Ion-pair Liquid Chromatography-mass Spectrometry Research Articles

Assay, Purity, and Impurity Profile of Phosphorothioate Oligonucleotide Therapeutics by Ion Pair-HPLC-MS.

The relatively large molecular size, diastereoisomeric nature, and complex impurity profiles of therapeutic phosphorothioate oligonucleotides create significant analytical challenges for the quality control laboratory. To overcome the lack of selectivity inherent to traditional chromatographic approaches, an ion pair liquid chromatography-mass spectrometry (LCMS) method combining ultraviolet and mass spectrometry quantification was developed and validated for >35 different oligonucleotide drug substances and products, including several commercialized drugs. The selection of chromatographic and spectrometric conditions, data acquisition and processing, critical aspects of sample and buffer preparation and instrument maintenance, and results from method validation experiments are discussed.

Specific Non-Reducing Ends in Heparins from Different Animal Origins: Building Blocks Analysis Using Reductive Amination Tagging by Sulfanilic Acid.

Heparins are linear sulfated polysaccharides widely used as anticoagulant drugs. Their nonreducing-end (NRE) has been little investigated due to challenges in their characterization, but is known to be partly generated by enzymatic cleavage with heparanases, resulting in N-sulfated glucosamines at the NRE. Uronic NRE (specifically glucuronic acids) have been isolated from porcine heparin, with GlcA-GlcNS,3S,6S identified as a porcine-specific NRE marker. To further characterize NRE in heparinoids, a building block analysis involving exhaustive heparinase digestion and subsequent reductive amination with sulfanilic acid was performed. This study describes a new method for identifying heparin classical building blocks and novel NRE building blocks using strong anion exchange chromatography on AS11 columns for the assay, and ion-pair liquid chromatography-mass spectrometry for building block identification. Porcine, ovine, and bovine intestine heparins were analyzed. Generally, NRE on these three heparins are highly sulfated moieties, particularly with 3-O sulfates, and the observed composition of the NRE is highly dependent on heparin origin. At the highest level of specificity, the isolated marker was only detected in porcine heparin. However, the proportion of glucosamines in the NRE and the proportion of glucuronic/iduronic configurations in the NRE uronic moieties greatly varied between heparin types.

Comprehensive Analysis of Umami Compounds by Ion‐Pair Liquid Chromatography Coupled to Mass Spectrometry

An ion-pair LC-ESI-MS method was developed capable of analyzing various reported umami or umami-enhancing compounds, including glutamic acid and 5'-ribonucleotides. The method was validated using tomato and potato samples and showed overall good analytical performance with respect to selectivity, detection limit, linearity, and repeatability. The method was applied to various tomato samples resulting in concentrations of glutamic acid and 5'-ribonucleotides that were in good comparison with literature. The methodology might also be used for the discovery of new umami (enhancing) compounds in an untargeted mode. This was to a certain extent demonstrated for tomato samples by correlating all peaks observed with the ion-pair liquid chromatography-mass spectrometry (LC-MS) method to sensory properties using multivariate statistics. This study describes the development and application of a LC-MS method, which can be used to quantify several known umami (enhancing) compounds in various foods. Furthermore, the method might be useful for the discovery of new umami (enhancing) compounds.

Characterization of Currently Marketed Heparin Products: Reversed-Phase Ion-Pairing Liquid Chromatography Mass Spectrometry of Heparin Digests

Here we report results from the analyses by enzymatic digestion and reversed-phase ion-pairing liquid chromatography mass spectrometry (RPIP-LC-MS) of active pharmaceutical ingredient (API) unfractionated heparins (UFHs) from six different manufacturers and one USP standard sample. We employed a reverse phase ion-pairing chromatography method using a C(18) column and hexylamine as the ion-pairing reagent with acetonitrile gradient elution to separate disaccharides generated from the digestion of the heparins by lyase I and III (E.C. 4.2.2.7 and 4.2.2.8) before introduction into an ion-trap mass spectrometer by an electrospray ionization (ESI) interface. Extracted ion chromatograms (EICs) were used to determine the relative abundance of the disaccharides by mass spectrometry. Eight disaccharides were observed and a similar composition profile was observed from digests of 20 UFH samples. The compositional profile determined from these experiments provides a measure of the norm and range of variation in "good" heparin to which future preparations can be compared. Furthermore, the profile obtained in the RPIP-LC-MS assay is sensitive to the presence of the contaminant, oversulfated chondroitin sulfate A (OSCS), in heparin.

Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography–electrospray ionization mass spectrometry

A method using ion-pairing liquid chromatography–mass spectrometry (MS) was developed for analyzing adenosine 5 ′-monophosphate (AMP), adenosine 5 ′-diphosphate (ADP), and adenosine 5 ′-triphosphate (ATP) in cellular extracts. Dimethylhexylamine (DMHA) was used as ion-pairing agent to retain and separate the analytes on a reversed-phase microbore column with a gradient program. Positive-ion electrospray ionization–MS was applied for the detection because of the use of the ion-pairing agent. Adduct ions of DMHA with AMP, ADP, and ATP were found to be the most intensive peaks and thus selected as quantitative ions. An external calibration method with linear ranges from 0.1 to 20 μM for AMP, 2 to 20 μM for ADP, and 2.5 to 20 μM for ATP was used for the quantitation. The method was applied to determine concentrations of AMP, ADP, and ATP in extracts of cultured rat C6 glioma cells that were pretreated with various concentrations of Zn. The detected levels of the adenosine nucleotides have been used to calculate total adenosine nucleotide and energy charge potential. Changes in cellular energy status upon exposure to increasing concentration of Zn in the culture medium were analyzed. The results indicated that the addition of Zn in a range of 40 to 120 μg/ml cause a gradual increased in energy charge potential of the cells.

Quantification of elastin cross-linking amino acids, desmosine and isodesmosine, in hydrolysates of rat lung by ion-pair liquid chromatography–mass spectrometry

The elastin cross-linking amino acids, desmosine (DES) and isodesmosine (IDE), in hydrolysates of rat lungs were quantified by ion-pair liquid chromatography–electrospray mass spectrometry. The column was a 2.0 mm i.d. × 150 mm Develosil UG3 (ODS) with a mobile phase A of 7 mM pentafluoropropionic anhydride (PFPA) using ultrapure water as the ion-pair reagent, and a mobile phase B of 7 mM PFPA in 80% methanol. The retention times of IDE and DES were 25.5 and 26.6 min, respectively. The mean concentrations of IDE and DES in the lung were 191.6±54.5 nmol/ g lung (dry tissue) (±SD) and 184.0±39.3 nmol/ g lung, respectively, and the IDE/DES ratio was 1.04, in Wistar Kyoto rats. Our results indicate that ion-pair liquid chromatography–mass spectrometry is a useful procedure for quantitation of DES and IDE in hydrolysates of rat lung.