LC UV Research Articles

Uncovering the lignin-degrading potential of Serratia quinivorans AORB19: insights from genomic analyses and alkaline lignin degradation

BackgroundLignin is an intricate phenolic polymer found in plant cell walls that has tremendous potential for being converted into value-added products with the possibility of significantly increasing the economics of bio-refineries. Although lignin in nature is bio-degradable, its biocatalytic conversion is challenging due to its stable complex structure and recalcitrance. In this context, an understanding of strain's genomics, enzymes, and degradation pathways can provide a solution for breaking down lignin to unlock the full potential of lignin as a dominant valuable bioresource. A gammaproteobacterial strain AORB19 has been isolated previously from decomposed wood based on its high laccase production. This work then focused on the detailed genomic and functional characterization of this strain based on whole genome sequencing, the identification of lignin degradation products, and the strain’s laccase production capabilities on various agro-industrial residues.ResultsLignin degrading bacterial strain AORB19 was identified as Serratia quinivorans based on whole genome sequencing and core genome phylogeny. The strain comprised a total of 123 annotated CAZyme genes, including ten cellulases, four hemicellulases, five predicted carbohydrate esterase genes, and eight lignin-degrading enzyme genes. Strain AORB19 was also found to possess genes associated with metabolic pathways such as the β-ketoadipate, gentisate, anthranilate, homogentisic, and phenylacetate CoA pathways. LC–UV analysis demonstrated the presence of p-hydroxybenzaldehyde and vanillin in the culture media which constitutes potent biosignatures indicating the strain’s capability to degrade lignin. Finally, the study evaluated the laccase production of Serratia AORB19 grown with various industrial raw materials, with the highest activity detected on flax seed meal (257.71 U/L), followed by pea hull (230.11 U/L), canola meal (209.56 U/L), okara (187.67 U/L), and barley malt sprouts (169.27 U/L).ConclusionsThe whole genome analysis of Serratia quinivorans AORB19, elucidated a repertoire of genes, pathways and enzymes vital for lignin degradation that widens the understanding of ligninolytic metabolism among bacterial lignin degraders. The LC-UV analysis of the lignin degradation products coupled with the ability of S. quinivorans AORB19 to produce laccase on diverse agro-industrial residues underscores its versatility and its potential to contribute to the economic viability of bio-refineries.

Neurobehavioral Effects of Fermented Rice Bran Extract in Zebrafish Larvae Model

Rice bran (RB) is a promising food ingredient that can improve biological function. In this study, we investigated the effects of RB, both unfermented (RB30) and fermented (RBF30), with five different microorganisms on the neurobehavioral activity in zebrafish larvae. Analytical methods such as LC–UV and LC–MS were performed for the analysis of RB30 and RBF30 extracts. Interestingly, niacin content, which is known to improve brain functions such as cognition and emotion, was found to be higher in RBF30 than in RB30. Furthermore, niacin content was highly increased in the RBF30-exposed fish, compared to those in the control fish. Therefore, we profiled behavioral patterns and various neurochemistry in zebrafish larvae following supplementation with RBF30 as well as performed calcium imaging on Tg (huC:GAL4-VP16); (UAS:GCaMP7a) zebrafish larvae to determine the correlation of neural activity. RBF30 revealed greater stimulation of locomotor activity without negatively affecting decision-making behavior in zebrafish larvae, as compared to RB30 or niacin. Its behavioral activation is mainly linked with the elevations of neural activity and several neurochemicals such as serotonergic and dopaminergic systems that are implicated in the control of anxiety and stress. Taken together, these results suggest that RBF30 could be a food material that improves the behavioral health by modulating neural activity and brain neurochemistry.

Polyvinyl alcohol-based hydrogel sorbent for extraction of parabens in human milk samples by in-tube SPME-LC-UV.

In this work, we developed an in-tube solid-phase microextraction (SPME) device consisting of a fused silica capillary modified with a polyvinyl alcohol (PVOH) hydrogel. Methylparaben, ethylparaben, propylparaben, and butylparaben were determined in human milk samples by using the in-tube SPME device coupled with liquid chromatography with spectrophotometric detection in the ultraviolet region (LC-UV). The inner surface of the fused silica capillary was silanized to allow covalent modification with the PVOH-hydrogel, using glutaraldehyde as cross-linking agent. The developed device was used up to 250 times with no reduction in the analytes' peak areas or carryover effect, besides having a low production cost. The human milk samples showed a significant matrix effect for parabens with higher logKo/w. Low limits of quantification (LLOQ) between 10.0 and 15.0ngmL-1 were obtained with RSD values in the range of 1.18 to 18.3%. For the intra- and inter-day assays, RSD values from 5.6 to 16.5% and accuracy from 74.5 to 128.8% were achieved. The PVOH-based hydrogel sorbent allowed the use of water as desorption solvent, eliminating the use of organic solvents, which follows the principles of green chemistry. The results showed a great application potential of the PVOH-based hydrogel sorbent for the extraction of organic compounds from high-complexity samples.

Quality Evaluation of Dietary Supplements for Weight Loss Based on Garcinia cambogia.

Food supplements of plant origin for weight control are increasingly being demanded by consumers as a way to promote good health. Among them, those based on Garcinia cambogia (GCFS) are widely commercialized considering their bioactive properties, mainly due to (-)-hydroxycitric acid ((-)-HCA). However, recently, controversy has arisen over their safety; thus, further research and continuous monitoring of their composition is required. Hence, in this work, a multi-analytical approach was followed to determine not only (-)-HCA but also other constituents of 18 GCFS, which could be used as quality markers to detect fraudulent practices in these samples. Discrepancies between the declared (-)-HCA content and that experimentally determined were detected by LC–UV in 33% of the samples. Moreover, GC–MS analyses of GCFS allowed the detection of different compounds not present in G. cambogia fruits and not declared on supplement labels, probably related to heat exposure or to the addition of excipients or other extracts. This multi-analytical methodology is shown to be advantageous to address different fraudulent practices affecting the quality of these supplements.

Nitrosamine impurities: from raw materials to final drug product.

Nitrosamine impurities: from raw materials to final drug product.

Etching of the seed cuticle by cold plasma shortens imbibitional leakage in Linum usitatissimum L.

Pre-sowing treatment of seeds with cold plasma, a reactive mixture of charged particles, is an emerging, environmentally friendly method, shown to improve water absorption, germination and seedling growth in many crop species. We studied how plasma affects different structural layers in flax seed coats, and focused on the effects on some of the earliest germination events, directly related to imbibition: solute leakage and mucilage extrusion. We characterized the ultrastructure of flax seed coats using scanning electron microscopy (SEM) and observed mucilage extrusion in situ, using environmental SEM. Time-courses of water uptake and solute leakage during germination were recorded, respectively, by weighing the seeds and semi-quantitative metabolite profiling of the exudates using GC–MS, LC–MS and LC–UV. Plasma treatment perforated the cuticle of flax seeds but did not visibly etch the underlying mucilage secretory cell (MSC) walls. Plasma treated seeds took up more water than untreated seeds during imbibition. Mucilage expansion of untreated seeds detached the distal MSC walls from the radial walls and extrusion occurred, relatively slowly, via sparse cracks in the distal cell wall layer. In plasma treated seeds, mucilage extrusion occurred fast, via rupture of distal cell walls of almost every single MSC, and the junctions between distal and radial MSC walls remained intact. Initially, imbibitional solute leakage was higher in plasma treated seeds but the leakage had quasi stopped after one hour of imbibition. In untreated seeds, the leakage continued amply more than two hours after the start of imbibition. Fragments of the lignan macromolecule, stored in parenchymatous cells under the MCS, were released more abundantly in the spermosphere of plasma treated than untreated flax seeds. It is concluded that plasma treatment etches the cuticle of flax seeds and weakens the underlying MSC cell walls, and that such treatment leads to significantly faster recovery of membranes during imbibition, reducing leakage time in germinating seeds.

Stability Indicating LC-MS Method Development and Validation for the Study of the Forced Degradation Behavior of Pimobendan Drug

Objectives: Analytical methods are necessary for the field of pharmaceuticals to discover the drug, determination of drugs and metabolites in biological matrices. The present study aimed to develop liquid chromatography-tandem mass spectrometry (LC-MS) and validation for quantification of Pimobendan in the formulation. Methods: The mobile phase composition was methanol, acetonitrile and 0.1 M phosphate buffers in the ratio of 45:15:40 (v/v) mixtures with isocratic elution. Prontosil ODS C18 column (250 × 4.6 mm, 5μ) was selected for separation with 264nm of LC UV detector wavelength. Ionization mass spectrophotometer was analyzed at nitrogen gas flow at 300 Psi and 300°C of source temperature. Mass spectrometry analysis of Pimobendan drug fragments was performed by introducing column eluting into equipment for Q1 and MS scan. Results: The drug was eluted at 3.56 min retention and the calibration curve was achieved at 0.5-250 μg/ml standard concentration. Different parameters of validation (linearity, specificity, precision, accuracy, selectivity and robustness) were performed and results confirm that all the parameters are in the acceptable limit. In the electrospray ionization spectra of Pimobendan [M+H], positive ions were observed at an m/z value of 335.42. The product-ions of 335.42 were observed at m/z value of 319.18, 287.19, 277.09, 261.08, 250.16, 223.30, 111.47 and 86.05. Conclusion: The proposed method is successfully achieved the acceptance criteria of all validation parameters and stability studies.

Valuable products from the flowers of lemon (Citrus limon (L.) Osbeck) and grapefruit (Citrus paradisi Macfad.) Italian trees

Citrus products are items of fundamental importance in the global market. The fruit of lemon (Citrus limon (L.) Osbeck) are mainly processed for the production of the juice, and of the peel essential oil, as a by-product. The same can be said for grapefruit (Citrus paradisi Macfad.), even if its production quantities are considerably lower. In the present study, the absolutes obtained from the flowers of Italian lemon and grapefruit plants were evaluated for their chemical composition and sensory properties. The GC analysis unveiled a volatile composition different from that of peel oils, being present compounds such as indole (0.04–0.22%) and methyl anthranilate (0.31–3.63%). The GC findings were correlated with sensory evaluation, carried out by a panel of trained assessors, that highlighted a pronounced floral character in grapefruit, while a predominant citrusy note was described in lemon. By means of an MDGC system, equipped with a chiral selector, a number of optical isomers were separated, allowing to define the enantiomeric ratios in these matrices for the first time. The nonvolatile fraction of the absolutes was investigated about its coumarin content by exploiting LC–UV and LC–MS methodologies. The predominant coumarins in grapefruit were epoxyaurapten (20,977.0±300.1mgL−1) and aurapten (3259.0±68.4mgL−1). A total amount of 9587.0±210.6mgL−1 was determined in lemon absolute, the highest content being that of bergamottin (2776.0±65.5mgL−1). The findings obtained in this study highlight the valuable properties of the absolutes of lemon and grapefruit flowers, as unique raw material for perfume and cosmetic industries.

Total Amino Acids by UHPLC–UV in Infant Formulas and Adult Nutritionals, First Action 2018.06

Abstract Background: An acid hydrolysis ultrahigh-performance LC–UV method was evaluated for the determination of total amino acids in infant formula and adult/pediatric nutritional formula. Objective: It was assessed for compliance against AOAC INTERNATIONAL Standard Method Performance Requirements (SMPR®) established by the Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN). Methods: A single-laboratory validation (SLV) study was conducted as a first step in the process to validate the method. In this SLV, 17 SPIFAN matrices representing a range of infant formula and adult nutritional products were evaluated for their amino acid content. Results: The analytical range was found to be within the needs for all products; some may require a dilution. Evaluation of trueness performed on Standard Reference Material 1849a (Infant/Adult Nutritional Formula) showed all compounds met the SMPR theoretical value, with exceptions for threonine and tyrosine. These may have a bias for the National Institute of Standards and Technology (NIST) data, depending on hydrolysis used in the determination of the NIST certificate of analysis. Conclusions: Based on the results of this SLV, this method met the SMPR and was approved as a First Action method by the AOAC Expert Review Panel on Nutrient Methods on August 28, 2018.

Assay of tipranavir capsules by a simple stability-indicating LC–UV method and cytotoxicity study of degraded samples

Tipranavir (TPV) is a protease inhibitor (PI) specially recommended for treatment-experienced patients who are resistant to other PI drugs. It was approved by FDA in 2005, but, until now, it has not been included in any official compendia. The objective of this study is to develop and validate a simple LC–UV method to assay TPV capsules, and to study the cytotoxicity of TPV and degraded samples over the cell viability. The optimized conditions were C8 Phenomenex® column (Luna®, 150 mm × 4.6 mm, 5 µm); mobile phase composed by methanol, acetonitrile, and acidified water pH 3.5 (40:31:29); flow rate 1.0 mL min−1 and detection at 254 nm. The cytotoxic effects of non-degraded and degraded TPV samples were evaluated in 3T3 cells by means of MTT viability assay. The method was linear in the range of 10–100 µg mL−1 (r = 0.9999) and specific, even in the presence of degradation products and impurities. The method showed suitable accuracy (mean recovery 100.02%), precision (RSD < 1.10%), and a two-level full factorial design indicated the robustness of the method. The degraded samples exhibited cytotoxicity patterns similar to TPV under controlled conditions. The method developed is appropriate for quality control of TPV capsules and stability studies.

Centrifugal partition chromatography as a fractionation tool for the analysis of lignocellulosic biomass products by liquid chromatography coupled to mass spectrometry

The conversion of lignocellulosic biomass into biofuels and bio-products leads to oxygenated matrices having a wide range of polarities and molecular weights. A complete analytical characterization of these complex mixtures is necessary to improve conversion processes. In this study, an innovative centrifugal partition chromatography (CPC) protocol was developed to fractionate aqueous biomass samples with a MTBE-water solvent system, by mixing elution, displacement and extrusion modes in the same run. This new protocol was validated on model molecules and applied to the water soluble phase of a fast pyrolysis bio-oil. It demonstrated a promising separation with a relevant selectivity on the most significant chemical families of biomass samples: carbohydrates, furans, carboxylic acids and phenols. CPC fractions of the sample were collected and analyzed comprehensively by HPLC-UV/MS (with ESI negative and positive ionization modes). This CPC x LC approach allowed more accurate attributions on the 217 peaks detected. The use of different detection modes gave a complete view of the water soluble phase of a fast pyrolysis bio-oil through 2D maps. Molecular characterization was enhanced by independent information: CPC retention time, LC retention time, UV and MS spectra. Concomitance of these different chemical information is of precious help for unambiguous identification.

Determination of lansoprazole, 5-hydroxylansoprazole, and lansoprazole sulfone in human plasma for CYP2C19 and CYP3A4 phenotyping

A simple and sensitive liquid chromatography method with absorbance or mass spectrometry detection was developed and validated for the determination of lansoprazole, lansoprazole sulfone, and 5-hydroxylansorpazole in human serum with omeprazole as an internal standard. Analytes were, after an extraction step with a mixture of isopropyl alcohol: dichloromethane (2:8, v/v), separated in a gradient elution (acetonitrile: 20 mM ammonium formate) and detected at 280 and 300 nm or by monitoring selective reactions in a triple quadrupole mass analyzer. The limits of detection were, for all analytes, below 2 ng/mL and 3 pg/mL in the LC UV and LC MS method, respectively. The combined linear dynamic range exceeded 5 orders of magnitude. The method was successfully applied in the assessment of CYP2C19 and CYP3A4 metabolic phenotypes in ST-elevated myocardial infarction patients receiving novel anticoagulant treatment.

Characterisation of folic acid by mass balance versus quantitative NMR

Two approaches were used to determine the mass fraction of a sample of folic acid, one indirect (mass balance) where the components other than folic acid were measured, and one direct, using quantitative nuclear magnetic resonance (qNMR). The procedures used for the mass balance were essentially those described in the European Pharmacopoeia monograph for folic acid. qNMR is a technique for quantifying the main component within an organic compound (Quantitative NMR (qNMR)—BIPM in https://www.bipm.org/en/bipm/chemistry/organic-analysis/qnmr/ , Santosh and Raja in Trends Anal Chem 35:5–26, 2012), whereas mass balance quantifies its minor components. The results obtained with the two approaches are independent. In this study, their joint application generated information that was used in a more than complementary fashion. The mass balance characterisation, in particular the results obtained by liquid chromatography with ultraviolet detection (LC–UV), allowed to improve the accuracy of the qNMR result by subtracting the interfering contribution of a related structure impurity. On the other hand, qNMR enabled concluding that the non-uniform response of UV detection at a single wavelength was not critically biasing the LC–UV results. In the end, the mass fractions obtained by mass balance and qNMR were not that different. It is concluded that mass balance and qNMR may be not only complementary but also synergistic. It was also observed that access to monographs of the European Pharmacopoeia, when available, gives skilled analysts an important tool for improved substance characterisation.

Exhaled breath condensate as an alternative sample for drug monitoring.

Exhaled breath condensate as an alternative sample for drug monitoring.

New LC–UV methods for pharmaceutical analysis of novel anti-diabetic combinations

New, simple, selective, and sensitive liquid chromatography–ultraviolet (LC–UV) methods have been developed and subsequently validated for simultaneous determination of linagliptin–empagliflozin combination and simultaneous determination of alogliptin benzoate–pioglitazone hydrochloride combination. Linearity was found to be acceptable over the concentration ranges of 2–50 μg mL−1, 4–100 μg mL−1, 0.5–25 μg mL−1, and 1–25 μg mL−1 for linagliptin (LNG), empagliflozin (EMG), alogliptin (ALG), and pioglitazone (PGN), respectively. All the methods were applied successfully to the analysis of the pharmaceutical dosage forms. The optimized methods were validated and proved to be robust and accurate for the quality control of the mentioned drugs in their different pharmaceutical dosage forms.

Structural characterization of dimethyldithiodenafil and dimethylthiocarbodenafil, analogs of sildenafil

Two analogs of sildenafil (compounds 1 and 2) were detected in three powder products acquired from online drug markets during an LC–UV–MS analysis of psychotropic drugs. Their structures were established by high-resolution mass spectrometry and NMR spectroscopy. Compound 1 was identified as 5-(5-(3,5-dimethylpiperazine-1-carbonothioyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidine-7(6H)-thione and named dimethyldithiodenafil. Compound 2 was identified as 5-(5-(3,5-dimethylpiperazine-1-carbonothioyl)-2-ethoxyphenyl)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one and named dimethylthiocarbodenafil. Compound 1 was subjected to the phosphodiesterase assay (IC50=0.20nM). The three powder products were found to contain 12–19mg of dimethyldithiodenafil and 1.4–3.9mg of dimethylthiocarbodenafil per tube.

Identification of acylation products in SHAPE chemistry

SHAPE chemistry (selective 2′-hydroxyl acylation analyzed by primer extension) has been developed to specifically target flexible nucleotides (often unpaired nucleotides) independently to their purine or pyrimidine nature for RNA secondary structure determination. However, to the best of our knowledge, the structure of 2′-O-acylation products has never been confirmed by NMR or X-ray data. We have realized the acylation reactions between cNMP and NMIA under SHAPE chemistry conditions and identified the acylation products using standard NMR spectroscopy and LC–MS/MS experiments. For cAMP and cGMP, the major acylation product is the 2′-O-acylated compound (>99%). A trace amount of N-acylated cAMP has also been identified by LC–UV–MS2. While for cCMP, the isolated acylation products are composed of 96% of 2′-O-acylated, 4% of N,O-diacylated, and trace amount of N-acylated compounds. In addition, the characterization of the major 2′-O-acylated compound by NMR showed slight differences in the conformation of the acylated sugar between the three cyclic nucleotides. This interesting result should be useful to explain some unexpected reactivity of the SHAPE chemistry.

Liquid chromatography-high-resolution mass spectrometry for regulated bioanalysis: bile acid and oligonucleotide quantitation as a reference.

Liquid chromatography-high-resolution mass spectrometry for regulated bioanalysis: bile acid and oligonucleotide quantitation as a reference.

Identification and characterization of naturally occurring inhibitors against human carboxylesterase 2 in White Mulberry Root-bark

White Mulberry Root-bark (WMR) is an edible Chinese herbal used for the treatment of inflammation, nephritis and asthma. This study aimed to investigate the inhibitory effects of ethanol extract from WMR against human carboxylesterase 2 (hCE2), as well as to identity and character natural hCE2 inhibitors in this herbal. Our results demonstrated that the ethanol extract of WMR displayed potent inhibitory effects against hCE2, while three major bioactive constitutes in WMR were identified on the basis of LC fingerprinting combined with activity-based screening of LC fractions. Three bioactive compounds including SD, KG and SC were efficiently identified by comparison of LC retention times, UV and MS spectral data, with the help of authentic standards. The inhibition potentials and inhibition types of these natural compounds against hCE2 were further investigated in human liver microsomes. The results demonstrated that these bioactive compounds are potent non-competitive inhibitors against hCE2, with the Ki values ranging from 0.76μM to 1.09μM. All these findings suggested that three abundant natural compounds in WMR displayed potent inhibitory effects against hCE2, which could be used as lead compounds to develop more potent hCE2 inhibitors for the alleviation of hCE2-mediated severe delayed-onset diarrhea.

Determination of Fenofibric Acid in Human Plasma by LC–MS/MS and LC–UV

A sensitive, high-throughput and economic liquid chromatographic method for determination of fenofibric acid in human plasma was developed and validated by ultraviolet detection and tandem mass spectrometry, then applied in pharmacokinetic study to investigate Lipanthyl™ 200 mg MC bioavailability under food and fasting conditions. Fenofibric acid with 2-chloro fenofibric acid-d6 (internal standard) was extracted from 100 µL of human plasma by acetonitrile in a single extraction step. 25 and 2 µL from supernatant were injected onto ACE column, 50 mm, 5 micron with 4.6 mm inner diameter for LC–UV and 2.1 mm for LC–MS/MS, and both systems were eluted isocratically by water:methanol:formic acid (35:65:0.1, v/v/v), with a constant flow rate of 1 mL min−1. The established calibration curve was linear between 0.05–20 µg mL−1, and the within- and between-day precisions were all below 13 % in both LC–MS/MS and LC–UV systems during validation, and accuracies ranged between 91 and 112 %. Twenty-eight healthy adult subjects participated in this clinical study, and the pharmacokinetic parameters including coefficient of variation were calculated and discussed. A dramatic decrease in C max and AUC0-72 (3.63- and 1.85-fold, respectively) were observed for Lipanthyl™ MC under fasting conditions with more variable inter subject measurements comparing to the fed state.