Ion-pair Reversed-phase High Performance Research Articles

Advancements in the characterisation of oligonucleotides by high performance liquid chromatography-mass spectrometry in 2021: A short review.

The first oligonucleotide therapeutic was approved by the Food and Drug Administration in 1998, and since then, 12 nucleic acids have been commercialised as medicines. To be approved, the oligonucleotides need to be identified and characterised as well as its related impurities. Different methods exist, but the most commonly used is ion-pairing reversed-phase liquid chromatography with tandem mass spectrometry. The separation obtained depends on the mobile phase and column used. Other methods have been developed, notably by using hydrophilic interaction chromatography and two-dimensional high performance liquid chromatography. Furthermore, ion-pairing reversed-phase high performance liquid chromatography ultra-violet spectroscopy detection and mass spectrometry has been optimised for the analysis of methylated nucleobases due to the utilisation of this modification in the drugs. This review covers the recent advancements in the analysis and characterisation of oligonucleotides in 2021 by high performance liquid chromatography mass spectrometry, notably by hydrophilic interaction chromatography and two-dimensional liquid chromatography but also the different parameters that influence the analysis by ion-pairing reversed-phase high performance liquid chromatography, the characterisation of methylated nucleobases, and the recent software developed for oligonucleotides.

A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems

Lipid nanoparticle (LNP)-formulated mRNA vaccines were rapidly developed and deployed in response to the SARS-CoV-2 pandemic. Due to the labile nature of mRNA, identifying impurities that could affect product stability and efficacy is crucial to the long-term use of nucleic-acid based medicines. Herein, reversed-phase ion pair high performance liquid chromatography (RP-IP HPLC) was used to identify a class of impurity formed through lipid:mRNA reactions; such reactions are typically undetectable by traditional mRNA purity analytical techniques. The identified modifications render the mRNA untranslatable, leading to loss of protein expression. Specifically, electrophilic impurities derived from the ionizable cationic lipid component are shown to be responsible. Mechanisms implicated in the formation of reactive species include oxidation and subsequent hydrolysis of the tertiary amine. It thus remains critical to ensure robust analytical methods and stringent manufacturing control to ensure mRNA stability and high activity in LNP delivery systems.

Quantification of Histidine-Containing Dipeptides in Dolphin Serum Using a Reversed-Phase Ion-Pair High-Performance Liquid Chromatography Method

The quantification of histidine-containing dipeptides (anserine, carnosine, and balenine) in serum might be a diagnostic tool to assess the health condition of animals. In this study, an existing reversed-phase ion-pair high-performance liquid chromatography (HPLC)–ultraviolet detection method was improved and validated to quantify serum anserine, carnosine, and balenine levels in the dolphin. The serum was deproteinized with trichloroacetic acid and directly injected into the HPLC system. Chromatographic separation of the three histidine-containing dipeptides was achieved on a TSK–gel ODS-80Ts (4.6 mm × 150 mm, 5 µm) analytical column using a mobile phase of 50 mmol/L potassium dihydrogen phosphate (pH 3.4) containing 6 mmol/L 1-heptanesulfonic acid and acetonitrile (96:4). The standard curve ranged from 0.1 µmol/L to 250 µmol/L. The average accuracy of the intra- and inter-analysis of anserine, carnosine, and balenine was 97–106%. The relative standard deviations of total precision (RSDr) of anserine, carnosine, and balenine in dolphin serum were 5.9%, 4.1%, and 2.6%, respectively. The lower limit of quantification of these compounds was 0.11–0.21 µmol/L. These results indicate that the improved method is reliable and concise for the simultaneous determination of anserine, carnosine, and balenine in dolphin serum, and may be useful for evaluation of health conditions in dolphins. Furthermore, this method can also be applied to other biological samples.

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.

Simultaneous Determination of Five Whitening Agents by Ion-Pair Reversed-Phase High Performance Liquid Chro- matography

ABSTRACT A gradient ion-air reversed-phase high performance liquid chromatography method was developed for simultaneous determination of 5 whitening agents (ascorbic acid (AA), magnesium ascorbyl phosphate (MAP), kojic acid (KA), arbutin (ART) and hydroquinone (HQ)). The gradient elution was performed using Inertsil ODS-3V column (250 × 4.6 mm, 5 μm). The analytes were detected by ultraviolet light absorption at the wavelength of 270 nm, the flow rate was set at 1.0 mL/min, and the sample injection volume was 10 μL. The buffer solution contains 50 mM sodium phosphate monobasic dihydrate and 2 mM n-hexadecyltrimethyl ammonium bromide at pH 4.5. The precision of the proposed method, expressed as relative standard deviation (RSD) (%), was determined by the analysis of each compound. The RSD range was from 0.23 to 0.55%. The calibration curves were found to be linear in 8.12-40.6 μg/mL (KA), 12.44-62.20 μg/mL (ART), 8.28-41.40 μg/mL (HQ), 8.16-40.80 μg/mL (MAP) and 4.25- 1.80 μg/mL (AA). The correlation coefficient of linear regression analysis (R2) was within the range of 0.9968 - 0.9997. The analytical results were satisfied for these 5 whitening agents. The recoveries ranging from 96.97% to 101.22% for lotion, and from 97.17 to 100.80% for cream, were obtained at both low and high concentrations. The proposed method is suitable for routine analysis of the 5 whitening agents in commercial cosmetic products, or examined the allowable amount of whitening agents for the Department of Health.

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.

A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography

We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.

Simultaneous assessment of atorvastatin-ezetimibe combination in tablets by an ion-pair rp-hplc

The purpose of this work was to develop and validate a new ion-pair reversed phase high performance liquid chromatographic method on a standard C18-type stationary phase with UV detection, for the analysis of the two antihyperlipidemic agents as a combination in tablets atorvastatin-ezetimibe. The mobile phase consisted of a 35% of cetyltrimethylammonium bromide (cetrimide) 10−3 M as the ion-pairing agent and 65% acetonitrile. The pH value of the mobile phase was adjusted if necessarily by ammonia solution at 10. The flow rate was 1.5 mL/min and the separation was performed at 40°. The method validation was based on linearity, accuracy, precision and specificity. This method exhibited good linearity and accuracy with mean recovery values between 95–105%, precision with relative standard deviations of the calculated concentrations less than 2% and specificity in the presence of excipients. These results indicates that the proposed method is applicable for the separation and determination of atorvastatin-ezetimibe combination in tablets and could be a relevant method to utilize in quality control laboratories.

Simultaneous Determination of Cysteamine and Cystamine in Cosmetics by Ion-Pairing Reversed-Phase High-Performance Liquid Chromatography

Cysteamine has been used in cosmetics as an antioxidant, a hair straightening agent, and a hair waving agent. However, recent studies indicate that cysteamine can act as an allergen to hairdressers. The objective of this study was to develop and validate a simple and effective reversed-phase high-performance liquid chromatography (RP-HPLC) method for the measurement of cysteamine and its dimer, cystamine. Sodium 1-heptanesulfonate (NaH-pSO) was used as an ion-pairing agent to improve chromatographic performance. Separation was performed on a Gemini C18 column (250 mm × 4.6 mm, 5 µm particle size) using a mobile phase composed of 85:15 (v/v) 4 mM NaHpSO in 0.1% phosphoric acid:acetonitrile. UV absorbance was monitored at 215 nm. The RP-HPLC method developed in this study was validated for specificity, linearity, limit of detection, limit of quantitation, precision, accuracy, and recovery. Cysteamine and cystamine were chromatographically resolved from other reducing agents such as thioglycolic acid and cysteine. Extraction using water and chloroform resulted in the recovery for cysteamine and cystamine ranging from 100.2–102.7% and 90.6–98.7%, respectively. This validated RP-HPLC method would be useful for quality control and monitoring of cysteamine and cystamine in cosmetics.

"Determination of nitrate and nitrite content in Zonar milk serum and derived dairy drinks using ion-pair reversed-phase high performance liquid chromatography "

"Determination of nitrate and nitrite content in Zonar milk serum and derived dairy drinks using ion-pair reversed-phase high performance liquid chromatography "

Novel Ion Pair HPLC Methods for the Assessment of Sitagliptin and Pioglitazone in Tablets

Objective: Developing simple, rapid and accurate ion-pair reversed phase high performance liquid chromatographic methods for the determination of pioglitazone hydrochloride, and sitagliptin phosphate in pure and tablet forms. These methods would be practical additional choices in quality control laboratories. Methods: pioglitazone hydrochloride and sitagliptin phosphateanalysis were performed depending on (Method A), the chromatographic conditions comprised of a classical C18-type stationary phase (250×4.6 mm, 5µm), The mobile phase consisting of 10-2 sodium hexane sulfonic acid, in a mixture of 550ml of deionized water and 450ml of acetonitrile, and apparent pH of 2.5 was adjusted with phosphoric acid. (Method B) is an additional method for pioglitazone hydrochloride analysis, the mobile phase consisting of 10-3 M cetrimide, in a mixture of 400 ml of deionized water and 600ml of acetonitrile, leading to measured pH=10. The flow rate was 1ml/min, the detection wavelengths were at 269nm for pioglitazone HCl (Method A,B), and 267nm for sitagliptin phosphate, the temperature was ambient temperature for both Methods A and B. Results: Two methods were validated in accordance to ICH guidelines. The developed methods given accepted linearity (R2>0.999) in the concentration range of (0.01875-0.21)mg/ml for pioglitazone hydrochloride and (0.1-0.6) mg/ml for sitagliptin phosphate, accuracy with mean recovery values between 98-102%, precision with relative standard deviations of the calculated concentrations were less than 2%. Conclusion: The results showed that the two developed methods were effective in separating and determining of pioglitazone HCl, sitagliptin phosphate in their pharmaceutical forms and routine analysis in quality control laboratories simply.

The content of selected vitamins and iodine in mare’s milk

The aim of the study was to contribute to scarce literature data on the content of selected vitamins and iodine in mare’s milk. The study focused on the content of selected lipophilic vitamins (A, E), hydrophilic vitamins B1, B2, B6) and iodine in milk samples obtained from 8 mares during 6 months of lactation. The content of micronutrients was evaluated according to the stage of lactation. Vitamins B2 and B6 were determined using ion-pair reversed phase high performance liquid chromatography with fluorescence detection, vitamin B1 by fluorescence detection by reversed-phase liquid chromatography, vitamins A and E by the ultra-high performance liquid chromatography method with ultraviolet and fluorescence detection and iodine by the spectrophotometric method. Mean concentrations of vitamins A (0.06 ± 0.08 mg/l), E (0.083 ± 0.14 mg/l), B1 (256.24 ± 44.19 µg/l), B2 (111.40 ± 81.88 µg/l), B6 (0.30 ± 0.12 mg/l) reached lower values in mare’s milk compared to cow’s milk. The mean value of iodine in mare’s milk reached 44.48 ± 54.45 µg/l. Lactation stage proved to be a significant factor for vitamin B6 and iodine.

Analysis of long dsRNA produced in vitro and in vivo using atomic force microscopy in conjunction with ion-pair reverse-phase HPLC.

Long double-stranded (ds) RNA is emerging as a novel alternative to chemical and genetically-modified insect and fungal management strategies. The ability to produce large quantities of dsRNA in either bacterial systems, by in vitro transcription, in cell-free systems or in planta for RNA interference applications has generated significant demand for the development and application of analytical tools for analysis of dsRNA. We have utilised atomic force microscopy (AFM) in conjunction with ion-pair reverse-phase high performance liquid chromatography (IP-RP-HPLC) to provide novel insight into dsRNA for RNAi applications. The AFM analysis enabled direct structural characterisation of the A-form duplex dsRNA and accurate determination of the dsRNA duplex length. Moreover, further analysis under non-denaturing conditions revealed the presence of heterogeneous dsRNA species. IP-RP-HPLC fractionation and AFM analysis revealed that these alternative RNA species do not arise from different lengths of individual dsRNA molecules in the product, but represent misannealed RNA species that present as larger assemblies or multimeric forms of the RNA. These results for the first time provide direct structural insight into dsRNA produced both in vivo in bacterial systems and in vitro, highlighting the structural heterogeneity of RNA produced. These results are the first example of detailed characterisation of the different forms of dsRNA from two production systems and establish atomic force microscopy as an important tool for the characterisation of long dsRNA.

Determination of Zoledronic Acid and Its Related Substances by High Performance Liquid Chromatography with Evaporative Light Scattering Detection.

A method has been developed and validated for analysis of zoledronic acid (ZOL) and its related substances by ion-pair reversed-phase high performance liquid chromatography with evaporative light scattering detection (ELSD). Chromatographic separation was achieved with gradient elution by using a C18 column, mobile phase containing 12 mM ammonium acetate buffer and 35 mM n-pentylamine, whose pH value is 7.0, and 5% acetonitrile. The mobile-phase flow rate was 1.0 mL/min. The calibration plot was linear in the range from 0.4 mg/mL to 6.0 mg/mL for ZOL and from 6.25 μg/mL to 100 μg/mL for its related substances. ZOL and its related substances, namely imidazole-1-yl-acetic acid, phosphate, phosphite and degradation products did not interfere with each other. The method was rapid, linear, accurate and reproducible. The high performance liquid chromatographic method that has been developed to determine the related substances and assay of ZOL can be used simultaneously to evaluate the quality of regular samples. It can be also used to test the stability samples of ZOL.

Arsenic speciation in tree moss by mass spectrometry based hyphenated techniques

A method based on ion-pair reversed phase high performance liquid chromatography (HPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) was developed for arsenic speciation in extract of tree moss. Under the optimal conditions, the limit of detection of eight arsenic species including arsenite (AsIII), arsenate (AsV), monomethylarsonic acid (MMA), dimethylarsonic acid (DMA), trimethylarsinoxide (TMAO), tetramethylarsonium (Tetra), arsenocholine (AsC) and arsenobetaine (AsB) is between 0.04 and 0.07 ng/mL, with a linear range of 0.2 − 500 ng/mL. Three unknown arsenic species (Unk1, Unk2 and Unk3) and six specific arsenic species (AsIII, AsV, DMA, TMAO, Tetra and AsB) were detected in the extract of tree moss. Unk3 was identified as a kind of arsenosugars (2,3-dihydroxypropyl-5-deoxy-5(dimethylarsenoso)furanoside, arsenosugar X) by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-qTOF-MS).

Separation of Glutathionate-Protected Gold Clusters by Reversed-Phase Ion-Pair High-Performance Liquid Chromatography

Recent studies have demonstrated that reversed-phase high-performance liquid chromatography (RP-HPLC) is a very effective means of separating thiolate (SR)-protected gold clusters (Aun(SR)m). In the present study, we applied RP-HPLC to the separation of hydrophilic glutathionate (SG)-protected Aun(SG)m clusters. To achieve such separation, an ion pair reagent was dissolved in the solution to form ion pairs with the functional groups on the cluster surfaces, improving the interaction between the cluster surfaces and the hydrophobic stationary phase, representing ion pair chromatography. This technique resulted in the high-resolution separation of Aun(SG)m clusters via RP-HPLC. Experiments at different solution pH values led to slight improvements in the resolution, showing that pH adjustment, which modifies the efficiency of the ion pair formation process, is a useful technique for improving this type of separation.

Reduced molecular size and altered disaccharide composition of cerebral chondroitin sulfate upon Alzheimer's pathogenesis in mice.

Alzheimer's disease (AD) is a progressive disorder leading to cognitive impairment and neuronal loss. Cerebral extracellular accumulation and deposition of amyloid ß plaques is a pathological hallmark of AD. Chondroitin sulfate (CS) is an extracellular component abundant in the brain. CS is a sulfated glycosaminoglycan covalently attached to a core protein, forming chondroitin sulfate proteoglycan. The structure of CS is heterogeneous with sulfation modification and elongation of the chain. The structural diversity of CS allows it to play various roles in the brain. Increasing evidence has shown that CS promotes aggregation of amyloid ß peptides into higher-order species such as insoluble amyloid ß fibrils. Difficulties in the structural analysis of brain CS, as well as its heterogeneity, limit the study of potential roles of CS in AD pathology. Here we established a microanalysis method with reversed-phase ion-pair high performance liquid chromatography and found that CS in the brains of Tg2576 AD model mice show a lower molecular size and an increased ratio of CS-B motif di-sulfated disaccharide. Our findings provide insight into the structural changes of cerebral CS upon Alzheimer's pathogenesis.

Structural peculiarity and antithrombin binding region profile of mucosal bovine and porcine heparins

The major compositional differences between bovine mucosal heparin (BMH) and the currently employed porcine mucosal heparin (PMH) have been reported to essentially consist of reduced 6-O-sulfation of the glucosamine residues in BMH and somewhat lower 2-O-sulfation of the iduronate residues in PMH. The present work is based on direct comparison of several BMH and PMH commercial preparations. A combined study by 2D (heteronuclear single quantum coherence, HSQC) NMR and ion-pair reversed-phase high performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) on the heparins, extended to the analysis of their heparinases digests and fractions separated by affinity chromatography on antithrombin (AT), confirmed the previously reported lower degree of 6-O-sulfation and showed lower 3-O-sulfated glucosamine content in BMH. More detailed studies allowed the identification of structural variants of AT-binding region (ATBR) structural variants, showing higher content of the N-sulfated components in BMH than in PMH.

Competitive adsorption of surfactant foaming agents to nanoclays added to cement foams for enhanced strength

The adsorption affinity of a surfactant foaming agent (SFA), α-olefin sulphonate (AOS)—used for generation of foam for low density concrete—to organically-modified montmorillonite (OMMT) has been investigated. OMMT has been proposed as an additive to cement and concrete for improved strength and durability. Similar thermodynamic processes are involved in the generation and stabilisation of foam and in the compatibilisation and stabilisation of organic particles in aqueous environments, so interaction between SFA and OMMT particles is likely. Association of foaming agent molecules with organoclay may lead to poor foaming performance and potential instability of the nanoparticles due to displacement of dispersants from the particle surface by foaming agent. Adsorption isotherms determined using a combination of ion-pair reverse phase high performance liquid chromatography (RP-HPLC) and gravimetric methods revealed that there is a relatively high affinity of AOS for the organoclay particles. This is a dynamic process, with smaller molecules adsorbing quickly but being displaced by larger molecules at higher surfactant loading. From the adsorption isotherm it was possible to calculate the minimum AOS addition that will ensure the full foaming performance in the cement formulation. Relative adsorption affinity and competitive adsorption at the particle surface of AOS with non-ionic and anionic surfactants commonly used as wetting and dispersing agents, was studied. The dispersants displayed considerably higher relative adsorption onto the organoclay than AOS, particularly in the case of the anionic species. There is evidence that some AOS adsorption takes place in particle systems stabilised by non-ionic dispersants; displacement of high adsorption affinity dispersants by the lower affinity AOS from the OMMT particle surface was not observed.

Simultaneous determination of five active components of compound α-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography

A simple and sensitive method for the simultaneous determination of five active components, D, L-α-hydroxymethionine calcium (HMACa), α-ketovaline calcium (KVCa), D, L-α-ketoisoleucine calcium (KILCa), α-ketoleucine calcium (KLCa) and α-ketophenylalanine calcium (KPACa) of compound α-ketoacid tablet in human urine by ion-pair reversed-phase high performance liquid chromatography (RP-HPLC) was developed and validated. The separation conditions, such as the concentration of ion-pair reagent, the pH value of the mobile phase and the concentration of the buffer were optimized. All the five analytes were separated well on a C18 column (250 mm x 4.6 mm, 5 µm) with diode array detection at 210 nm and the column temperature of 35 °C. The mobile phases were acetonitrile and 20 mmol/L phosphate buffer (containing 15 mmol/L tetrabutylammonium hydroxide; pH 7) at the flow rate of 1. 0 mL/min with gradient elution. The calibration curves for the five components were linear in the range from 20 to 200 mg/L (r≥ 0. 9990). The limits of detection (LODs, S/N= 3) were 3.0, 5. 0, 3. 6, 5.7 and 2. 5 mg/L, and the limits of quantification (LOQs, S/N= 10) were 9. 6, 16.7, 12.0, 19.0 and 8.3 mg/L for HMACa, KVCa, KILCa, KLCa and KPACa, respectively. The intra-day and inter-day precisions were less than 7%, and the average recoveries were between 86.79% and 112. 00% in the human urine with RSDs lower than 9% (n= 5). The method proved precise, specific and reproducible, and can be used for the determination of the five components in urine.