direct determination of amino acids by hydrophilic interaction liquid chromatography (HILIC) was developed. A dual gradient simultaneously varying the pH 3.2 ammonium formate buffer concentration and level of acetonitrile (ACN) in the mobile phase was employed. Using a charged aerosol detector (CAD) and a 2(nd) order regression analysis, the fit of the calibration curve showed R(2) values between 0.9997 and 0.9985 from 1.5mg/mL to 50μg/mL (600ng to 20ng on column). Analyte chromatographic parameters such as the sensitivity of retention to the water fraction in the mobile phase values (mHILIC) were determined as part of method development. A degradation product of glutamine (5-pyrrolidone-2-carboxylic acid; pGlu) was observed and resolved chromatographically with no method modifications. The separation was used to quantitate amino acid content in acid hydrolysates of various protein samples.

Dual-opposite-injection capillary electrophoresis (DOI-CE) is a separation technique that utilizes both ends of the capillary for sample introduction. Depending on the individual electrophoretic mobilities of the analytes of interest and the migration distance that each analyte travels to the detection window, the elution order of analytes in a DOI-CE separation can vary widely. One consequence of a variable elution order is that co-detection of oppositely charged analytes becomes possible under certain experimental conditions. While several approaches currently exist to avoid co-detection, they are often time consuming and inefficient. This paper describes a systematic approach to easily avoid co-detection by analyzing the net mobilities of the analytes of interest to aid in choosing appropriate capillary lengths for a particular separation. Simulated electropherograms that highlight various aspects of this new approach are shown. An electropherogram from the literature is replicated and the derived relationship is applied to show the potential advantages of this new approach.

Numerous combinations of one-, two-, and three-chiral-component microemulsions have been previously prepared in our group, using N-dodecoxycarbonylvaline (DDCV), 2-hexanol, and ethyl acetate, dibutyl tartrate, or diethyl tartrate. A few results of the various formulations investigated suggested the possible presence of minor impurities in one or more components of the microemulsion. In this study, the purity of the current lots of R- and S-surfactant were measured, as was the subsequent effect of minor impurities on the relevant chromatographic figures of merit (CFOMs) that describe a chiral separation, i.e., efficiency, enantioselectivity, retention, migration window (elution range), and resolution. Two related methods are proposed for correcting enantioselectivities measured in the presence of chiral impurities in the chiral microemulsion.

Micellar liquid chromatography (MLC) remains hindered by reduced chromatographic efficiency compared to reversed phase liquid chromatography (RPLC) using hydro-organic mobile phases. The reduced efficiency has been partially explained by the adsorption of surfactant monomers onto the stationary phase, resulting in a slow mass transfer of the analyte within the interfacial region of the mobile phase and stationary phase. Using an array of 12 columns, the effects of various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, superficially porous and perfluorinated, were evaluated to determine their impact on efficiency in MLC. Additionally, each stationary phase was evaluated using 1-propanol and 1-butanol as separate micellar mobile phase alcohol additives, with several columns also evaluated using 1-pentanol. A simplified equation for calculation of A' and C' terms from reduced plate height (h) versus reduced velocity (nu) plots was used to compare the efficiency data obtained with the different columns and mobile phases. Analyte diffusion coefficients needed for the h versus nu plots were determined by the Taylor-Aris dispersion technique. The use of a short alkyl chain, wide-pore silica column, specifically, Nucleosil C4, 1000A, was shown to have the most improved efficiency when using a micellar mobile phase compared to a hydro-organic mobile phase for all columns evaluated. The use of 1-propanol was also shown to provide improved efficiency over 1-butanol or 1-pentanol in most cases. In a second series of experiments, column temperatures were varied from 40 to 70 degrees C to determine the effect of temperature on efficiency for a subset of the stationary phases. Efficiency improvements ranging from 9% for a Chromegabond C8 column to 58% for a Zorbax ODS column were observed over the temperature range. Based on these observed improvements, higher column temperatures may often yield significant gains in column efficiency, assuming the column is thermally stable.

The effect of deuterated solvents in size-exclusion chromatography (SEC) was studied by comparing intrinsic viscosity measurements, SEC calibration curves, and column efficiency using water-soluble polymers. For aqueous SEC, the use of deuterium oxide slightly increases the SEC elution volume. To verify that adsorption onto the packing was absent, data from exclusion experiments were compared at 35 and 50 degrees C. Our results indicate that adsorption is not occurring for pullulan or polyethylene glycol (PEG)/poly(ethylene oxide) (PEO); for the latter, however, the elution volume increased using both D2O and H2O, indicative of slight hydrodynamic volume contraction of PEG/PEO at higher temperatures. A moderate increase in band broadening (moderate decrease in column efficiency) was observed using D2O. Finally, the effects of chloroform versus deuterated chloroform were evaluated, but no hydrodynamic volume changes were observed.

Sertraline is a basic compound and of pharmaceutical application for antidepressant treatment. The compound has two chiral centers. Separation of the three enantiomeric impurities from the parent compound is challenging. In this study, we successfully separated all four stereoisomers by electrokinetic chromatography using highly sulfated gamma-cyclodextrin and highly sulfated alpha-cyclodextrin as the chiral selectors. The two chiral selectors provided different selectivity and therefore affected the overall separation profiles. This may be due to the size difference between the dichlorophenyl moiety end and naphthalenamine moiety end, resulting in two different types of inclusion complexes with the different cyclodextrins. For routine analysis, highly sulfated gamma-cyclodextrin was better than highly sulfated alpha-cyclodextrin. For each stereoisomeric impurity, the method using sulfated gamma-cyclodextrin provided a limit of quantitation at or lower than 0.1% of the drug substance with adequate resolution. The critical resolution at this concentration level was not less than 4.0. Experimental data suggested that an internal standard was necessary for the purpose of quantitation, and the practical linearity range for analysis of sertraline stereoisomeric impurities was of about two orders of magnitude.

Current polymer formulations contain mixtures of copolymers to tailor the performance needs. Reversed-phase liquid chromatography is commonly used to separate polymers according to their chemical composition by adsorption, partition or precipitation mechanisms if retention is not influenced by molecular mass. Liquid chromatography–mass spectrometry (LC–MS) has mainly been used to identify low molecular mass polymers and additives. In this paper, we report on the use of LC–MS for the quantitative analysis of copolymer composition of several high-molecular-mass polymers by monitoring the low-mass fragments formed by thermal decomposition and electron impact ionization when using a particle beam interface. The fragment ions produced are proportional to the comonomers present and are quantitatively related to the copolymer composition. Area ratio calibration with copolymers of known composition is used to determine the composition of unknown copolymers of similar structure.

Separations of very hydrophobic neutral analytes were achieved using hydrophobic interaction electrokinetic chromatography (HI-EKC). Alkyl aryl ketone homologues dodecanophenone (C18), tetradecanophenone (C20), hexadecanophenone (C22) and octadecanophenone (C24) were separated via hydrophobic interactions between free sodium dodecyl sulfate (SDS) monomers and the analytes. The first running buffer consisted of 50 mM SDS and 50% acetonitrile (pH 7.0). A complete reversal in the elution order of these analytes was obtained with the second running buffer, 20 mM cetyltrimethylammonium bromide (CTAB) and 50% acetonitrile (pH 2.8). With the second running buffer, electroosmotic flow was suppressed and the free CTAB monomers migrated toward the detector. Through hydrophobic interactions between the free CTAB monomers and the analytes, separations of these very hydrophobic alkyl aryl ketones were obtained in less than 10 min; analysis times were less than 5 min with the SDS-based separations.

Improved empirical equations based on the graphically measurable parameters peak height (hp), width (W) and asymmetry (b/a) at 10, 25, 50, 71, 73, 75, 77 and 79% of the peak height have been derived. These equations were developed using the exponentially modified Gaussian function as an asymmetric peak model. Equations are reported for the following chromatographic figures of merit: τ, σG, area, variance, third and fourth statistical moments, excess, and skew. The accuracy and precision of the equations are discussed, along with their applicability to fronted and overlapped peaks.

Two different types (low-pressure mixing and high-pressure mixing) of high-performance liquid chromatographic (HPLC) solvent delivery systems were evaluated for their flow-rate accuracy using three common hydro-organic mobile phases: methanol-water, acetonitrile-water and tetrahydrofuran-water. Both systems delivered flow-rates 2–12% lower than the desired (set) values, depending on the mobile phase composition. Further investigations revealed that these errors in flow-rate were due in part to non-ideal mobile phase behavior (non-zero volumes of mixing, non-zero compressibilities, pressure dependent viscosities, and deviations from Darcy's law) and non-ideal stationary phase behavior (column expansivity and packing compression). An approach to correct for the systematic errors in flow-rate is described for binary solvent mixtures. The approach utilizes correction factors based on non-ideal mobile and stationary phase behavior and has been experimentally confirmed for the mobile phases and columns we examined. Although approximate in nature, our approach reduces the systematic errors in flow-rate about ten-fold, and provides a means for estimating the instrumental contribution to flow-rate error as well. This leaves only an instrumental bias which can be measured and then factored out for all further experiments with that instrument. The resulting improvements in flow-rate accuracy provided by our approach will be extremely useful to researchers who need very accurate retention data for physicochemical measurements as well as to HPLC manufacturers and users for quality control and troubleshooting of these solvent delivery systems.

A novel reversed-phase approach is described for the separation of phenol, the cresols, the xylenols, and other methylated phenols in real samples such as shale oil. The approach, based on the fundamental optimization of mobile phase pH to provide enhanced selectivity, enables m-cresol and p-cresol and many xylenol isomers to be baseline resolved for the first time in a reversed-phase analysis of shale oil. Selectivities obtained with our reversed-phase method for the difficult-to-separate isomers are compared with those observed using other reversed-phase methods. The moderate oxidation potentials of the phenols allows them to be detected selectively at the unusually high (but optimum) pH of 10.82 in the presence of redox-active and redox-inactive interferents. Although unnecessary with our chromatographic method, m-cresol and p-cresol can also be resolved electrochemically via judicious control of the working electrode potential.

A method for the accurate calculation of statistical moments, excess and skew is described. Based on peak width, asymmetry and peak height measurements this method is applicable to both fully resolved and overlapping chromatographic peaks. IT is also useful for the deconvolution of overlapping chromatographic peaks. The advantages of this method over the traditional approach to measuring peak statistical moments, excess and skew are discussed.