Charged Aerosol Detector Research Articles

Synthesis of HDAC Inhibitor Libraries via Microscale Workflow.

An integrated workflow has been established that enables the synthesis, purification, and subsequent biological testing of compound libraries on a microgram scale. This approach utilizes mass directed preparative HPLC in conjunction with charged aerosol detection (CAD) to generate solutions of investigational compounds at high purity and standardized concentrations, facilitating high fidelity biological testing. This new workflow successfully delivered libraries of histone deacetylase (HDAC) inhibitors that afforded biological data consistent with that obtained from standard scale parallel medicinal chemistry techniques. The advantages of this new approach to library synthesis include greatly reduced material requirements and amenability to high-throughput experimentation.

Multiplexed small molecule impurity monitoring in antibody-based therapeutics by mixed-mode chromatography paired with charged aerosol detection

With advanced genetic engineering technologies and better understanding of disease biology, antibody-based therapeutics are emerging as promising new generation biopharmaceuticals. These novel antibody formats are carefully designed to possess desired features such as enhanced selectivity. However, their high level of structural complexity with multiple components often leads to long development and complex multi-step manufacturing processes, through which a variety of potential small molecule impurities can be introduced. In this work, an in-process assay was developed in which mixed-mode chromatography coupled with charged aerosol detection was utilized for multiplexed detection of nine reagents commonly used in development and manufacturing of antibody-based therapeutics: isopropyl β-d-1-thiogalactopyranoside, methionine sulfoximine, ampicillin, guanidine, dehydroascorbic acid, glutathione, tris(2-carboxyethyl)phosphine, N-acetyl cysteine, and arginine. This method utilized a mixed-mode column with ion-exchange properties operated in the hydrophilic interaction chromatography mode. Various parameters were systematically optimized and under optimal conditions, the method demonstrated excellent specificity, sensitivity, linearity, precision, accuracy, and was successfully applied to determine residual impurities in multiple samples from antibody-derived molecules.

Analytical development of seamless procedures on cation-exchange chromatography and ion-pair chromatography with high-precision mass spectrometry for short-chain peptides

We report comprehensive analytical procedures for characterization of underivatized short-chain peptides by wet chemical treatment and ion-pair liquid chromatography with electrospray ionization–mass spectrometry (LC/ESI–MS). The chromatographic separation via a porous graphitic carbon stationary column eluted using an acetonitrile solvent gradient revealed a positive correlation between the retention time and the degree of hydrophobicity reflected in a distinct relationship between methylene (–CH2–) elongation of amino acids. We also validated the chromatographic separation utilizing an InertSustain PFP column and high-resolution mass spectrometry of the Orbitrap system, which supports the analytical accuracy for identification of monoisotopic mass. Using the quantitative analysis on a Corona charged aerosol detector, the recovery of the underivatized short-chain peptide fraction was >94.0 ± 4.9% (n = 10) after purification via cation-exchange chromatography. By eliminating the inorganic and organic matrix effect, we can avoid ion suppression and enhancement to demonstrate accurate analytical evaluations with robust mass spectroscopic responses. The developed method provides new insight into short-chain peptide chemistry that can be utilized for numerous interdisciplinary research applications.

Comprehensive ethoxymer characterization of complex alcohol ethoxy sulphate products by mixed-mode high-performance liquid chromatography coupled to charged aerosol detection

The present work describes a simultaneous mixed-mode high performance liquid chromatography (HPLC) method combined with a universal and non-selective-response detector for the complete ethoxymer profiling of alcohol ethoxy sulphate mixtures. The optimized HPLC methodology combines the dual hydrophilic (HILIC) and reversed-phase selectivity of a surfactant-type column in order to render a comprehensive and simultaneous separation of more than 50 endogenous ethoxymers in a single analysis. Furthermore, an accurate quantitation of every single analyte was achieved using a final universal charged aerosol detector (CAD) including specific mathematical processing tools. Results obtained helped describing a complete alkyl chain and ethoxymer distribution of the investigated AES samples. Method validation evidences provided reliability of the individual ethoxymer contributions determined with the proposed HPLC−CAD methodology. Regarding accuracy including independent nuclear magnetic resonance (NMR) experiments, an excellent correlation was found between the structural information provided by a COSY NMR spectrum and the CAD results regarding the mono/polyethoxylated and the non-ethoxylated/ethoxylated distribution. Additional calculations including the average molecular weight and the degree of ethoxylation for the reference AES sample showed minimum differences (relative error < 1 %) between the two considered techniques. An outstanding precision and linearity along the working concentration range (r2>0.999) was also observed. The individual limit of detection for the target sulphate ethoxymers was determined to be in the low ppm range. Further validated distribution profiles for a large number of AES samples demonstrated the applicability of the optimized HPLC−CAD methodology to routine surfactant screenings. Therefore, the hereby developed methodology provided extensive information regarding the detailed individual ethoxymer profile of AES formulations, which can be extremely useful for the surfactant industry in order to gain information on specific synthesis routes and/or detergency properties.

A Direct Method to Monitor Glutathione Stability in High Concentration Protein Formulations

Due to its antioxidant properties and favorable safety profile, glutathione (GSH) finds use in protein formulations by improving overall protein stability. Once degraded, primarily by oxidation into glutathione disulfide (GSSG), the protecting effect of GSH is lost. A simple, direct method using reversed-phase separation and charged-aerosol detection (RP-CAD) to quantitate GSH is described in this paper. The analytical methodology is also capable of monitoring several by-product degradants of GSH, both oxidative and non-oxidative. For high-concentration protein formulations, the method provides direct analysis of GSH and its degradants in the presence of protein at up to 225 mg/mL simply through a dilution of the sample. Quantitation of many amino acids typically included in pharmaceutical protein formulations is also possible. Use of an online diverting valve in the method prevents interference in the detector from the high protein concentration in formulation. Accuracy and effectiveness of this method is demonstrated through monitoring the stability of GSH in high-concentration protein formulations through confirmation of GSH concentration and mass-balance of its loss over time. Monitoring GSH stability in protein formulations is necessary, as GSH concentration is indicative of protein stability.

Investigations of polymer samples of polyamide 11 concerning the content of monomer, oligomers, and the oxidation stabilizer Irganox 1098 by utilizing inverse gradient HPLC in combination with a triple detection system (diode array detection/mass spectrometry/charged aerosol detection)

The aim of this work was to develop a method to comprehensively characterize extracts from polyamide 11 powder samples used in the laser sintering process. Firstly, high resolution mass spectrometry was applied to identify low molecular compounds in the material. Secondly, by means of an inverse gradient high-performance liquid chromatography (HPLC) in combination with a triple detection system consisting of a diode array detector (DAD), a mass spectrometer (MS), and a charged aerosol detector (CAD), a method was established to determine the amount of monomer and oligomers up to the linear tetramer and cyclic pentamer. Furthermore, the oxidation stabilizer identified as Irganox 1098 was determined. The DAD was utilized for the quantification of the UV-active oxidation stabilizer. Thus, to be able to extract the low molecular weight compounds, various protocols were studied and finally accelerated solvent extraction was used. As there is a lack of commercial standards of polyamide 11 related oligomers, these compounds were detected with the CAD, taking advantage of its uniform response behavior in conjunction with an inverse gradient HPLC protocol. In addition, the MS was utilized as a qualitative detector for peak identification. The information regarding low molecular weight compounds are required as the powder is subject to the thermal loading of the sintering process, which influences the composition of the low molecular weight compounds. Thus, the method was applied to new and oven-aged polyamide 11 samples. Finally, it was possible to monitor the change of low molecular weight compounds in stressed polyamide 11 powder samples.

Polyacetylene (9Z,16S)-16-hydroxy-9,17-octadecadiene-12,14-diynoic acid in Dendropanax morbifera leaves

Dendropanax morbifera extracts were obtained from three different tissues: the heartwood, stem bark, and leaves. The 95% ethanol extract from leaf showed the strongest inhibitory activity (88% inhibition at 50 μg/mL with HepG2 cells) against triglyceride (TG) biosynthesis. Bioactivity-guided fractionation and metabolite investigation of the leaf extracts resulted in the separation and identification of three polyacetylene derivatives that inhibited newly synthesized TG in HepG2 cells. A method for the profile and contents analyses of the leaf extracts were measured using QTOF-MS with a charged aerosol detector (CAD), including (9Z,16S)-16-hydroxy-9,17-octadecadiene-12,14-diynoic acid (1), which was decomposed during the temperature and storage studies. All metabolites decomposed at different temperatures, especially the polyacetylene, which was rapidly oxidized when the isolated form was exposed to air. However, it was fairly stable in the extract at low temperature <–20 °C. The aim of this study was to provide a way to protect the active ingredients in the process of developing and distributing functional foods containing D. morbifera. The application of this quality control/quality assurance step may improve the chemistry, manufacturing, and control for raw materials.

Identification and determination of fructooligosaccharides in snow chrysanthemum ( Coreopsis tinctoria nutt.)

Objective: Small molecules in snow chrysanthemum such as flavonoids, phenolic compounds and amino acids have been extensively investigated. No study to date has focused on water-soluble oligosaccharides. The objective of this study is identification and determination of water-soluble oligosaccharides in snow chrysanthemum. Methods: The oligosaccharides in snow chrysanthemum were identified by high performance thin layer chromatography (HPTLC), liquid chromatography-mass spectrometry (LC-MS) combined MS library and methylation analysis for the first time. Subsequently the oligosaccharides were determined by high performance liquid chromatography with a charged aerosol detector (HPLC-CAD). Results: The oligosaccharides in snow chrysanthemum were identified as inulin-type fructooligosaccharides (FOS). The yield of FOS (DP3~DP13) in the first extraction was over 97.6%. The RSDs of repeatability in three sample amount levels (0.08 g, 0.1 g, 0.12 g) are lower than 4.8% and the RSDs of stability are less than 3.5%. The recoveries of FOS (DP3~13) were ranging from 96.9% to 105.6%. The contents of FOS (DP3~DP13) in flowers of snow chrysanthemum from different regions of China were greatly variant. Conclusion: This is the first time to identify and quantify FOS in snow chrysanthemum which is helpful for its performance in the in the fields of biomedical, agriculture and functional food industry as well as development of the quality control methods. In addition, the identification approach developed in this work can also be used for screening potential natural sources containing FOS..

UHPLC Coupled with Charged Aerosol Detector for Rapid Separation of Steviol Glycosides in Commercial Sweeteners and Extract of Stevia Rebaudiana

Natural sweeteners are in high demand as a part of a healthy lifestyle. Among them, sweeteners with decreased caloric value and suitability for diabetes patients are most requested. Extension in their consumption extends the need for their quality control. A fast gradient UHPLC coupled with charged aerosol detection enabling quantitation of stevioside, rebaudioside A-D, and steviolbioside in commercial sweeteners and Stevia rebaudiana plant extracts has been developed. The method was developed to achieve high efficiency, simplicity, versatility, and low solvent consumption. All steviol glycosides were baseline-separated in less than 4 min with a total run time of 7 min. Buffer-free eluents were used in the separations and only 2.45 mL solvent were needed per analysis. The state of the art column Luna Omega Polar featuring polar modification of C18 stationary phase was used for the excellent separation of polar steviol glycosides. Critical pair of glycosides, stevioside and rebaudioside A, were baseline separated with a resolution of 2.41. The universal charged aerosol detector allowed quantitation of steviol glycosides with a limit of detection and quantitation 0.15 and 0.5 µg/mL, respectively. Method intra-day precision was less than 2% (RSD), and the recovery was 89.6-105.0% and 93.8-111.4% for plant material and sweetener tablets, respectively. The quantity of steviol glycosides in three out of four commercial sweeteners was 3.0-12.3% higher than declared. The content was about 12.4% less than declared in one sample. The most abundant glycoside detected in sweeteners was stevioside followed by rebaudioside A. A leaf-to-stem ratio describing the dominant accumulation of steviol glycosides in leaves affected the differences in the amount of steviol glycosides among plant samples.

Power function setting in charged aerosol detection for the linearization of detector response – optimization strategies and their application

Charged aerosol detection (CAD) is an universal technique in liquid chromatography that is increasingly used for the quality control of drugs. Consequently, it has found its way into compendial monographs promoted by its simple and robust application. However, the response of CAD is inherently nonlinear due to its principle of function. Thus, easy and rapid linearization procedures, in particular regarding compendial applications, are highly desirable. One effective approach to linearize the detector's signal makes use of the built-in power function value (PFV) setting of the instrument. The PFV is basically a multiplication factor to the power law exponent of the equation describing the CAD's response, thereby altering the detector's signal output to optimize the quasi-linear range of the response curve. The experimental optimization of the PFV for a series of analytes is a time-consuming process, limiting the practicability of this approach. Here, two independent approaches for the determination of the optimal PFV based on an empirical model and a mathematical transformation in each case, are evaluated. Both approaches can be utilized to predict the optimal PFV for each analyte solely based on the experimental results of a series of calibration standards obtained at a single PFV. The approaches were applied to the HPLC-UV-CAD impurity analysis of the drug gabapentin to improve the observed nonlinear response of the impurities in the range of interest. The predicted optimal PFV of both approaches were in good agreement with the experimentally obtained optimal PFV of the analytes. As a result, the accuracy of the method was significantly improved when using the optimal PFV (90 – 105% versus 81 – 115% recovery rate for quantitation by either single-point calibration or linear regression) for the majority of the analytes. The final method with a PFV adjusted to 1.30 was validated with respect to ICH guideline Q2(R1).

Simple method for simultaneous quantification of two new derivatives of betulinic and ursolic acids with antimalarial activity by RP-HPLC-UV-CAD

Endemic in more than 90 countries and territories, malaria is the most widely, populational, and geographically, parasitic disease in the world. Plasmodium sp. resistance to available drugs is one of the biggest problems for malaria eradication. In this study, we develop a method for the simultaneous determination of two new derivatives of betulinic and ursolic acids with antimalarial activity designated 3-O-butanoylbetulinic and 3-O-butanoylursolic acids. An analytical method was developed by high-performance liquid chromatography coupled, in series, to ultraviolet (UV) and charged aerosol (CAD) detectors. The chromatographic system, operated isocratically by reversed-phase, consisted in a mobile phase composed of acetonitrile: water pH 3.0 adjusted with formic acid (85:15, v/v), flow rate of 1.2 mL/min and a PhenoSphere Next octadecylsilane column (250 mm x 4.6 mm, 5 μm particle size). Chromatograms were recorded simultaneously in UV and CAD, at a concentration of 50 µg mL-1, an injection volume of 20 μL at a controlled temperature of 50 °C. The method was found to be selective, linear (r &gt; 0.99), precise (RSD &lt; 2.0%), accurate, and robust for both analytes, and considered statistically validated, and can be applied to the identification and quantification of these new drug candidates.

Использование высокоэффективной жидкостной хроматографии с детектором заряженных аэрозолей для определения четвертичных аммониевых соединений в дезинфицирующих средствах

Cationic surfactants based disinfectants have been widely used in practice for over 20 years. Despite the significant advances in analytical chemistry during this period, the determination of mixtures of several quaternary ammonium compounds in one disinfectant still causes certain difficulties. This work describes the separation in the combined presence of alkyldimethylbenzylammonium chloride, alkyldimethyl (ethylbenzyl) ammonium chloride, and dimethyldioctylammonium chloride in disinfectants using high performance liquid chromatography with a charged aerosol detector. The method was tested on model solutions and real disinfectants. The linearity range for alkyldimethylbenzylammonium chloride was from 0.03 to 0.17 %, for alkyldimethyl (ethylbenzyl) ammonium chloride – from 0.03 to 0.15 %, and from 0.01 to 0.11 % for dimethyldioctylammonium chloride. The calculated relative error of the method did not exceed 9 %. Thus, the proposed method can be used to analyze preparative forms of disinfectants containing these and other cationic surfactants.

Comparison between charged aerosol detector and evaporative light scattering detector for analysis of sugar in Zhusheyong Yiqi Fumai and study on accuracy of methods

Evaporative light scattering detector(ELSD) and charged aerosol detector(CAD) methods were established in this study for the content determination of four kinds of sugars in Zhusheyong Yiqi Fumai(YQFM), and the factors affecting the accuracy of CAD methods were discussed. HPLC-ELSD chromatographic separation was performed on a Shodex Asahipak NH2 P-50 column with acetonitrile-water(75∶25)as the mobile phase, with a flow rate of 0.8 mL·min~(-1), drift tube temperature of 80 ℃. The analysis by HPLC-CAD was performed on the same column with acetonitrile-water as mobile phase for gradient elution, with a flow rate of 0.8 mL·min~(-1), a neb temperature of 45 ℃, and power function(PF) of 1.3. The samples of YQFM were detected by ELSD and CAD respectively. It was found that YQFM was composed of fructose, glucose, sucrose and maltose. The linear relationship of the two methods was good, and the recoveries, reproducibility and stability of these four kinds of sugars measured by the two methods satisfied the requirements of methodology. Both CAD and ELSD detectors were accurate and reliable in detecting saccharides components in YQFM. In addition, it was revealed in this study for the first time that the PF parameter of CAD had an important influence on the accuracy of sugar determination and acted as the key parameter of CAD method. It was also found that for CAD, a non-linear detector, there was no significant difference between the results of linear regression and logarithmic regression.

Corona Charged Aerosol Detector in studying retention and β-cyclodextrin complex stability using RP-HPLC

Binding between cyclodextrin (CD) cavity and guest molecule in Reversed Phase High-Performance Liquid Chromatography (RP-HPLC) is dynamic process. In general, increasing CD concentration is inducing inclusion complex formation, leading to reduction of analyte’s retention time. Consequently, the shortness in retention time is a measure of complex stability in HPLC. However, under certain experimental conditions, the retention of some analytes could be prolonged even when concentration of CD in the mobile phase is increased. In order to reveal the cause of this unexpected retention behavior, the present study was carried on. The model mixture consisted of risperidone, olanzapine and their related impurities, while β-CD was selected among CDs, as in the previous study. In order to achieve fast equilibrium between free analyte and β-CD-analyte complex, β-CD was not added to the mobile phase, but only to the sample. Detection was performed with Corona Charged Aerosol Detector (CAD), suitable for non-chromophoric β-CD. When analyzing olanzapine impurity B-β-CD sample, three peaks were detected, namely free β-CD, β-CD-analyte complex and free analyte. The complex stability constant was calculated employing a modification of the Benesi-Hildebrandt equation and CAD has proven to be useful in complex stability constants assessment if retention of free analyte and β-CD-analyte complex is distinguished. For all other analytes only two peaks could be detected, because free analyte and formed complex are eluting at the same retention time. Under such circumstances, the authors proposed the methodology for calculating stability constants and confirmed its applicability to studied model mixture.

Universal response method for the quantitative analysis of multi-components in josamycin and midecamycin using liquid chromatography coupled with charged aerosol detector

Josamycin and midecamycin are consisted of three groups of components with different ultraviolet maximum absorption wavelengths (λmax), which are 231 nm, 280 nm and 205 nm. The quantitative analysis of all these components is challengeable due to the absence of the respective reference substances. To address this problem, universal and reliable methods were developed using high performance liquid chromatography coupled with charged aerosol detector (HPLC-CAD) for the quantitative analysis of components in josamycin and midecamycin. The chromatographic conditions and CAD parameters setting were optimized. Subsequently, the components were identified using HPLC coupled with ion trap/time-of-flight mass spectrometry (IT/TOF MS). The developed methods were validated by assessing linearity, limit of quantitation (LOQ), accuracy, precision and robustness. Good separations were achieved for all components and the adjustment of the filter valve and power function value efficiently improved sensitivity. The developed methods were more comprehensive than current HPLC-UV method. The experimental results demonstrated good linearity with coefficients of determination (R2) greater than 0.999 in the range of 0.002−0.30 mg mL−1. The limits of detection (LOD) were ranging from 1.8 to 2.0 μg·mL−1. The intra-day and inter-day RSD values were less than 2.0 % (n = 6) and 5.6 % (n = 9) respectively. The recoveries were 95.0 %–124.0 % at the spiked concentration levels of 0.05 %, 0.50 %, 0.10 % and 2.5 % with relative standard deviations (RSDs, n = 3) lower than 2.0 %. Finally, the developed methods were successfully applied to the quantitative analysis of minor components and used main components (leucomycin A3 and midecamycin A1) as alternative reference substance of minor components. The overall results demonstrated that the HPLC-CAD was a good alternative for the quantitative analysis of multi-components in 16-membered macrolides.

Identification and target-pathway deconvolution of FFA4 agonists with anti-diabetic activity from Arnebia euchroma (Royle) Johnst

FFA4 is a novel therapeutic target for the treatment of metabolic diseases, such as type II diabetes. However, there are still few ligands with structural diversity, selectivity and high potency, and the signaling pathway downstream of FFA4 remains to be poorly characterized. In this study, a high performance liquid chromatography-corona charged aerosol detector (HPLC-CAD) combined with label-free dynamic mass redistribution (DMR) method was introduced to guide the discovery of FFA4 agonists from Arnebia euchroma (Royle) Johnst. Ten compounds were identified as FFA4 agonists and structure-activity relationship was obtained. Among them, shikonin displayed the most potent activity with pEC50 value of 6.02 ± 0.19. The activity of shikonin was confirmed by FLIPR (fluorometric imaging plate reader) assay. Signaling pathways of FFA4 were explored in HT-29 cells endogenously expressing FFA4 using shikonin and known FFA4 agonists α-linolenic acid (ALA) and TUG891. Multiple pathways included Gq/11-PLC-Ca2+-PKC, RohA, JNK, p38 MAPK, Gi/o and PI3K signaling but may not involve Gs signaling triggered by shikonin, ALA and TUG891. Besides, shikonin, TUG891 and ALA could induce ERK1/2 and AKT phosphorylation in HT-29 cells. Moreover, anti-diabetes effects of shikonin were evaluated on the glucose intolerance in diabetic db/db mice. Shikonin reduced plasma glucose level, suggesting that it had the potential in treatment of type II diabetes. The agonists identified in this study provided structure guidance for FFA4 drug design. This study was also useful for understanding FFA4 pharmacology and its biological function.

Hyphenation of supercritical fluid chromatography with different detection methods for identification and quantification of liamocin biosurfactants

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.

Investigating the Ionization of Dissolved Organic Matter by Electrospray.

Electrospray ionization (ESI) operating in the negative mode coupled to high-resolution mass spectrometry is the most popular technique for the characterization of dissolved organic matter (DOM). The vast molecular heterogeneity and the functional group diversity of this complex mixture prevents the efficient ionization of the organic material by a single ionization source, so the presence of uncharacterized material is unavoidable. The extent of this poorly ionizable pool of carbon is unknown, is presumably variable between samples, and can only be assessed by the combination of analysis with a uniform detection method. Charged aerosol detection (CAD), whose response is proportional to the amount of nonvolatile material and is independent from the physicochemical properties of the analytes, is a suitable candidate. In this study, a fulvic acid mixture was fractionated and analyzed by high-pressure liquid chromatography–mass spectrometry in order to investigate the polarity and size distributions of highly and poorly ionizable material in the sample. Additionally, DOM samples of terrestrial and marine origins were analyzed to evaluate the variability of these pools across the land–sea aquatic continuum. The relative response factor values indicated that highly ionizable components of aquatic DOM mixtures are more hydrophilic and have lower molecular weight than poorly ionizable components. Additionally, a discrepancy between the samples of terrestrial and marine origins was found, indicating that marine samples are better represented by ESI than terrestrial samples, which have an abundant portion of hydrophobic poorly ionizable material.

Development of an ultra-high-performance liquid chromatography-charged aerosol detection/UV method for the quantitation of linear polyethylenimines in oligonucleotide polyplexes.

Linear polyethylenimines are polycationic excipients that have found many pharmaceutical applications, including as a delivery vehicle for gene therapy through formation of polyplexes with oligonucleotides. Accurate quantitation of linear polyethylenimines in both starting solution and formulation containing oligonucleotide/polyethylenimine polyplexes is critical. Existing methods using spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry time-of-flight, or nuclear magnetic resonance are either complex or suffer from low selectivity. Here, the development and performance of a simple analytical method is described whereby linear polyethylenimines are resolved by ultra-high-performance liquid chromatography and quantified using either a charged aerosol detector or an ultraviolet detector. For formulated oligonucleotide/polyethylenimine polyplexes, sample preparation through decomplexation/digestion by trifluoroacetic acid was necessary to eliminate separation interference. The method can be used not only to support formulation development but also to monitor the synthesis/purification and characterization of linear polyethylenimines.

Development of a comprehensive method combining UHPLC-CAD fingerprint, multi-components quantitative analysis for quality evaluation of Zishen Yutai Pills: A step towards quality control of Chinese patent medicine

Zishen Yutai Pills (ZYP) is a well-known Chinese patent medicine which has been used to prevent recurrent miscarriage and treat threatened abortion in China. In this study, a comprehensive strategy combining ultrahigh performance liquid chromatography coupled with charged aerosol detector (UHPLC-CAD) fingerprint and multi-components quantitative analysis was developed and validated for quality evaluation of ZYP. For fingerprint analysis, a total of 52 characteristic peaks were selected to evaluate the similarities of 16 batches of ZYP. In addition, combining the chemical fingerprint profile with an advanced hybrid LTQ-Orbitrap mass spectrometer, 281 compounds were identified or tentatively identified in ZYP based on chemical standards, accurate mass and fragmentation information. Moreover, 18 chemical markers were simultaneously determined within 13 min by ultra-performance liquid chromatography couple to tandem mass spectrometry (UPLC-MS/MS) with positive-negative conversion multiple reaction monitor (+/-MRM) technique. This method has been validated and exhibited satisfactory sensitivity, precision, reproducibility and accuracy. The validated quantitative method was successfully applied to the analysis of 16 batches of ZYP samples. The combination of UHPLC-CAD fingerprint and multi-components quantification has been proved to be an efficient and reliable strategy for quality control of ZYP and could be considered as a reference for quality evaluation of Chinese patent medicine.