Reversed-phase UHPLC Research Articles

MICROBIAL REPRESENTATION ASSOCIATION WITH LEVEL OF SHORT CHAIN FATTY ACIDS IN SMALL BOWEL

Abstract BACKGROUND Small bowel microbiome and associated postbiotics understanding is still in its infancy. In intestinal bowel disease treatment, surgical interventions leading to an ostomy are often the last resort. The ileostomates and the colostomates lack the whole or part of the colonic function, respectively. In these populations, fecal effluents represent aspects of the small bowel microbial communities and associated small molecules not available in traditional stool collections. METHODS We completed a cross-sectional collection of samples from participants with digestive resections were recruited including 26 ileostomates and 23 colostomates. Additionally, 22 individuals with unaltered digestive tracts were enrolled for a total of 71 participants. Stool were self-collected in transport and storage medium. Identification and quantitation of short chain fatty acids were performed using LC MS/MS equipped with a C18 reversed phase UHPLC column. Concentrations for acetic, propionic, butyric, isobutyric, valeric, 2-methylbutyric, isovaleric, and hexanoic acid were obtained. Analysis of covariance (ANCOVA) was used to evaluate the difference in the mean SCFA concentration across the groups. Microbial membership identification was performed using 16 rDNA sequencing for a subset of 59 samples. A table of amplicon sequence variants (ASV) counts per sample was derived using the dada2 R package. Kruskal-Wallis and Post-hoc test Dunn with Benjamini-Hochberg multiple test correction were used to compare the abundance of bacteria producing and utilizing specific short chain fatty acids between the three groups. RESULTS For the eight measured short chain fatty acids, all were significantly decreased in ileostomates compared to colostomates and control group (p < 0.001). We observed significant positive correlations between microbial metabolite producers and corresponding metabolites for propionic acid, butyric acid, isobutyric acid, and isovaleric acid. For example, at the level of the population, a positive correlation was found between the abundance of butyric acid microbial producers and the butyric acid level (R=0.5, p = 7.3e-05). Conversely, a significant negative correlation between levels of acetate producers and fecal acetic acid was found in the whole population. CONCLUSION The presence of a partial colon, in colostomate, did not drastically alter the detection of the different short chain fatty acids compared to the individuals with an intact colon. In contrast the absence of a colon was associated with a significant drop in level of detected postbiotics. These findings underline the biogeographical distribution of both the microbiome and small molecules along the digestive tract.

Isolation and characterization of pseudo degradation products of acalabrutinib using ESI-HRMS/MS and NMR: Formation of possible geometrical isomers

A forced degradation study of acalabrutinib (ACB), used to treat relapsed mantle cell lymphoma, was performed to identify and characterize all possible major degradation products formed under different stress conditions. The degradation products (DP) were separated using reverse phase UHPLC system on Kinetex EVO C18 column. Major DPs formed were isolated using semi-preparative HPLC and characterized by LC-ESI-HRMS/MS and NMR. ACB degraded to form seven major degradants (DP-I to DP-VII). DP-I and DP-V were formed under alkaline stress condition, whereas DP-II, DP-III, DP-VI and DP-VII were formed under both acidic and basic conditions. Further, DP-IV was formed when ACB drug was exposed to hydrogen peroxide stress condition. ACB was found to be stable when subjected to aqueous (neutral pH), thermal and UV radiation of 254 nm, as it has not shown any significant degradation under these conditions. Interestingly, two pairs of pseudo geometrical isomeric DPs (DP-II and DP-III, DP-VI and DP-VII) were observed. The plausible degradation pathway of ACB and fragmentation patterns of both ACB and major DPs were discussed.

Characterization of the major degradation products of the praziquantel API by mass spectrometry: Development and validation of a stability-indicating reversed phase UPLC method

Praziquantel was subjected to forced degradation studies under various conditions of hydrolysis (acidic, alkaline, and neutral/water), oxidation, photolysis, and thermal as prescribed by International Conference on Harmonization guideline Q1A (R2). A short and simple reversed phase UHPLC method was developed for the Praziquantel API. The method was developed on a Shimadzu, Shimpack Velox, SP-C18, 100 × 2.1 mm, 1.8 µm column. The isocratic mobile phase was a blend of water and acetonitrile in the ratio of 70:30 (v/v), respectively. The flow rate of the mobile phase was 0.5 mL/min. The developed method was validated using ICH guidelines. The parameters considered for method validation were solution stability, specificity, DL/QL, linearity, accuracy, precision, and robustness. The drug showed significant degradation in acidic and alkaline conditions while slight degradation was observed in water and oxidative conditions. The drug was found to be stable in photolytic and thermal conditions. The characterization of four major degradation products (DP1, DP2, DP3, and DP4) was done with LC–Q-TOF-MS/MS in combination with accurate mass measurements. The most probable mechanisms for the formation of DPs have been proposed on the basis fragmentation pattern.

A Qbd Driven Approach for Development and Validation of an RP-UHPLC Method for Simultaneous Estimation of Ofloxacin and Fluconazole: An Application to Pharmaceutical Analysis

A simple reverse-phase UHPLC (RP-UHPLC) analytical method was developed and validated according to International Council on Harmonization (ICH) guidelines for simultaneous estimation of ofloxacin (OFX) and fluconazole (FCZ) using a Response surface aided central composite design (CCD, Design-Expert®, Version 11.1.2.0). The method was validated for linearity, selectivity, sensitivity, accuracy, precision, limits of detection and quantification, solution stability and robustness. OFX and FCZ separation were achieved using Agilent 1290 Infinity II UHPLC system encompassed with Waters Atlantis® T3 (150 × 4.6 mm2 i.d., 3 µm particle size) column, water containing 0.1% v/v formic acid and methanol as isocratic mobile phase. OFX and FCZ eluted at 4.1 and 10.5 min with no interference of the excipients used in formulation at λmax 260 nm. The developed method was envisaged to determine total drug content, in vitro drug release study and ex-vivo corneal permeation study of conventional as well as novel drug delivery formulations. The proposed method is simple, cost-effective, dependable and reproducible and could be used for the regular analysis of OFX and FCZ in bulk and quality controls in the research and pharmaceutical industries.

Investigation into reversed-phase chromatography peptide separation systems part V: Establishment of a screening strategy for development of methods for assessment of pharmaceutical peptides' purity

The paper describes a simple and rapid reversed-phase UHPLC method development screening strategy for the purity determination of peptide-based pharmaceuticals. The protocol utilises five disparate column and six volatile or non-volatile mobile phases (i.e., 30 combinations). The method development strategy has been demonstrated to be highly effective in identifying conditions which generate complementary selectivity and good peak shape. Columns with varying degrees of charge (positive and negative), in addition to their differing hydrophobic character, were used in combination with mobile phases within the pH range of 2.3 to 5.1. The novel ion-pair / chaotropic reagent ammonium hexafluorophosphate at pH 2.3 was shown to be an extremely useful mobile phase additive in that it produced excellent complementary separation and good peak shape. Methanesulfonic acid was demonstrated to be a good alternative to the ubiquitously employed trifluoroacetic acid which failed to generate optimum separation for the peptides investigated highlighting the importance of screening disparate mobile phase additives. Both ammonium hexafluorophosphate and methanesulfonic acid were shown not to adversely affect the stability of C18 columns or demonstrated any irreversible adsorption / memory effects. No pH hysteresis effects were demonstrated with any of the stationary phases on mobile phase pH cycling. No major problems have been observed with the novel mobile phase additives ammonium hexafluorophosphate and methanesulfonic acid, however, it is recommended that they be used with caution until long-term routine use has been established.

Validated Stability-Indicating Assay UHPLC Method for Simultaneous Analysis of Saxagliptin and Metformin in Fixed-Dose Combinations

A Saxagliptin (SAX) and Metformin (MET) fixed-dose combination (FDC) can improve the chances of achieving optimal glycaemic control in type 2 diabetes mellitus subjects. Accordingly, the scientific novelty of design work was to develop a specific and precise stability-indicating UHPLC assay method for simultaneous quantification of cited drugs in extended-release FDC's. The reversed-phase UHPLC resolution was analyzed with the assistance of UPLC BEH C18 (150 mm × 2.1 mm) with 1.7 µm particle size column at ambient temperature using a solvent system in a proportion of (85:15 % v/v) potassium dihydrogen orthophosphate buffer and acetonitrile, with 0.4 mL/min rate of flow of a solvent system. The analytes were supervised at 220 nm by employing photodiode array recognition. The retention times of analytes SAX and MET were 2.687 ± 0.022 min and 1.856 ± 0.03 min, respectively. The SAX and MET have confirmed the linearity ranges of 10 – 60 μg/mL and 100 – 600 μg/mL, with 0.9989 and 0.9979 determination coefficients. The UHPLC method was effectually validated concerning the accuracy, precision, sensitivity, robustness, ruggedness, selectivity, and specificity. Moreover, the anticipated UHPLC method's capability to analyzed the SAX and MET with no obstruction from degradation products.

Novel UHPLC DAD Method for Simultaneous determination of Vildagliptin and Metformin in Bulk and its Tablet formulation

A robust and rapid reversed-phase UHPLC method for routine determination of vildagliptin (VILDA) and metformin (MET) was developed and validated with DAD detection (207 nm). Chromatographic analysis was carried out with isocratic separation by C18 column (150mm x 2.1mm, 5μm) using a mobile phase with a mixture of 1.36 g phosphate buffer (pH 4.2) set to phosphoric acid and acetonitrile (80:20, v/v) with 0.6mL/min flow rate. The procedure was validated as per International Conference on Harmonization (ICH) guidelines for linearity (correlation coefficient r2 = 0.999), accuracy, intermediate precision, and robustness. Accuracy was 99.79%±0.54% and 100.07 %±0.34% for VILDA and MET, respectively. For precision, the %RSD was founded to be less than 2% for three concentrations analyzed from three replicates. The proposed method was used for successful determination of VILDA and MET in a pharmaceutical preparation.

Untargeted UHPLC-ESI-QTOF-MS/MS analysis with targeted feature extraction at precursor and fragment level for profiling of the platelet lipidome with ex vivo thrombin-activation

Lipids play a major role in platelet signaling and activation. In this study, we analyzed the platelet lipidome in an untargeted manner by reversed-phase UHPLC for lipid species separation coupled to high-resolution QTOF-MS/MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) for compound detection. Lipid identification and peak picking was supported by the characteristic regular elution pattern of lipids differing in carbon and double bond numbers. It was primarily based on post-acquisition targeted feature extraction from the SWATH data. Multiple extracted ion chromatograms (EICs) from SWATH data of diagnostic ions on MS1 and MS2 level from both positive and negative ion mode allowed to distinguish between poorly resolved isomeric lipids based on their distinct fragment ions, which were used for relative quantification at a molecular lipid species level. It supports assay specificity for relative lipid quantitation via multiple quantifiably ions unlike to data-dependent acquisition methods which rely on precursor ions only. This approach was used to analyze human platelet samples. 457 lipids were annotated. Concentrations of lipids were estimated by stable isotope-labelled lipid class-specific internal standards as surrogate calibrants. Heatmaps of lipid concentrations in dependence on carbon and double bond numbers for the distinct lipid classes revealed a snapshot of the platelet lipidome in the resting state with lipid species distributions within classes supporting some functional interpretations. As expected, activation of the platelets by thrombin has led to significant alterations in the platelet lipidome as proven by univariate (volcano plot) and multivariate (PLS-DA) statistics. Several lipids were significantly up-regulated (lysophosphatidylinositols, oxylipins such as thromboxane B2 (TXB2), hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE), hydroxyoctadecadienoic acid (HODE), sphingoid-bases, (very) long chain saturated fatty acids) or down-regulated (lysophosphatidylethanolamines, polyunsaturated fatty acids, phosphatidylinositols). Several of them are well known as biomarkers of platelet activation while others may provide some further insights into pathways of platelet activation and platelet metabolism.

Sensitive determination of gentamicin in plasma using ion-exchange solid-phase extraction followed by UHPLC-MS/MS analysis

BackgroundTherapeutic drug monitoring (TDM) of gentamicin sulfate (GEN) is usually recommended, particularly in critical patients. Only a few reports had described the determination of GEN in plasma or plasma using LC-MS/MS. ObjectiveThis study aimed to develop and validate a sensitive ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) assay for the quantification of GEN in small volumes of human plasma. ResultsThe use of a very low concentration of the ion-pairing agent HFBA allowed significant retention of the very polar GEN forms in a reversed phase UHPLC column. The solid-phase extraction (SPE) procedure allowed clean extracts, with no interferences detected in blank samples, and high sensitivity. The assay was linear on the range of 0.2–40 mg L−1 of GEN complex. The combined GEN complex had inter-assay CV of 8.8–10.0%, intra-assay CV of 10.2–11.0%, and accuracy of 96.8–104.0%. The assay was applied to 17 clinical samples obtained from neonate patients. Measured concentrations were in the range of 0.15–3.57 mg L−1 for GEN C1, 0.12–3.55 mg L−1 for GEN C1a, 0.20–5.77 mg L−1 for GEN C2, and 0.47–12.88 mg L−1 for the GEN complex, all within the linear range of the assay. ConclusionA sensitive assay for the quantification of gentamicin in plasma using anion-exchange SPE and UHPLC-MS/MS was validated. The assay can be used for TDM of gentamicin, particularly in centers with access to proper instrumentation and with a low demand for gentamicin measurements, where immunoassays are not cost-effective.

Microextraction of Reseda luteola-Dyed Wool and Qualitative Analysis of Its Flavones by UHPLC-UV, NMR and MS.

Detailed knowledge on natural dyes is important for agronomy and quality control as well as the fastness, stability, and analysis of dyed textiles. Weld (Reseda luteola L.), which is a source of flavone-based yellow dye, is the focus of this study. One aim was to reduce the required amount of dyed textile to ≤50 μg for a successful chromatographic analysis. The second aim was to unambiguously confirm the identity of all weld flavones. By carrying out the extraction of 50 μg dyed wool with 25 μL of solvent and analysis by reversed-phase UHPLC at 345 nm, reproducible chromatographic fingerprints could be obtained with good signal to noise ratios. Ten baseline separated peaks with relative areas ≥1% were separated in 6 min. Through repeated polyamide column chromatography and prepHPLC, the compounds corresponding with the fingerprint peaks were purified from dried weld. Each was unequivocally identified, including the position and configuration of attached sugars, by means of 1D and 2D NMR and high-resolution MS. Apigenin-4′-O-glucoside and luteolin-4′-O-glucoside were additionally identified as two trace flavones co-eluting with other flavone glucosides, the former for the first time in weld. The microextraction might be extended to other used dye plants, thus reducing the required amount of precious historical textiles.

Determination of Total Proteinogenic Amino Acids and Taurine by Pre-column Derivatization and UHPLC: Single Laboratory Validation, First Action Official MethodSM 2019.09.

A method has been developed and validated for selective, accurate, and precise determination of total proteinogenic amino acids and taurine from infant formula and adult/pediatric nutritional formulas (powders, ready-to-feed liquids, and liquid concentrates). The method was reviewed by the AOAC INTERNATIONAL SPIFAN Expert Review Panel (ERP) during the 133rd AOAC Annual Meeting and Expo on September 7, 2019 in Denver, CO, USA, and was recommended to First Action Official MethodsSM status. The method involves protein hydrolysis to amino acids, a simple pre-column derivatization of amino acids and separation of derivatized amino acids by UHPLC. The quantification of amino acids is performed by multi-point calibration using norvaline as the internal standard. The analytical method is capable of quantitative determination for 22 proteinogenic amino acids, but cannot be used to quantitate tryptophan, which is destroyed during the acid hydrolysis step. Asparagine is determined as aspartic acid and glutamine as glutamic acid. Cystine and cysteine are converted to S-2-carboxyethylthiocysteine (CYSx) and the derivative is separated from the other amino acids. Citrulline, which is present in some matrixes, is separated from other amino acids and is not included in the method performance evaluation in the single-laboratory validation (SLV). The proposed method met all the performance requirement limits set in Standard Method Performance Requirement (SMPR®) 2014.013 for total proteinogenic amino acids and taurine. The correlation coefficient of multi-point calibration was not less than 0.999 for any amino acids at any point in the SLV study, confirming the validity of linear dynamic range (LDR) and linearity of the method. The individual amino acids in the chromatogram were identified by absolute retention time and relative retention time (RRT) with respect to the internal standard norvaline. There were no significant (S/N <10) interferences from the reagents or byproducts of derivatization and targeted matrixes. The method demonstrated high selectivity. Accuracy of the method was validated using standard reference materials (SRMs; NIST SRM 1869 and 1849a) and spike recovery studies. The amino acid results in the SRMs were within the ranges of Reference Mass Fraction Values. The accuracy of the method was corroboratively validated by spike recovery studies. The average spike recovery ranged between 93 and 107% ensuring the accuracy of the method for amino acids and compliance with AOAC SMPR 2014.013. Precision data of the method demonstrate that it meets the stakeholder requirements as per the SMPR. The mean RSDr for all the amino acids for 17 matrixes selected for the SLV were not more than 4%. The method is very sensitive and the LOQ can go down to approximately ten times lower than the SMPR requirements. The sensitivity of method is a direct reflection of its signal-to-noise ratio which ensures guaranteed method performance at the lower levels of amino acids in these matrixes. Taurine (aminoethane sulfonic acid), unlike the other amino acids, is a beta-sulfonic amino acid that is not used in protein synthesis but is found as a free amino acid in tissues. The acidic functional group (-COOH) in common amino acids is replaced with a sulfonic acid (-SO3H) group in taurine. The method offers baseline separation of citrulline which is an alpha amino acid generally present in infant formula and adult/pediatric nutritional products. The separation of citrulline eliminates the risk of interference of this compound with other amino acids. The method can also separate and quantitate hydroxyproline, an important component of collagen that is often used to quantitate collagen. The method is simple and does not include any proprietary chemicals or instruments, and it can be performed on any basic reverse phase UHPLC system with UV detection (1-6).

Development of a selective and sensitive analytical method to detect isomerized aspartic acid residues in crystallin using a combination of derivatization and liquid chromatography mass spectrometry

The isomerization of amino acids in peptides and proteins induces structural changes and aggregation. The isomerization rate of aspartic acid (Asp) is high and causes various serious diseases including Alzheimer's disease and cataract. Herein, a method for the comprehensive separation and sensitive detection of isomerized crystallin containing Asp (l-α-Asp, l-β-Asp, d-α-Asp, and d-β-Asp) was developed using chiral derivatization and reversed-phase UHPLC separation. Of three candidate derivatization reagents tested for the separation of peptides containing isomerized aspartic acid, 2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazine-2-yl) pyrrolidine-2-carboxylate (DMT-(R)-Pro-OSu) was the most suitable reagent for separating isomerized peptides and improved the sensitivity of mass spectrometry by 50-fold. This method was applied to analyze heat-denatured crystallin. Asp58 and Asp151 residues in αA-crystallin (AAC) exhibited the highest isomerization rate in heated crystallin. Furthermore, the analysis of α-crystallin extracted from bovine eye lens identified isomerized Asp residues (Asp24/35, Asp58, and Asp151 in AAC and Asp140 in αB-crystallin (ABC)). These results indicate that the newly developed method using chiral derivatization provides selective and sensitive analysis of isomerized Asp sites in α-crystallin protein. This novel method will allow for the identification and quantification of isomerized amino acids in crystallin proteins.

Separation and Quantification of Superwarfarin Rodenticide Diastereomers-Bromadiolone, Difenacoum, Flocoumafen, Brodifacoum, and Difethialone-in Human Plasma.

Superwarfarins, second-generation long-acting anticoagulant rodenticides, are 4-hydroxycoumarin analogues of warfarin that contain a large hydrophobic side chain. These compounds contain two chiral centers and are synthesized for commercial use as two pairs of diastereomer. To support studies of superwarfarin pharmacokinetics and other efforts to improve clinical care for poisoning victims, a quantitative assay was developed for the measurement of diastereomer of bromadiolone, difenacoum, flocoumafen, brodifacoum, and difethialone in human plasma. Based on ultrahigh-pressure liquid chromatography-tandem mass-spectrometry (UHPLC-MS/MS), this method was validated according to U.S. Food and Drug Administration (FDA) guidelines. Sample preparation involved simple protein precipitation followed by reversed phase UHPLC, which resolved all five pairs of cis/trans diastereomer in less than 10 min. Superwarfarins were measured using negative ion electrospray followed by selected-reaction monitoring on a triple quadrupole mass spectrometer. Calibration curves covered 3-4 orders of magnitude with linear regression coefficients of >0.999. The lower limits of quantitation were from 0.013 to 2.41 ng/mL, and intra-day and inter-day accuracy and precision coefficients of variation were <12%. A 10-min UHPLC-MS/MS assay was developed and validated for the separation and quantitative analysis of the pairs of diastereomer of five superwarfarins in human plasma. This method was used to identify and measure superwarfarins and their cis/trans diastereomers in plasma obtained from patients treated for coagulopathy following consumption of contaminated synthetic cannabinoid products.

Quantitation of a Novel Engineered Anti-infective Host Defense Peptide, ARV-1502: Pharmacokinetic Study of Different Doses in Rats and Dogs.

The designer proline-rich antimicrobial peptide (PrAMP) Chex1-Arg20 amide (ARV-1502) is active against Gram-negative and Gram-positive pathogens in different murine infection models when administered parenterally and possesses a wide therapeutic index. Here we studied the pharmacokinetics of ARV-1502 for the first time when administered intramuscularly or intravenously (IV) in Sprague Dawley rats and Beagle dogs. First, a specific and robust quantitation method relying on parallel reaction monitoring (PRM) using a high-resolution hybrid quadrupole-Orbitrap mass spectrometer coupled on-line to reversed-phase uHPLC was established and validated. The limit of detection was 2 ng/mL and the limit of quantitation was 4 ng/mL when spiked to pooled rat and dog plasma. When ARV-1502 was administered IV at doses of 75 and 250 μg/kg in dogs and rats, the plasma concentrations were 0.7 and 3.4 μg/mL 2 min post-administration, respectively. ARV-1502 plasma concentrations declined exponentially reaching levels between 2 and 4 ng/mL after 2 h. Intramuscular administration of 0.75 mg/kg in dogs and 2.5 mg/kg in rats resulted in a different pharmacokinetics profile. The plasma concentrations peaked at 15 min post-injection at 1 μg/mL (dogs) and 12 μg/mL (rats) and decreased exponentially within 3 h to 4 and 16 ng/mL, respectively. The initial plasma concentrations of ARV-1502 and the decay timing afterwards indicated that the peptide circulated in the blood stream for several hours, at some point above the minimal inhibitory concentration against multidrug-resistant Enterobacteriaceae, with blood concentrations sufficient to suppress bacterial growth and to modulate the immune system.

In-depth profiling, characterization, and comparison of the ginsenosides among three different parts (the root, stem leaf, and flower bud) of Panax quinquefolius L. by ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometry.

Despite Panax quinquefolius L. serving as a crucial source for food additives, healthcare products, and herbal medicines, unawareness of the metabolome differences among three parts (root, PQR; stem leaf, PQL; flower bud, PQF) seriously restricts its quality control. Ultra-high performance liquid chromatography/quadrupole-Orbitrap mass spectrometry (UHPLC/Q-Orbitrap-MS) was fully utilized to comprehensively identify and compare the ginsenoside compositions among PQR, PQL, and PQF. Metabolite profiling and characterization were performed by coupling reversed-phase UHPLC (a CSH C18 column) and improved untargeted data-dependent MS2 acquisition in the negative mode. A novel vehicle, "Ginsenoside Sieve," was proposed by developing fixed tolerance (± 10mDa), discrete mass defect filtering (MDF) based on the m/z features of 499 known ginsenosides, which assisted in the screening of 71 (from 3453 ions), 89 (from 6842), and 84 (from 7369) target precursor masses for PQR, PQL, and PQF, respectively. The newly established data-dependent acquisition (DDA) approach exhibited 14% improvement in characterization of targeted components (using a PQL sample), and comparable performance in identifying the unknown, compared with conventional DDA. We could characterize 347 saponins (147 from PQR, 173 from PQL, and 195 from PQF), and 157 thereof not ever-isolated from the Panax genus. These potentially new saponins have 63 unknown masses. Subsequent untargeted metabolomics analysis unveiled 20 marker saponins, among which m-Rb1, Rb1, Ro, m-Rb2, and m-Rb1 isomer are the most important diagnostic for differentiating the three parts. Conclusively, the established improved DDA represents a potent ginsenoside characterization strategy, and the results obtained in this work would benefit better quality control of P. quinquefolius. Graphical abstract.

Development and validation of a sensitive reverse-phase UHPLC fluorescence method for the quantification of citreoviridin in rice, corn and wheat

In the present study, a sensitive and new reverse-phase UHPLC fluorescence method is developed and validated for the quantification of citreoviridin (CTV) in rice, corn and wheat. The analysis was carried out isocratically on a C18 column (2.1 mm × 100 mm × 1.8 µm particles) utilizing a solvent system of acetonitrile:water:formic acid (55:45:0.1, v/v). The flow rates were set to 0.2 ml min‒1 and 0.3 ml min‒1, at 0.00–4.00 min and 4.01–10.00 min, respectively. 5 µl of sample was injected into the UHPLC column. A fluorescence detector was adjusted to 220 nm excitation and 340 nm emission wavelengths. The linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), stability and robustness variables were tested for the validation of the developed UHPLC method. A good linearity was achieved in the concentration interval of 2 ng ml‒1–200 ng ml‒1 for CTV. The intra-day and inter-day assay variability was less than 3% for the CTV. The LOD and LOQ values were 0.38 and 1.16 ng ml‒1, respectively. The developed UHPLC method was successfully applied for the determination of CTV in food.

Multiple metabolic pathways are predictive of ricin intoxication in a rat model

IntroductionExposure to ricin can be lethal and treatments that are under development have short windows of opportunity for administration after exposure. It is therefore essential to achieve early detection of ricin exposure to provide the best prognosis for exposed individuals. Ricin toxin can be detected in clinical samples via several antibody-based techniques, but the efficacy of these can be limited due to the rapid processing and cellular uptake of toxin in the body and subsequent low blood ricin concentrations. Other diagnostic tools that perform, in an orthogonal manner, are therefore desirable.ObjectivesTo determine time-dependent metabolic changes in Sprague–Dawley rats following intravenous exposure to ricin.MethodsSprague–Dawley rats were intravenously exposed to ricin and multiple blood samples were collected from each animal for up to 48 h following exposure in two independent studies. Plasma samples were analysed applying HILIC and C18 reversed phase UHPLC–MS assays followed by univariate and multivariate analysis.ResultsIn Sprague–Dawley rats we have demonstrated that metabolic changes measured in blood can distinguish between rats exposed intravenously to ricin and controls prior to the onset of behavioral signs of intoxication after 24 h. A total of 37 metabolites were significantly altered following exposure to ricin when compared to controls. The arginine/proline, bile acid and triacylglyceride metabolic pathways were highlighted as being important with two triacylglycerides at 8 h post exposure giving an AUROC score of 0.94. At 16 h and 24 h the AUROC score increased to 0.98 and 1.0 with the number of metabolites in the panel increasing to 5 and 7, respectively.ConclusionsThese data demonstrate that metabolites may be a useful tool to diagnose and detect ricin exposure, thus increasing the effectiveness of supportive therapy and future ricin-specific medical treatments.

Simultaneous separation of small interfering RNA and lipids using ion-pair reversed-phase liquid chromatography

RNA interference offers a novel approach for the development of new therapeutics for targets that are otherwise “undruggable” using traditional modalities. The safety and efficacy of siRNA-based therapy mainly rely on lipid or polymer-based nanocarriers to overcome inherent barriers to a systemic delivery of siRNA. A multicomponent lipid nanoparticle (LNP) system is a promising delivery platform, typically consisting of a cationic lipid, phospholipid, PEG-containing short-chain lipid, and cholesterol. Characterization and chemical analysis of the LNP formulation is important to assure drug product stability, a key consideration for chemistry, manufacturing and control strategy. Here we report an ion-pair reversed phase UHPLC method capable of simultaneously separating both siRNA and functional lipids in LNPs with a minimal retention gap for two classes of biologically essential yet chemically distinct molecules. Key chromatographic parameters critical to the separation are discussed, including the structure of the ion-pair agent, stationary phase chemistry, column temperature and an organic additive. The results showed that the retention time of siRNA is tunable by using various ion-pair reagents. The retention factor of the siRNA exhibited a first order relationship with the number of carbons in the alkyl chain of the ion-pair reagents. In contrast, the type of ion-pair reagent has no significant impact on the separation of phospholipids. Separations using a BEH phenyl column and dibutylammonium acetate as the ion-pair reagent showed satisfactory selectivity for a range of double-stranded siRNAs and phospholipids, key components for lipid nanoparticle formulations. Furthermore, the method was applied to the separation of an experimental LNP formulation, demonstrating good selectivity for siRNA, functional lipids and their potential degradation products.

Analysis of the retention of tetracyclines on reversed-phase columns: Chemometrics, design of experiments and quantitative structure-property relationship (QSPR) study for interpretation and optimization

In this study, design of experiments was applied for the analysis of 6 reversed phase U-HPLC columns used for the separation of four tetracyclines (TCs): tetracycline, doxycycline, chlortetracycline and oxytetracycline in different elution conditions. In a first part, a fractional factorial design (24-1) was used to study the influence of four chromatographic parameters: column temperature, pH, flow rate and composition of the mobile phase (i.e. nature of the solvent used as the organic modifier), on the quality of the separation, which was evaluated in terms of peak width and resolution between two pairs of TCs. This experimental design revealed that the nature of the solvent: acetonitrile (ACN) or methanol (MeOH), and the mobile phase flow rate were the two main factors actually having the most influence on the quality of the separation. Moreover, these two factors presented an antagonistic influence according to the response considered: peak width or peak resolution. In order to understand this behavior, a Doehlert design was performed in the second part. It consisted in modeling the evolution of responses as a function of the two main factors: nature of the composition of the mobile phase (mix of ACN and MeOH, from 100% ACN to 100% MeOH) and mobile phase flow rate (from 0.3 to 0.8 mL min−1). For all the reversed phase columns studied, an inversion of the elution order of TCs and an increase of the retention factors was observed according to the composition of the organic mixture at the end of the gradient. To understand the modification of the interactions implied in the various retention modes related to the selectivity of the organic solvents used, a quantitative structure-property relationship (QSPR) study was achieved. In this final study, the molecular descriptors of each TCs were connected to its retention factor.

Reversed phase UHPLC/ESI-MS determination of oxylipins in human plasma: a case study of female breast cancer.

The ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method was optimized and validated for the determination of oxylipins in human plasma using the targeted approach with selected reaction monitoring (SRM) in the negative-ion electrospray ionization (ESI) mode. Reversed phase UHPLC separation on an octadecylsilica column enabled the analysis of 63 oxylipins including numerous isomeric species within 12-min run time. The method was validated (calibration curve, linearity, limit of detection, limit of quantification, carry-over, precision, accuracy, recovery rate, and matrix effect) and applied to 40 human female plasma samples from breast cancer patients and age-matched healthy volunteers (control). Thirty-six oxylipins were detected in human plasma with concentrations above the limit of detection, and 21 of them were quantified with concentrations above the limit of quantitation. The concentrations determined in healthy controls are in a good agreement with previously reported data on human plasma. Quantitative data were statistically evaluated by multivariate data analysis (MDA) methods including principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). S-plot and box plots showed that 13-HODE, 9-HODE, 13-HOTrE, 9-HOTrE, and 12-HHTrE were the most upregulated oxylipin species in plasma of breast cancer patients.