Separations of very hydrophobic neutral analytes were achieved using hydrophobic interaction elec... more Separations of very hydrophobic neutral analytes were achieved using hydrophobic interaction electrokinetic chromatography (HI-EKC). Alkyl aryl ketone homologues dodecanophenone (C,,), tetradecanophenone (C,,), hexadecanophenone (C,,) and octadecanophenone (C,,) 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 CT’AB 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.
An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, I... more An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, Ibuprofen, and Flurbiprofen) and three of the four pairs of enantiomers in one run is reported. The NSAID samples were prepared with 1 mg/mL of Ketoprofen, Naproxen, Ibuprofen and Flurbiprofen, and 0.5 mg/mL thiourea as a t0 marker in a sample solvent consisting of 1: 1 acetonitrile and 0.2% formic acid. The separation of the enantiomers of the NSAIDs was investigated using a chiral column in two different lengths (5-cm, 10-cm) packed with Amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel and using a tandem column that consists of a biphenyl column connected by a zero dead volume connector to a 5-cm chiral column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel. Both isocratic and step-gradient methods were investigated, and the results of the different methods were compared to determine the benefits and drawbacks of each approach, with an emphasis on the resolution and analysis time.
An approach is described for determining if there is an intrinsic advantage, from a selectivity a... more 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 (∆Rs,crit) using TC-LC gradually decreased from about 0.70 for m = 5 to 0.18 for m = 32 components. The average improvement in critical resolution achieved by switching from SC-LC to TC-LC was also lower when a smaller number of columns and column combinations were available to explore, as would be the case for a finite column inventory in a real laboratory. Nevertheless, on average there does appear to be an intrinsic advantage of tandem-column liquid chromatography, however small, which can be amplified by using high efficiency columns.
Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new... more Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new developments in the use of aptamers, antisense, and small interfering ribonucleic acid (siRNA) for the treatment of different human diseases. The development of chromatographic separation methods for the quantitative and sensitive analysis of oligonucleotides is a critical part for the advancement of this research area. A typical therapeutic oligonucleotide sample is a short ribonucleic acid (RNA) molecule with about 19–21 nucleotide length (~ 7–8 kDa), or a 21-mer base pair length in double-stranded siRNA (~ 16 kDa). Due to their relatively large sizes and multiple negative charges compared with typical, small molecule pharmaceutical compounds, oligonucleotides can be difficult to analyze with conventional reversed-phase liquid chromatography (RPLC) methods. In this chapter, we provide an overview of liquid chromatographic (LC) separation of oligonucleotides, including anion-exchange, ion-pair reversed-phase, and mixed-mode LC.
RNA interference offers a novel approach for the development of new therapeutics for targets that... more 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.
Vesicle electrokinetic chromatography (VEKC) using vesicles synthesized from the oppositely charg... more Vesicle electrokinetic chromatography (VEKC) using vesicles synthesized from the oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) and from the double-chained anionic surfactant bis(2-ethylhexyl)sodium sulfosuccinate (AOT) was applied to the indirect measurement of octanol-water partition coefficients (log Po/w). A variety of small organic molecules with varying functional groups, pesticides, and organic acids were evaluated by correlating log Po/w and the logarithm of the retention factor (log k') and comparing the calibrations. A linear solvation energy relationship (LSER) analysis was conducted to describe the retention behavior of the vesicle systems and compared to that of octanol-water partitioning. The solute hydrogen bond donating behavior is slightly different with the vesicle interactions using CTAB-SOS vesicles as compared to the octanol-water partitioning model. The AOT vesicle and octanol-water partitioning systems showed similar partitioning characteristics. VEKC provides rapid separations for determinations of log Po/w in the range of 0.5 to 5 using CTAB-SOS vesicles and 0 to 5.5 using AOT vesicles.
An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, I... more An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, Ibuprofen, and Flurbiprofen) and three of the four pairs of enantiomers in one run is reported. The NSAID samples were prepared with 1 mg/mL of Ketoprofen, Naproxen, Ibuprofen and Flurbiprofen, and 0.5 mg/mL thiourea as a t0 marker in a sample solvent consisting of 1: 1 acetonitrile and 0.2% formic acid. The separation of the enantiomers of the NSAIDs was investigated using a chiral column in two different lengths (5-cm, 10-cm) packed with Amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel and using a tandem column that consists of a biphenyl column connected by a zero dead volume connector to a 5-cm chiral column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel. Both isocratic and step-gradient methods were investigated, and the results of the different methods were compared to determine the benefits and drawbacks of each approach, with an emphasis on the resolution and analysis time.
Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new... more Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new developments in the use of aptamers, antisense, and small interfering ribonucleic acid (siRNA) for the treatment of different human diseases. The development of chromatographic separation methods for the quantitative and sensitive analysis of oligonucleotides is a critical part for the advancement of this research area. A typical therapeutic oligonucleotide sample is a short ribonucleic acid (RNA) molecule with about 19–21 nucleotide length (~ 7–8 kDa), or a 21-mer base pair length in double-stranded siRNA (~ 16 kDa). Due to their relatively large sizes and multiple negative charges compared with typical, small molecule pharmaceutical compounds, oligonucleotides can be difficult to analyze with conventional reversed-phase liquid chromatography (RPLC) methods. In this chapter, we provide an overview of liquid chromatographic (LC) separation of oligonucleotides, including anion-exchange, ion-pair reversed-phase, and mixed-mode LC.
RNA interference offers a novel approach for the development of new therapeutics for targets that... more 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.
RNA interference offers a novel approach for the development of new therapeutics for targets that... more 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.
A stochastic approach was utilized to estimate the probability of a successful isocratic or gradi... more A stochastic approach was utilized to estimate the probability of a successful isocratic or gradient separation in conventional chromatography for numbers of sample components, peak capacities, and saturation factors ranging from 2 to 30, 20-300, and 0.017-1, respectively. The stochastic probabilities were obtained under conditions of (i) constant peak width ("gradient" conditions) and (ii) peak width increasing linearly with time ("isocratic/constant N" conditions). The isocratic and gradient probabilities obtained stochastically were compared with the probabilities predicted by Martin et al. [Anal. Chem., 58 (1986) 2200-2207] and Davis and Stoll [J. Chromatogr. A, (2014) 128-142]; for a given number of components and peak capacity the same trend is always observed: probability obtained with the isocratic stochastic approach<probability obtained with the gradient stochastic approach≤probability predicted by Davis and Stoll < probability predicted by Martin...
A chromatographic analytical method for the direct determination of amino acids by hydrophilic in... more 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.
Separations of very hydrophobic neutral analytes were achieved using hydrophobic interaction elec... more Separations of very hydrophobic neutral analytes were achieved using hydrophobic interaction electrokinetic chromatography (HI-EKC). Alkyl aryl ketone homologues dodecanophenone (C,,), tetradecanophenone (C,,), hexadecanophenone (C,,) and octadecanophenone (C,,) 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 CT’AB 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.
An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, I... more An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, Ibuprofen, and Flurbiprofen) and three of the four pairs of enantiomers in one run is reported. The NSAID samples were prepared with 1 mg/mL of Ketoprofen, Naproxen, Ibuprofen and Flurbiprofen, and 0.5 mg/mL thiourea as a t0 marker in a sample solvent consisting of 1: 1 acetonitrile and 0.2% formic acid. The separation of the enantiomers of the NSAIDs was investigated using a chiral column in two different lengths (5-cm, 10-cm) packed with Amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel and using a tandem column that consists of a biphenyl column connected by a zero dead volume connector to a 5-cm chiral column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel. Both isocratic and step-gradient methods were investigated, and the results of the different methods were compared to determine the benefits and drawbacks of each approach, with an emphasis on the resolution and analysis time.
An approach is described for determining if there is an intrinsic advantage, from a selectivity a... more 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 (∆Rs,crit) using TC-LC gradually decreased from about 0.70 for m = 5 to 0.18 for m = 32 components. The average improvement in critical resolution achieved by switching from SC-LC to TC-LC was also lower when a smaller number of columns and column combinations were available to explore, as would be the case for a finite column inventory in a real laboratory. Nevertheless, on average there does appear to be an intrinsic advantage of tandem-column liquid chromatography, however small, which can be amplified by using high efficiency columns.
Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new... more Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new developments in the use of aptamers, antisense, and small interfering ribonucleic acid (siRNA) for the treatment of different human diseases. The development of chromatographic separation methods for the quantitative and sensitive analysis of oligonucleotides is a critical part for the advancement of this research area. A typical therapeutic oligonucleotide sample is a short ribonucleic acid (RNA) molecule with about 19–21 nucleotide length (~ 7–8 kDa), or a 21-mer base pair length in double-stranded siRNA (~ 16 kDa). Due to their relatively large sizes and multiple negative charges compared with typical, small molecule pharmaceutical compounds, oligonucleotides can be difficult to analyze with conventional reversed-phase liquid chromatography (RPLC) methods. In this chapter, we provide an overview of liquid chromatographic (LC) separation of oligonucleotides, including anion-exchange, ion-pair reversed-phase, and mixed-mode LC.
RNA interference offers a novel approach for the development of new therapeutics for targets that... more 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.
Vesicle electrokinetic chromatography (VEKC) using vesicles synthesized from the oppositely charg... more Vesicle electrokinetic chromatography (VEKC) using vesicles synthesized from the oppositely charged surfactants cetyltrimethylammonium bromide (CTAB) and sodium octyl sulfate (SOS) and from the double-chained anionic surfactant bis(2-ethylhexyl)sodium sulfosuccinate (AOT) was applied to the indirect measurement of octanol-water partition coefficients (log Po/w). A variety of small organic molecules with varying functional groups, pesticides, and organic acids were evaluated by correlating log Po/w and the logarithm of the retention factor (log k&#39;) and comparing the calibrations. A linear solvation energy relationship (LSER) analysis was conducted to describe the retention behavior of the vesicle systems and compared to that of octanol-water partitioning. The solute hydrogen bond donating behavior is slightly different with the vesicle interactions using CTAB-SOS vesicles as compared to the octanol-water partitioning model. The AOT vesicle and octanol-water partitioning systems showed similar partitioning characteristics. VEKC provides rapid separations for determinations of log Po/w in the range of 0.5 to 5 using CTAB-SOS vesicles and 0 to 5.5 using AOT vesicles.
An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, I... more An HPLC method that separates four Non-Steroidal Anti-Inflammatory Drugs (Ketoprofen, Naproxen, Ibuprofen, and Flurbiprofen) and three of the four pairs of enantiomers in one run is reported. The NSAID samples were prepared with 1 mg/mL of Ketoprofen, Naproxen, Ibuprofen and Flurbiprofen, and 0.5 mg/mL thiourea as a t0 marker in a sample solvent consisting of 1: 1 acetonitrile and 0.2% formic acid. The separation of the enantiomers of the NSAIDs was investigated using a chiral column in two different lengths (5-cm, 10-cm) packed with Amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel and using a tandem column that consists of a biphenyl column connected by a zero dead volume connector to a 5-cm chiral column packed with amylose tris(3,5-dimethylphenylcarbamate) coated on silica gel. Both isocratic and step-gradient methods were investigated, and the results of the different methods were compared to determine the benefits and drawbacks of each approach, with an emphasis on the resolution and analysis time.
Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new... more Abstract Synthetic oligonucleotides have become increasingly important molecules as a part of new developments in the use of aptamers, antisense, and small interfering ribonucleic acid (siRNA) for the treatment of different human diseases. The development of chromatographic separation methods for the quantitative and sensitive analysis of oligonucleotides is a critical part for the advancement of this research area. A typical therapeutic oligonucleotide sample is a short ribonucleic acid (RNA) molecule with about 19–21 nucleotide length (~ 7–8 kDa), or a 21-mer base pair length in double-stranded siRNA (~ 16 kDa). Due to their relatively large sizes and multiple negative charges compared with typical, small molecule pharmaceutical compounds, oligonucleotides can be difficult to analyze with conventional reversed-phase liquid chromatography (RPLC) methods. In this chapter, we provide an overview of liquid chromatographic (LC) separation of oligonucleotides, including anion-exchange, ion-pair reversed-phase, and mixed-mode LC.
RNA interference offers a novel approach for the development of new therapeutics for targets that... more 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.
RNA interference offers a novel approach for the development of new therapeutics for targets that... more 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.
A stochastic approach was utilized to estimate the probability of a successful isocratic or gradi... more A stochastic approach was utilized to estimate the probability of a successful isocratic or gradient separation in conventional chromatography for numbers of sample components, peak capacities, and saturation factors ranging from 2 to 30, 20-300, and 0.017-1, respectively. The stochastic probabilities were obtained under conditions of (i) constant peak width ("gradient" conditions) and (ii) peak width increasing linearly with time ("isocratic/constant N" conditions). The isocratic and gradient probabilities obtained stochastically were compared with the probabilities predicted by Martin et al. [Anal. Chem., 58 (1986) 2200-2207] and Davis and Stoll [J. Chromatogr. A, (2014) 128-142]; for a given number of components and peak capacity the same trend is always observed: probability obtained with the isocratic stochastic approach<probability obtained with the gradient stochastic approach≤probability predicted by Davis and Stoll < probability predicted by Martin...
A chromatographic analytical method for the direct determination of amino acids by hydrophilic in... more 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.
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