Reversed-phase Ion-pair Chromatography Research Articles

Impurity profiling of siRNA by two-dimensional liquid chromatography-mass spectrometry with quinine carbamate anion-exchanger and ion-pair reversed-phase chromatography

A short RNA with the sequence of the antisense strand of Patisiran has been selected as test material for the investigation of its common impurities using three different two-dimensional liquid chromatography (2D-LC) platforms. On the one hand, a quinine (QN) carbamate-based weak anion-exchange (AX) stationary phase (QN-AX) and a classical C18 reversed phase (RP) stationary phase in ion-pair (IP) mode with tripropylammonium acetate, respectively, have been used in the first dimension (1D) to provide the selectivity for impurities formed during the synthesis of the RNA. In the next step, certain peaks of interest from 1D have been transferred by multiple-heart-cutting (MHC) into a 2D in which an ESI-MS-compatible non-ionpairing RP method has been used for desalting via a diverter valve to remove non-volatile phosphate buffer components and ion-pair agents, respectively. Thus, a sensitive electrospray-ionization quadrupole time of flight mass spectrometry (ESI-TOF-MS) analysis of resolved impurity peaks of the siRNA has become possible under MS-friendly conditions. With both 2D-LC setups, peak purity of the ON has been evaluated by selective comprehensive (high resolution) sampling of the main peak. In a third MHC 2D-LC approach, the QN-AX LC mode was online coupled with the IP-RPLC in the 2D using UV detection. It allows the separation of additional impurities which coeluted in the first dimension. The potential of these methods for comprehensive impurity profiling of ON therapeutics is illustrated and discussed.

Isolation and Characterization of Nitrate Reducing Bacteria for Conversion of Vegetable-Derived Nitrate to ‘Natural Nitrite’

In the US, sodium nitrate is used as a preservative and curing agent in processed meats and is therefore a regulated ingredient. Nitrate reducing bacteria (NRB) can convert vegetable nitrate into nitrite allowing green/clean label status in the US as per the USDA-FSIS definition of ‘natural nitrite’. The current ‘in-liquid’ test tube assay for detecting nitrite is not suitable for screening mixtures of bacteria nor is commercial nitrate broth suitable for growth of many Gram (+) bacteria. M17 broth was therefore used to develop M17-nitrate broth to be inclusive of Gram (+) bacteria. An ‘on-agar’ colony-screening assay was developed to detect the conversion of nitrate to nitrite on agar plates and could detect one NRB+ colony among ~300–500 colonies on a single plate. Samples that might have NRB were spread-plated on M17 agar plates, sandwiched with nitrate agar, and after incubation followed with sequential agar overlays containing the reagents used in the nitrate reduction assay; the appearance of red color zones above colonies indicated the presence of nitrite. NRB derived from various samples were confirmed for nitrate conversion and both nitrate and nitrite were quantified by C8 reversed-phase (RP) ion-pairing high performance liquid chromatography (HPLC) analysis (1 ppm limit of detection). Staphylococcus carnosus, a strain commonly used for nitrate reduction, was able to convert 1100 ppm M17-nitrate broth to 917 ppm nitrite. Staphylococcus caprae and Panteoa agglomerans, NRB isolated using the M17-nitrate agar assay, were also able to ferment the same broth to 916 ppm and 867 ppm nitrite, respectively. This is the first report of an on-agar colony screening assay for the detection and isolation of nitrite reducing bacteria allowing NRB to be readily isolated. This may allow for the identification of new bacteria that may have a more efficient process to generate nitrite, and possibly concomitant with production of additional natural antimicrobials, as vegetable nitrite becomes more widely used to prevent spore germination.

Dynamic Inventory of Intermediate Metabolites of Cyanobacteria in a Diurnal Cycle.

SummaryCyanobacteria are gaining importance both as hosts for photoautotrophic production of chemicals and as model systems for studies of diurnal lifestyle. The proteome and transcriptome of cyanobacteria have been closely examined under diurnal growth, whereas the downstream effects on the intermediary metabolism have not received sufficient attention. The present study focuses on identifying the cellular metabolites whose inventories undergo dramatic changes in a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801. We identified and quantified 67 polar metabolites, whose inventory changes significantly during diurnal growth, with some metabolites changing by 100-fold. The Calvin-Benson-Bassham cycle intermediates peak at midday to support fast growth. The hitherto unexplored γ-glutamyl peptides act as reservoirs of amino acids. Interestingly, several storage molecules or their precursors accumulate during the dark phase, dispelling the notion that all biosynthetic activity takes place in the light phase. Our results will guide metabolic modeling and strain engineering of cyanobacteria.

Use of a Mixed Cationic-Reverse Phase Column for Analyzing Small Highly Polar Metabolic Markers in Biological Fluids for Multiclass LC-HRMS Method

The determination of small highly polar metabolites at low concentrations is challenging when reverse-phase (RP) chromatography is used for multiclass analysis. A mixed cationic-RP column coupled to high-resolution tandem MS (HR-MS/MS) was tested for highly polar compounds in biological fluids, i.e., trimethylamine N-oxide (TMAO) and the isobaric molecules beta-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB). The efficient retention and separation of the above compounds were obtained with common and MS-friendly RP conditions, reaching high selectivity and sensitivity. The method was firstly assessed in plasma and urine, showing good linearity in the range 50–1000 µg/L and 500–10,000 µg/L for TMAO and both BMAA and DAB, respectively. Excellent precision (RDS < 3%) and good accuracies (71–85%) were observed except for BMAA in plasma, whose experimental conditions should be specifically optimized. Preliminary tests performed on compounds with biological relevance and a wider range of polarities proved the effectiveness of this chromatographic solution, allowing the simultaneous analysis of a larger panel of metabolites, from very small and polar compounds, like trimethylamine, to quite lipophilic molecules, such as corticosterone. The proposed LC-HRMS protocol is an excellent alternative to hydrophilic interaction liquid chromatography and ion-pairing RP chromatography, thus providing another friendly analytical tool for metabolomics.

Development and validation of an ion-pair HPLC chromatography for simultaneous determination of lactone and carboxylate forms of SN-38 in nanoparticles

A simple and reproducible reversed-phase ion-pair high performance liquid chromatography (HPLC) method using isocratic elution with UV absorbance detection was developed and validated for the simultaneous determination of carboxylate (C) and lactone (L) forms of SN-38 (active metabolite of irinotecan) in nanoparticles (NPs). Reversed phase (RP) chromatography was performed on a C18 column with an ion-pair solution, KH2PO4 buffer solution and acetonitrile, under a mobile phase 30:30:40 (v/v) and at a flow rate of 1 mL/min. Detection was performed at 265 nm and sharp peaks were obtained for C and L forms of SN-38 at retention times of 2.9 ± 0.01 and 5.1 ± 0.01 min respectively. Linear regression analysis data for the calibration plot showed a good linear relationship between response and concentration in the range of 4 to 100 μg/mL; the regression coefficient was 0.9999 for L form and 0.9998 for C form. The optimized method showed good performance in terms of specificity, linearity, detection and quantitation limits, precision and accuracy in accordance with the International Conference on Harmonization (ICH) Q2 (R1) guidelines. This assay was demonstrated to be applicable for routine quantitation of SN-38 in PLGA NPs.

Simultaneous determination of bromhexine HCl and baicalin in Chinese compound medicine by a reversed-phase ion-pair HPLC

In this article, a reversed-phase ion-pair high-performance liquid chromatography (HPLC) method was established for simultaneous separation and quantitative analysis of bromhexine HCl (BHH) and baicalin in Chinese Ke-Chuan-Ling compound capsule. The influence of the mobile phase composition on the retention of the analytes was investigated. The optimal determination conditions consisted of acetonitrile/water/phosphoric acid/triethylamine (22/78/0.1/0.1, v/v/v/v) mobile phase with the addition of tetra-n-butylammonium bromide (TBA) (0.7 mmol L-1, terminal concentration) as pairing ion, and a detection wavelength at 225 nm. In this case, baicalin and bromhexine HCl were eluted at 21 min and 27 min respectively and were separated successfully from other ingredients. Excellent linear relationship between the peak area and concentration was obtained in the range of 0.2-15.0 μg mL-1 for bromhexine HCl and 0.1-12.0 μg mL-1 for baicalin. The limit of detection of BHH was 0.1 μg mL-1 and it was 0.05 μg mL-1 for baicalin. The average recoveries of bromhexine HCl and baicalin were 97.7% and 99.1%, respectively. From the results it can be concluded that this method is simple, versatile, and reliable for the quality control of Chinese traditional medicines containing baicalin and/or bromhexine HCl.

Multiple heart-cutting mixed-mode chromatography-reversed-phase 2D-liquid chromatography method for separation and mass spectrometric characterization of synthetic oligonucleotides

Until today, ion-pair reversed-phase chromatography is still the dominating method for analytical characterization of synthetic oligonucleotides. Its hyphenation with mass spectrometry, however, has some drawbacks such as ion-suppression in electrospray ionization. To overcome this problem, we present in this work a multiple heart-cutting (MHC) two-dimensional liquid chromatography (2D-LC) method with ultra-violet (UV) and electrospray ionization (ESI) mass spectrometry (MS) detection. A reversed-phase/weak anion-exchange (RP/WAX) stationary phase in the first dimension (1D) provides the selectivity for separation of structurally closely related oligonucleotide sequences and deletions (shortmers), respectively, using a mixed pH/triethylammonium phosphate buffer gradient at constant organic modifier content. Heart cuts of the oligonucleotide peaks are transferred to the second dimension (2D) via a multiple heart-cutting valve which is equipped with two loop decks. The 2D RP column is used for desalting via a diverter valve. Active solvent modulation enables to refocus the oligonucleotide peak into a sharp zone by 2D RP entirely free of non-volatile buffer components and ion-pair agents. Oligonucleotides can thus be sensitively detected by ESI-QTOF-MS under MS-compatible conditions.

A high-resolution measurement of nucleotide sugars by using ion-pair reverse chromatography and tandem columns.

N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese hamster ovary (CHO) cells are typicallyglycosylatedduring biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donorsand impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on theuse of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed. Graphical abstract.

Characterization of Antisense Oligonucleotide Impurities by Ion-Pairing Reversed-Phase and Anion Exchange Chromatography Coupled to Hydrophilic Interaction Liquid Chromatography/Mass Spectrometry Using a Versatile Two-Dimensional Liquid Chromatography Setup.

Determination of phosphorothioate oligonucleotide purity and impurity profile is commonly performed by ion-pairing reversed-phase liquid chromatography (IPRP) with a mobile phase containing triethylamine (TEA) and hexafluoro-2-propanol (HFIP). However, ion-suppressing effects of TEA hamper mass spectrometry (MS) instrumentation sensitivity and HFIP can affect the robustness of the mass spectrometer due to its corrosive nature. Anion exchange chromatography (AEX) is an orthogonal separation mode to IPRP but typically cannot be directly coupled to MS. In this work, we developed a multiple heart-cutting IPRP-, AEX-hydrophilic interaction liquid chromatography(HILIC)/MS method for quantification and high sensitivity identification of antisense oligonucleotide (ASO) impurities using a Q-Exactive mass spectrometer. Notably, both AEX-HILIC and IPRP-HILIC modes could be operated on a versatile two-dimensional liquid chromatography (2D-LC) setup including several column selectors. The HILIC mobile phase contained 25 mM ammonium acetate and allowed identifying impurities at levels down to 0.3%. Careful selection of the sample loop volume and the 2D HILIC column dimension allowed straightforward coupling of HILIC for both IPRP and AEX without the need to use any solvent modulation. Overall, the 2D HILIC allowed online desalting of AEX and IPRP modes and further separation of additional impurities.

Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates

Glycosaminoglycans (GAGs) are polysaccharides produced by most mammalian cells and involved in a variety of biological processes. However, due to the size and complexity of GAGs, detailed knowledge about the structure and expression of GAGs by cells, the glycosaminoglycome, is lacking. Here we report a straightforward and versatile approach for structural domain mapping of complex mixtures of GAGs, GAGDoMa. The approach is based on orthogonal enzymatic depolymerization of the GAGs to generate internal, terminating, and initiating domains, and nanoflow reversed-phase ion-pairing chromatography with negative mode higher-energy collision dissociation (HCD) tandem mass spectrometry (MS/MS) for structural characterization of the individual domains. GAGDoMa provides a detailed structural insight into the glycosaminoglycome, and offers an important tool for deciphering the complexity of GAGs in cellular physiology and pathology.

Impurity profiling of l-aspartic acid and glycine using high-performance liquid chromatography coupled with charged aerosol and ultraviolet detection

For the compendial related substances test of l-aspartic acid (Asp) and glycine (Gly), two separate reversed-phase ion-pair high-performance liquid chromatography methods coupled with charged aerosol and ultraviolet detection were developed. Separation of all putative impurities, in particular of the related carboxylic and amino acids, was achieved using volatile perfluorocarboxylic acids as ion-pairing reagents on a polar embedded C18 stationary phase. It was shown that an adjustment of the evaporation temperature of the charged aerosol detector (CAD) was an efficient strategy for meeting the required quantitation limits, when dealing with non-volatile analytes. It was also demonstrated that the usage of a two detector setup can be beneficial for extending the detection range and providing accurate quantitation of low level impurities (LOQs from 5 to 50 ng on column). Both methods were validated with accordance to ICH guideline Q2(R1) assessing specificity, linearity, accuracy, precision, and robustness. Several batches of Asp and Gly were tested for related substances using the developed methods. The purity of each sample was higher than 99.7 %. Coupled charged aerosol and UV detection proved to be a more simple, robust and selective alternative to established derivatization procedures such as the Amino-Acid-Analyser (AAA) for the impurity profiling of amino acids, and should thus be considered for implementation into pharmacopoeial monographs in the future.

Determination of isotope fractionation of Cr(iii) during oxidation by LC/low-resolution MC-ICPMS

A robust and sensitive method for the precise isotopic analysis of 53Cr/50Cr during Cr(iii) oxidation by coupling ion pair reversed-phase chromatography and Multicollector Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS) is presented.

Two-dimensional liquid chromatography coupled to mass spectrometry for impurity analysis of dye-conjugated oligonucleotides

Two-dimensional liquid chromatography coupled to mass spectrometry (2D-LC/MS) has been successfully implemented for several biopharmaceutical applications, but applications for oligonucleotide analysis have been relatively unexplored. When analyzing oligonucleotides in one-dimension, selecting an ion-pairing agent often requires a balance between acceptable chromatographic and mass spectrometric performance. When oligonucleotides are modified or conjugated to include extremely hydrophobic groups, such as fluorophores, the separation mechanism is further complicated by the impact the fluorophore has on retention. Triethylamine (TEA) buffered in hexafluoroisopropanol (HFIP) is the most commonly used ion-pairing agent for analyses requiring mass spectrometry, but the elution order of dye-conjugated failed sequences relative to the main peak is not length-based compared to what would be predicted for unconjugated oligonucleotides having the same sequence. Hexylammonium acetate (HAA) offers more efficient ion-pairing for a length-based separation, but MS response is compromised due to ion suppression. In this study, 2D-LC/MS is used to show that dye-conjugated oligonucleotide failed sequences can be resolved from the parent oligonucleotide using a strong ion-pairing agent in the first-dimension and further identified using a weaker but MS compatible ion-pairing agent in the second-dimension, results that are not achievable in a one-dimensional analysis. More specifically, a heart-cut configuration using ion-pair reversed-phase chromatography in both the first and second dimension (IP-RP – IP-RP) is used to transfer the n-1 impurity from a length-based separation in the first-dimension to a second-dimension analysis for identity confirmation using a single quadrupole detector. Identical C18 column chemistry is used in both the first and second dimension to exploit changes in selectivity that are due to mobile phase selection. The n-1 impurity from the two-dimensional analysis can be detected at low nanogram levels, comparable to results achieved in a one-dimensional dilution series, which approaches the limit of detection of the instrumentation. This work has future applicability to more complex impurity profiling using high-resolution instrumentation, where a more extensive set of impurities could not be evaluated using one-dimensional techniques.

Salt-free fractionation of complex isomeric mixtures of glycosaminoglycan oligosaccharides compatible with ESI-MS and microarray analysis

Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization.

Separation and indirect ultraviolet detection of piperidinium cations by using imidazolium ionic liquids in liquid chromatography

A method for the separation and indirect ultraviolet detection of piperidinium cations by using imidazolium ionic liquids in liquid chromatography was developed. This method uses the most common ultraviolet detector and C18 reversed-phase chromatographic column. The influences of imidazolium ionic liquids, ion-pair reagents, organic solvents, etc. on the separation and detection of piperidinium cations were investigated. The imidazolium ionic liquids in mobile phase are both the background ultraviolet absorption reagent of indirect ultraviolet detection and the active components to improve the separation of piperidinium cations. Compared with reversed-phase chromatography, reversed-phase ion-pair chromatography is a better method for the determination of piperidinium cations. The determination of N-methyl-N-propylpiperidinium (PMPI) and N-butyl-N-methylpiperidinium (BMPI) was achieved by reversed-phase ion-pair chromatography with indirect ultraviolet detection using 0.5 mmol/L 1-ethyl-3-methylimidazolium tetrafluoroborate–0.1 mmol/L sodium 1-heptanesulfonate aqueous solution/methanol (volume ratio 70–30) as mobile phase. The retention times of PMPI and BMPI were 1.96 and 2.48 min, respectively. The limits of detection for PMPI and BMPI were 0.29 and 0.53 mg/L, respectively. The linear range of the method for determination of PMPI and BMPI was 2.0–100.0 mg/L. The relative standard deviation of the method is less than 2.0%. The method has been applied to the determination of piperidinium ionic liquid samples synthesized by chemistry laboratory with satisfactory accuracy and precision. This method is simple, rapid and easy to popularize.

New Evidence of the Bidentate Binding Mode in 3-MBA Protected Gold Clusters: Analysis of Aqueous 13-18 kDa Gold-Thiolate Clusters by HPLC-ESI-MS Reveals Special Compositions Aun(3-MBA)p, (n = 48-67, p = 26-30).

Gold clusters protected by 3-MBA ligands (MBA = mercaptobenzoic acid, –SPhCO2H) have attracted recent interest due to their unusual structures and their advantageous ligand-exchange and bioconjugation properties. Azubel et al. first determined the core structure of an Au68-complex, which was estimated to have 32 ligands (3-MBA groups). To explain the exceptional structure-composition and reaction properties of this complex, and its larger homologs, Tero et al. proposed a “dynamic stabilization” via carboxyl O–H––Au interactions. Herein, we report the first results of an integrated liquid chromatography/mass spectrometer (LC/MS) analysis of unfractionated samples of gold/3-MBA clusters, spanning a narrow size range 13.4 to 18.1 kDa. Using high-throughput procedures adapted from bio-macromolecule analyses, we show that integrated capillary high performance liquid chromatography electrospray ionization mass spectrometer (HPLC-ESI-MS), based on aqueous-methanol mobile phases and ion-pairing reverse-phase chromatography, can separate several major components from the nanoclusters mixture that may be difficult to resolve by standard native gel electrophoresis due to their similar size and charge. For each component, one obtains a well-resolved mass spectrum, nearly free of adducts or signs of fragmentation. A consistent set of molecular mass determinations is calculated from detected charge-states tunable from 3− (or lower), to 2+ (or higher). One thus arrives at a series of new compositions (n, p) specific to the Au/3-MBA system. The smallest major component is assigned to the previously unknown (48, 26); the largest one is evidently (67, 30), vs. the anticipated (68, 32). Various explanations for this discrepancy are considered. A prospective is given for the various members of this novel series, along with a summary of the advantages and present limitations of the micro-scale integrated LC/MS approach in characterizing such metallic-core macro-molecules, and their derivatives.

HPLC methods for purity evaluation of man-made single-stranded RNAs

Synthetic RNA oligos exhibit purity decreasing as a function of length, because the efficiency of the total synthesis is the numerical product of the individual step efficiencies, typically below 98%. Analytical methods for RNAs up to the 60 nucleotides (nt) have been reported, but they fall short for purity evaluation of 100nt long, used as single guide RNA (sgRNA) in CRISPR technology, and promoted as pharmaceuticals. In an attempt to exploit a single HPLC method and obtain both identity as well as purity, ion-pair reversed-phase chromatography (IP-RP) at high temperature in the presence of an organic cosolvent is the current analytical strategy. Here we report that IP-RP is less suitable compared to the conventional ion-exchange (IEX) for analysis of 100nt RNAs. We demonstrate the relative stability of RNA in the denaturing/basic IEX mobile phase, lay out a protocol to determine the on-the-column stability of any RNA, and establish the applicability of this method for quality testing of sgRNA, tRNA, and mRNA. Unless well resolving HPLC methods are used for batch-to-batch evaluation of man-made RNAs, process development will remain shortsighted, and observed off-target effects in-vitro or in-vivo may be partially related to low purity and the presence of shorter sequences.

Simultaneous selenium and sulfur speciation analysis in cultivated Pleurotus pulmonarius mushroom

Selenium (Se) and sulfur (S) speciation analysis in edible and medicinal Se enriched P. pulmonarius extracts was performed. Mycelium, colonized substrate, and fruiting bodies at different harvesting times were analyzed using ion-pairing reversed-phase chromatography coupled to an ICPMS/MS detector. Extraction efficiencies in enzymatically digested and aqueous extracts were between 45.3 and 109% for Se, depending on the sample type. Selenomethionine (Se-Met) was found to be the major Se-compound, together with a number of unknown Se-species. Cystine (Cys2), methionine (Met), and sulfate were also detected and quantified in all samples. Most of the Se-Met (84.0%) and Met (75.8%) were found to be in free form in the fruiting body, in contrast with the mycelium where 53.4% of Se-Met and 80.5% of Met is incorporated into proteins.

Reversed-phase ion-pair chromatography of hydroxyl functionalized imidazolium ionic liquid cations and its application in analysis of environmental water and measurement of hydrophobicity constants

A reversed-phase ion-pair chromatography with ultraviolet detection method for the determination of 1‑hydroxyethyl‑3‑methyl imidazolium cation ([HOEtMIm]+) and 1‑hydroxypropyl‑3‑methyl imidazolium cation ([HOPrMIm]+) was developed. The method has been applied to the analysis of hydroxyl functionalized imidazolium ionic liquid cations in Songhua River water samples and the determination of hydrophobicity constants. [HOEtMIm]+ and [HOPrMIm]+ were not completely separated in the reversed-phase chromatography, but complete separation was realized in the reversed-phase ion-pair chromatography. The effects of ion-pair reagents, organic solvents, column temperatures and ultraviolet detection wavelengths on the separation and detection of hydroxyl functionalized imidazolium ionic liquid cations in reversed-phase ion-pair chromatography were investigated. Ordinary imidazolium ionic liquids do not interfere with the determination of hydroxyl functionalized imidazolium ionic liquids. The suitable ion-pair reagent was sodium octanesulfonate. Using reversed-phase C18 chromatographic column, the mobile phase consisted of 0.5 mmol/L sodium octanesulfonate solution/methanol (85/15, v/v) with column temperature of 40 °C, ultraviolet detection wavelength of 210 nm and flow rate of 1.0 mL/min, [HOEtMIm]+ and [HOPrMIm]+ can be achieved baseline separation within 20 min. The detection limits were 0.36 and 0.50 mg/L, respectively. The relative standard deviations of peak area and retention time were less than 0.80%. Recoveries of sample addition were between 97.3% and100.5%. The method was simple, accurate, reliable, satisfying the requirement of quantitative analysis, and has good practicability.

High resolution fingerprinting of single and double-stranded RNA using ion-pair reverse-phase chromatography

The emergence of new sustainable approaches for insect management using RNA interference (RNAi) based insecticides has created the demand for high throughput analytical techniques to fully characterise and accurately quantify double stranded RNA (dsRNA) prior to downstream RNAi applications. In this study we have developed a method for the rapid characterisation of single stranded and double stranded RNA using high resolution RNase mapping in conjunction with ion-pair reverse-phase chromatography utilising a column with superficially porous particles. The high resolution oligoribonucleotide map provides an important ‘fingerprint’ for identity testing and bioprocess monitoring. Reproducible RNA mapping chromatograms were generated from replicate analyses. Moreover, this approach was used to provide a method to rapidly distinguish different RNA sequences of the same size, based on differences in the resulting chromatograms. Principal components analysis of the high resolution RNA mapping data enabled us to rapidly compare multiple HPLC chromatograms and distinguish two dsRNA sequences of different size which share 72% sequence homology. We used the high resolution RNase mapping method to rapidly fingerprint biomanufactured dsRNA across a number of different batches. The resulting chromatograms in conjunction with principal components analysis demonstrated high similarity in the dsRNA produced across the different batches highlighting the potential ability of this method to provide information for batch release in a high throughput manner.