High-performance Liquid Chromatography System Research Articles

Enantiomer-Specific Stable Carbon and Nitrogen Isotopic Analyses of Underivatized Individual l- and d-Amino Acids by HPLC + HPLC Separation and Nano-EA/IRMS.

We developed a new method for stable carbon and nitrogen isotopic (δ13C and δ15N) analysis of underivatized amino acid (AA) enantiomers simultaneously, based on high-performance liquid chromatography (HPLC) separation and off-line isotopic measurement. l- and d-Enantiomers of each AA were isolated using a ReproSil Chiral-AA column, purified by wet chemical procedure, and analyzed for δ13C and δ15N values with a nanomol-scale elemental analyzer/isotope-ratio mass spectrometry (nano-EA/IRMS) system. We successfully achieved the separation of l- and d-enantiomers of 15 proteinogenous AAs, with all l-enantiomers eluting before respective d-enantiomers. The δ13C and δ15N values of AA enantiomers were consistent before and after HPLC separation, demonstrating that this analytical method conserves isotopic information. By coupling this column with a multidimensional HPLC system for isolating individual AAs, we analyzed l- and d-AAs in a natural sample, peptidoglycan isolated from Gram-positive bacterium Bacillus subtilis. Results show a surprisingly large 15N-depletion, up to 20‰, in d-glutamic acid relative to its l-counterpart. The first example, to our knowledge, of δ13C and δ15N analyses of underivatized AA enantiomers is expected to contribute to various research areas in the future.

Development and Validation of Stability Indicating RP-HPLC Method for the Estimation of Pirtobrutinib and Characterization of its Degradants by LC - MS

Background: Pirtobrutinib is a novel non-covalent BTK inhibitor used to treat adult patients with relapsed/refractory mantle cell lymphoma and B-cell leukemias. The mechanism of action involves binding and inhibition of Bruton's tyrosine kinase (BTK). Objective: The main goal of the current work was to create a selective liquid chromatographic method (RP-HPLC) that is easy to use, accurate and exact for quantifying Pirtobrutinib and its degradation products, thereby seeking insight into the drug’s degradation behaviour. Methods: Using an isocratic mode and a 50:50 mixture of acetonitrile and buffer solution (0.1% orthophosphoric acid) as the mobile phase, a High Performance Liquid Chromatographic System with a PDA detector and an X-Bridge Phenyl column (150 x 4.6mm, 3.5μm) at a flow rate of 1.0 ml/min was able to achieve good chromatographic separation. At 219 nm, the detection was carried out. A retention time of 2.271 minutes was discovered. To ascertain the drug's degrading properties and stability, forced degradation tests were carried out, leading to the development of the RPHPLC method. The chemical structures of the degradation products were clarified and their fragmentation mechanisms were suggested using LC-MS. Results: The suggested approach demonstrated a linearity within the 25-150% (2.5 to 15μg/mL) concentration range, with a correlation coefficient of 0.9999. The precision of the system (measured by % RSD=0.49) and the method (measured by % RSD=0.86) were all within the acceptable limits set by ICH guidelines, with % RSDs less than 1% and less than 2% for system precision and method precision, respectively. The LOD was 0.3μg/mL, and the LOQ was1μg/mL. Pirtobrutinib underwent rigorous tests for forced degradation under the specified conditions outlined in ICH Q1 (R2) guidelines. Pirtobrutinib was primarily broken down in acidic, alkaline, peroxide, and thermal conditions. It was found to remain stable under reduction, photolytic, and hydrolytic conditions. Through LC-MS analysis, the chemical structures of the resulting degradation by-products were identified, along with the proposed pathways of their fragmentation. Conclusion: The current study gives insight into Pirtobrutinib’s degradation behaviour. Pirtobrutinib was stable in reduction, photolytic, and hydrolytic conditions but degraded more readily in acidic, alkaline, peroxide, and thermal environments. The degradation products were characterized as 4-carbamoyl- 5-chloro-3(4-((5-fluoro-2-methoxybenzamido)methyl)phenyl)-4,5-dihydro-1H-pyrazol-5-aminium (acid impurity, DP1), N-(4-(4,5-diamino-1H-pyrazol-3-yl)benzyl)-5-fluoro-2-hydroxybenzamide (alkali impurity, DP2), 5-amino-3-(4-((s-fluro-2-methoxybenzamido)methyl))-4,5dihydro-1H-pyrazol-5-aminium (peroxide impurity, DP3) and N-(4-(4-acetyl-5-amino-4,5-dihydro-1H-pyrazol-3-yl)benzyl)-5-fluoro-2- methoxybenzamide compound with λ1-oxidane (1:1) (thermal impurity, DP4) and their fragmentation pathways were proposed. This study presents the first ever reported method for the quantification of Pirtobrutinib. It ensures precise quantitation of Pirtobrutinib, a new Bruton's Tyrosine Kinase inhibitor, and its degradation products. The method's enhanced sensitivity and regulatory compliance ensure its consistent use in the quantification of Pirtobrutinib.

Metabolism of Furanocoumarins by Three Recombinant CYP9A Proteins From the Polyphagous Cotton Bollworm Helicoverpa armigera.

Furanocoumarins are a class of chemical compounds with phototoxic properties. For herbivores, efficient detoxification of such defense compounds is the prerequisite to feed successfully on furanocoumarin-containing plants. The cotton bollworm Helicoverpa armigera is a very important polyphagous pest in agriculture, but how it copes with toxic furanocoumarins in some of its host plants is not well understood. Given that cytochrome P450s are well known for their capacity in xenobiotic metabolism, this study attempted to explore the potential roles of cytochrome P450s in furanocoumarin transformation in this pest. Our data showed that two linear structures (psoralen and xanthotoxin) could be metabolized by three recombinant CYP9A enzymes, but no detectable depletion was observed for the linear one with the 8-dimethylallyloxy substituent on the coumarin moiety (imperatorin) and the angular furanocoumarin (angelicin). Initial epoxidation of the double bond connecting C2' and C3' of the furano ring following by cleavage of the epoxidated furan ring, leading to the formation of more soluble, less reactive and nonphotosensitizing metabolites, was identified as a common mechanism of linear furanocoumarin metabolism using a quadrupole/time-of-flight (Q-TOF) mass spectrometry interfaced with a high performance liquid chromatography (HPLC) system. Our data demonstrated that multiple P450s were involved in the detoxification of linear furanocoumarins in the cotton bollworm. These findings contribute to a better understanding of the biochemical basis of adaptation to plant defense chemicals in this economically important pest.

Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Dalbavancin in Plasma of Pediatric and Young Adult Patients.

Dalbavancin, an antimicrobial lipoglycopeptide, is authorized in Europe for treating acute bacterial infections of the skin and skin structures in adults and pediatric patients aged 3 months and older. However, off-label dosing regimens have been proposed for various indications beyond acute bacterial infections of the skin and skin structures. This study presents a novel bioanalytical method using liquid chromatography-tandem mass spectrometry to quantify dalbavancin in low-volume plasma samples (50 μL). The method underwent validation in accordance with international guidelines for bioanalytical method validation and was applied to 9 clinical samples obtained from pediatric and young adult patients undergoing dalbavancin therapy. Liquid chromatography-tandem mass spectrometry analyses were conducted at the G. Gaslini Institute in Genoa, Italy, utilizing an Ultimate 3000 ultra high performance liquid chromatography system coupled to a TSQ Quantiva Triple Quadrupole system (Thermo Fisher Scientific, Milan, Italy). The analytical procedure involved the addition of deuterated dalbavancin as internal standard and a rapid extraction from 50 µL of human plasma, followed by chromatographic separation on a Thermo Scientific Accucore Polar Premium column. Accurate quantification of the analyte was achieved through multiple reaction monitoring detection. The assay exhibited linearity within the concentration range of 0.66-400 mcg/mL in plasma, demonstrating accuracy and reproducibility in the absence of matrix effects. Stability testing was conducted on both quality controls and real samples to establish a robust protocol under real-life conditions. This fast and reliable dalbavancin quantitation method could improve current pediatric clinical practice by enabling data collection for future dose recommendations in special patient populations.

Evaluation of azo dyes degradation potential of Staphylococcus strains: A strategy for dye-waste management

The current investigation aimed to identify the bacterial isolates that could prove helpful in the degeneration of harmful azo dyes from wastewater. The bacterial strains 1b, 1 m, and 4v could decolorize azo dyes up to 81 %, 79 %, and 87 % within 5 days. The degraded products by thin layer chromatography (TLC) showed Rf values of 0.89, 0.95, 0.90, 0.92, and 0.98 while the control showed an Rf value of 0.94. The comparison of the retention time of control and treated samples by the high-performance liquid chromatography (HPLC) system convinced that remarkable decolorization had occurred by the bacterial strains. The Fourier transform infrared spectroscopy (FTIR) analysis of the control and degraded samples was proof that the bond stretching occurred in the treated samples due to the action of bacterial strains. The release of compounds by bacteria i.e., 3-Aminobutanoic acid, pyrrolo pyrazine-1, 4-dione, and palmitic acid was inspected by Gas Chromatography-Mass Spectroscopy (GC-MS) analysis. No clear zones showed that the bacterial dye-degraded wastewater had no harm to the normal flora. At last, phytotoxicity was studied on Vigna radiata which had negative results. Given their pollutant degrading capabilities, these bacterial isolates are a good bioresource for green chemistry to exterminate azo dyes from the environment.

Study on determination of cynarin in dietary supplement containing artichoke by high performance liquid chromatography

There has been no announcement on the standardization and testing of cynarin in dietary supplements containing artichokes in Vietnam. The high-performance liquid chromatography (HPLC) method was developed to determine cynarin in dietary supplements containing artichokes. The sample was extracted twice with 80% methanol solvent and ultrasonicated at 50°C for 20 minutes. Centrifuge at 6000 rpm for 5 minutes, filter, and inject into HPLC system with XBridge C18 column (250 mm x 4,6 mm, 5 µm) and corresponding pre-column, mobile phase consisting of two solvents phosphoric acid 0,1% and acetonitrile eluted according to gradient program, flow rate 1mL/min, detection wavelength 322nm. The calibration curves for cynarin had R2 = 1, and the validation parameters met AOAC standards. The method detection limits and the limit of quantification were 3.00 mg/kg and 10.0 mg/kg, respectively. The validated method was applied to determine cynarin in 15 dietary supplement samples. The content of cynarin varied depending on the manufacturer

Microdialysis-integrated HPLC system with dual-electrode detection using track-etched membrane electrodes for in vivo monitoring of dopamine dynamics

A capillary high-performance liquid chromatography (HPLC) system equipped with a dual-electrode detector utilizing track-etched membrane electrodes (TEMEs) was combined with a microdialysis sampling setup. The electrochemical detector benefits from the high electrolysis efficiency of TEMEs, allowing for calibration-free coulometric detection and simplifying data analysis to determine the dopamine recovery through a dialysis probe. Additionally, this system was used for in vivo monitoring of dopamine in the right striatum of a mouse brain. Temporal changes in dopamine levels, including an exponential decay immediately after the dialysis probe insertion and an excess release of dopamine induced by a high concentration of potassium ions, confirmed the system’s proper operation. Furthermore, subsequent measurements following the intraperitoneal injection of mirtazapine showed no increase in dopamine levels in the right dorsal striatum. The dual-electrode system displayed characteristic dopamine detection behavior, with anodic and cathodic peak pairs indicative of reversible electrochemical reactions. This capability facilitated the identification of the dopamine peak within the complex chromatogram of the mouse brain dialysate. The consistency between dopamine collection efficiency from standard solutions and dialysate indicated the absence of interfering electroactive substances overlapping with the dopamine peak in the chromatogram. This integrated analysis system successfully tracked temporal fluctuations in dopamine concentration within the mouse brain.

Analytical quality by design-based stability-indicating high performance liquid chromatography method for the estimation of evogliptin tartrate in bulk and tablet dosage form.

The present study utilized Analytical Quality by Design (AQbD) approach to develop a stability-indicating high-performance liquid chromatography (HPLC) method for estimating evogliptin tartrate using design expert software. The key parameters were methodically optimized, contours were plotted, and stability was evaluated using various forced degradation conditions. Using an Agilent HPLC system with a photo diode array (PDA) detector along with Fortis C18 column (250 × 4.6mm, 5μm) effectively separated the drug from its degradants. The mobile phase used was methanol: water (pH adjusted to 3.0, 76:24; v/v) at 0.8mL/min flow rate. Evogliptin was eluted at 2.98 min, at a detection wavelength of 267 nm. The proposed method was found to be specific, precise, linear and robust. The drug was sensitive to acidic, basic, oxidative, thermal, and photodegradation resolving six degradation products. Thus, the developed AQbD-based stability-indicating HPLC method is applicable in analyzing evogliptin in bulk, tablet dosage form and stability samples.

Study of phenolic compounds of umbellate wintergreen herb and their influence on biochemical indicators of blood and urine in the rat model of chronic kidney disease

Diseases of the kidneys and urinary tract are a common problem in people of all ages. Kidneys filter blood, removing water-soluble waste from the body, maintain water-salt balance, stabilize blood pressure and PH level. Intoxication, hypothermia, injuries and other causes lead to problems with the kidneys - inflammatory disease, urolithiasis, etc. Therefore, the development of effective herbal remedies that affect the etiopathogenic factors of diseases is urgent. The aim. Study of the profile of phenolic compounds of umbellate wintergreen, study of antioxidant and nephroprotective properties of umbellate wintergreen extract on the model of chromate-induced renal failure (chronic kidney disease) in rats. Materials and methods. Phenolic compounds were analyzed by high-performance liquid chromatography (HPLC) using a Waters e2695 Alliance HPLC system in combination with a 2998 PDA detector (Waters, Milford, MA, USA). Study of in vitro antioxidant activity by HPLC method for 50 % extract of umbellate wintergreen herb. A Waters 2695 chromatograph (Waters, USA) equipped with a Waters 996 diode-matrix detector was used. To determine the nephroprotective effect, 60 outbred sexually mature rats (males), divided into six groups, were studied. Based on the results of biochemical studies, creatinine clearance and urea clearance were calculated in experimental animals. Results. 8 phenolic compounds - apigenin, hyperoside, quercitrin, rutin, quercetin, gallic acid, guaiaverine and isoquercetin - were identified and quantified in umbellate wintergreen by HPLC. Based on the results of a preliminary assessment of the antioxidant contribution of individual phenolic compounds to the overall effectiveness of the umbellate wintergreen grass extract, a significant effect of caffeic acid derivatives and quercetin was determined. The use of Chimaphila umbellata extract in experimental animals was marked by the normalization of diuresis, a decrease in the total protein content of urine by 1.8 times. The effect of the use of umbellate wintergreen extract was expected to be dose dependent. Conclusions. The profile of umbellate wintergreen phenolic compounds was investigated by HPLC, and their antioxidant effect was determined. In the conditions of the development of renal failure in rats, the studied extract of Chimaphila umbellata improved the physical condition of the animals, reduced their mortality, improved the excretory function of the kidneys, normalized nitrogen and protein metabolism, contributed to the protection of the structure of the kidney tissue, and therefore had a positive effect on the course of nephropathy

Efficacy of the peak area method of high-performance liquid chromatography (HPLC) analysis: The separation, identification, and quantification of phytochemical compounds of Moringa oleifera

The quantification and identification of phytochemical compounds in Moringa oleifera have received significant attention due to their potential health benefits. High-Performance Liquid Chromatography (HPLC) has become a widely used method for analyzing these phytochemicals due to its speed, adaptability, and effectiveness. However, there is a need to improve these methods to achieve more accurate quantification and understanding of these compounds. This study aims to assess the effectiveness of the peak area method in HPLC for the separation, identification, and quantification of phytochemical compounds in Moringa oleifera leaves. The main goal of the study is to use the peak area method within HPLC to measure the concentration of various phytochemical compounds in Moringa oleifera leaves. This method offers a more reliable measurement than peak height, allowing for comparison of different components' concentrations within or between samples, which is important for trace analysis in various applications. The methodology involved collecting and preparing Moringa oleifera leaves from five regions: Nigeria, Ghana, Haiti, India, and Texas, USA. The leaves were dried, ground, and extracted using methanol and ethanol. The extracts were then analyzed using HPLC, with the peak area method utilized to quantify the phytochemical compounds. The HPLC system utilized a Nucleosyl C18 column with a UV-vis diode-array detector, and the separation process was monitored at 332 nm. Key findings of the study indicated that chlorogenic acid, rutin, ferulic acid, luteolin, apigenin, and kaempferol were successfully separated, identified, and quantified from Moringa oleifera leaves using the peak area method in HPLC. Chlorogenic acid had a retention time of 15.759 minutes and a peak area of 170,844, indicating it is more polar and present in significant quantities. Rutin followed with a retention time of 16.919 minutes and a peak area of 115,599. Ferulic acid was the most abundant compound, with a peak area of 422,129 and a retention time of 17.965 minutes. Luteolin, with a peak area of 235,655 and a retention time of 19.777 minutes, was also present in considerable amounts. Apigenin and kaempferol showed retention times of 20.840 minutes and 21.039 minutes with peak areas of 343,378 and 197,585, respectively. The study also compared the extraction efficiencies of 70% ethanol and 80% methanol, finding that methanol was significantly more effective, especially for highly polar compounds like chlorogenic acid, luteolin, and rutin, with peak areas being 74% to over 1100% higher than those observed with ethanol. The study concluded that the peak area method in HPLC is a highly effective tool for the separation, identification, and quantification of phytochemical compounds in Moringa oleifera leaves. It recommended further optimization of the HPLC method and exploration of pharmacological properties of these phytochemical compounds for nutraceuticals and pharmaceuticals.

Sorption experiments as efficient as possible: Mini-column experiments (MCE) using HPLC-ICP-MS coupling with a new data analysis approach to determine sorption parameters

To quantify retention and sorption parameters, diffusion-, batch- or column experiments are common practice. However, these are either not close to nature, extremely time-consuming or material intensive. A promising approach to eliminate these problems is the performance of mini-column experiments (MCE). Due to dynamic retention and realistic solid–liquid ratios, these are close to nature and resource-efficient. The aim of this work is to perform highly controllable MCE using a HPLC (high-performance liquid chromatography) system and HPLC-ICP-MS (inductively coupled plasma mass spectrometry) coupling. With this approach, ultra-fast sorption experiments are possible only using 100–150 mg adsorbent and 160 mL eluent. That means, sorption experiments are even more time- and cost-efficient and also more controllable than with conventional methods. The presented method is based on a newly developed MCE-HPLC-ICP-MS coupling, which was applied to investigate the sorption of Eu(III) on kaolinite and sand in 10 mM NaCl. With that it was possible to carry out dynamic sorption experiments in just 5–15 h and thus determine e.g. maximum loading capacity (qmax) in a very short amount of time. Furthermore, a quantification method for eluates of high ionic strength is presented. Here, the sorption of Cs(I) on calcium silicate hydrate (C-S-H) phases in 10 mM and 100 mM NaCl was investigated. For validation, the sorption distribution ratios (Rd) obtained were compared with those from batch experiments. These agreed very well with each other and with those from the literature. Overall, with MCE more sorption parameters can be determined much faster, more accurately and more efficient than with the conventional methods.

Greener method proposal for the determination of phenolic compounds and coumarins in aged sugarcane spirits using green bio-based solvents in HPLC-DAD-FLD system

High Performance Liquid Chromatography (HPLC) has been the most used analytical technique for analyzing phenolic compounds and coumarins in aged sugarcane spirit samples. It requires a significant amount of organic solvents, often toxic, as the mobile phase. The use of green bio-based solvents is a good alternative in the implementing more eco-friendly methods. Therefore, this work aims to propose a greener chromatographic method for the determination of phenolic compounds and coumarins in sugarcane spirit samples aged in different woods, using bio-based organic solvents (ethanol and acetic acid) in HPLC system with diode array and fluorescence detectors. Different metric tools (AGREE, ComplexMoGAPI, BAGI and Analytical Eco-scale) were used to evaluate the greenness of the analytical method. The proposed method was rigorously validated: linearity (R2 ≥ 0.9901); LOD (0.05 to 0.55 mg l-1); LOQ (0.25 to 5.00 mg l-1); precision (0.13 % ≤ RSD ≤ 18.6 %). The concentrations of total phenolic compounds and coumarins were up to 7.22 mg l-1. The sugarcane spirit aged in umburana presented the highest concentration of total phenolic compounds and coumarins with emphasis on trans-cinnamic acid (4.74 ± 0.01 mg l-1) and catechin (1.22 ± 0.02 mg l-1). All samples presented coumarin concentrations (<5.00 mg l-1) within the limit acceptable by legislation (10 mg l-1). The AGREE score (0.72) showed that the proposed method was greener and sustainable compared to other methods. The application of new mobile phase using green bio-based solvents proved to be advantageous from a Green Analytical Chemistry point of view.

Novel Insights into Hb Shaare Zedek Associated with β0-Thalassemia: Molecular Characteristics, Genetic Origin and Diagnostic Approaches.

Hemoglobin Shaare Zedek (Hb SZ) is a rare structural α-Hb variant. Characterizing its genotype-phenotype relationship and genetic origin enhances diagnostic and clinical management insights. We studied a proband and six family members using high-performance liquid chromatography (HPLC), capillary electrophoresis (CE), PCR, and sequencing to analyze α- and β-globin genes and α-globin haplotypes. Pathogenicity predictions and a rapid diagnostic method were developed. The proband, his father, grandfather, and aunt had Hb migrating to the HbH-zone on CE and elevated fetal hemoglobin (HbF) on HPLC. Direct sequencing identified an A to G mutation at codon 56 of the α2-globin gene, characteristic of Hb SZ. Additionally, the proband carried a β-globin gene mutation [HBB.52A>T]. Mild thalassemia-like changes were observed in the proband, whereas individuals with only the Hb SZ variant did not exhibit these changes. Pathogenicity predictions indicated that Hb SZ is benign. The variant can be identified using restriction fragment length polymorphism (RFLP) and allele-specific PCR. The Thai variant of Hb SZ is associated with the haplotype [- - M - - - -]. Hb SZ is a non-pathological variant that minimally affects red blood cell parameters, even when it coexists with β0-thalassemia. HPLC and CE systems cannot distinguish it from other Hbs, necessitating DNA analysis for accurate diagnosis.

Toward a new classification Method: Fractal dimension applied to chromatographic wine data

A study of Argentine wines was carried out by direct injection of the samples without previous treatment in a high-performance liquid chromatography system coupled to a diode array detector (HPLC-DAD). The collected data, corresponding to six different varietals (Bonarda, Cabernet Sauvignon, Malbec, Merlot, Syrah, and Tempranillo), were processed with a new chemometric approach: determining the fractal dimension of principal components or fractal dimension analysis (FDA). A comparative study of the efficiency of the new proposed model to discriminate wine samples according to botanical origin was made with methods based on a linear multivariate resolution curve-alternating least squares model (MCR-ALS) previously reported. The results show that the fractal dimension analysis of the data improves the discrimination between all the varietals studied, allowing better discrimination in relation to other reported methodologies. Finally, a comparative analysis of the method’s sustainability was carried out using the Analytical Eco-Scale indicator. This analysis demonstrated that the method based on fractal analysis is a simple procedure that meets most of the Green Analytical Chemistry requirements.

Quantification of Glycyrrhizic Acid in FLEGMEN-SIP Capsules Using High-Performance Liquid Chromatography for CNS Disorders

Background: Neurological diseases affect over one billion people globally, presenting a significant health burden, especially for vulnerable populations. The FLEGMEN-SIP capsule was developed as a treatment option for central nervous system disorders, containing glycyrrhizic acid as its primary active ingredient. This study aims to quantify the glycyrrhizic acid content in FLEGMEN-SIP capsules using high-performance liquid chromatography (HPLC) to ensure compliance with therapeutic standards. Methods: The study utilized HPLC to quantify glycyrrhizic acid in FLEGMEN-SIP capsules. The capsules contain a dry extract derived from Phlomis regelii, Leonurus turkestanicus, Mentha piperita, and Glycyrrhiza glabra, with glycyrrhizic acid comprising 1.39% of the dry extract. Samples were prepared by dissolving 0.050 g of the drug in the mobile phase and filtered using a 0.45 µm membrane filter. The HPLC system included a Zorbax column and UV detection at 254 nm. The analysis was repeated five times for accuracy. Results: The glycyrrhizic acid content in FLEGMEN-SIP capsules was determined to be 0.55%, surpassing the minimum regulatory requirement of 0.4%. The chromatographic method demonstrated high reproducibility and reliability, confirming the quality and therapeutic efficacy of the capsules. Conclusion: The study validated the use of HPLC as an effective method for quantifying glycyrrhizic acid in FLEGMEN-SIP capsules. The results confirm the capsules meet regulatory standards, ensuring their suitability for treating central nervous system disorders.

Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method

Psilocybin is a psychedelic compound found in some hallucinogenic “magic mushrooms”. Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

Comparison of Maceration and Ultrasound-Assisted Extraction Method in Determining Quercetin Content of Clitoria ternatea L. Flowers

Butterfly pea flowers (Clitoria ternatea L.) contain the compound quercetin which has been studied extensively. Butterfly pea flowers have shown various pharmacological activities. The extraction method has an impact on the amount of active compound extracted. The aim of this research was to determine the effect of different extraction methods on quercetin content of C. ternatea L flowers using the High-Performance Liquid Chromatography (HPLC). The HPLC system uses an ODS-3 column stationary phase; a mobile phase mixture of 0.1 % orthophosphoric acid and methanol (36:64); a flow rate of 1.2 mL/min; and a PDA-UV detector. The extraction methods used were maceration, and ultrasound-assisted extraction (UAE) using methanol as a solvent. The results showed that the quercetin content of C. ternatea L flower extract with maceration method results was 1.0669 ± 0.0283 mg/g and with UAE method was 1.3915 ± 0.1789 mg/g. Statistical test results showed that differences in both extraction method did not have a significant effect on quercetin content. However, the UAE method is considered more efficient in terms of extraction time, so the UAE method can be used as an alternative for extracting quercetin from C. ternatea L. flowers.

Microbial Protein Production Using Lignocellulosic Biomass (Switchgrass) and Klebsiella oxytoca M5A1—A Nitrogen Fixer

The expanding global population has increased the demand for sustainable protein sources, and microbial protein (MP) has emerged as a promising alternative. However, conventional carbon (glucose) and nitrogen (ammonia, urea) sources needed for MP production pose environmental and economic issues. This study aims to produce protein using lignocellulosic biomass (LCB) as a carbon source and the nitrogen fixation ability of Klebsiella oxytoca M5A1 as a nitrogen source. The study investigates the pretreatment of LCB (switchgrass), enzymatic hydrolysis, protein quantification, nitrogen fixation, glucose utilization and organic acids production. K. oxytoca M5A1 harnessed free nitrogen from the atmosphere and used abundant, cheap glucose from LCB to produce MP and organic acids as by-products. Protein production occurred in two phases: first within the initial 8 h and secondly, within the last 16 h. The highest protein concentration was at 40 h, with approximately 683.46 µg/mL protein content. High-performance liquid chromatography system (HPLC) analysis revealed a dynamic profile of glucose utilization and organic acids (Lactic acid, Propionic acid, Acetic acid, and Succinic acid) production. K. oxytoca M5A1 exhibited an early high rate of glucose consumption, and conversion to organic acids, that were later used for second-phase protein production. The acids profile revealed intra-conversion from one acid to another via metabolic pathways (glycolysis and tricarboxylic acid cycle). Overall, leveraging LCB and the nitrogen-fixing ability of K. oxytoca M5A1 for MP production offers an eco-friendly and cost-effective alternative to traditional protein sources, contributing to a sustainable circular economy.

A microchip based Z-cell absorbance detector integrating micro-lenses and slits for portable liquid chromatography

A miniaturized microchip-based absorbance detector was developed for portable high-performance liquid chromatography (HPLC) to test glycated hemoglobin (HbA1c). The microchip integrating a Z-shaped cell, two collimating micro-lenses and two ink-filled optical slits is small in size (30 mm × 15 mm × 7 mm). The Z-shaped cell has a cross-sectional size of 500 μm × 500 μm and a physical optical path length of 2 mm. Two collimating micro-lenses were inserted in empty grooves on both sides of the cell, one micro-lens for collimating the initial light and the other for focusing the transmitted light. Optical slits on each end of the cell were used to block the stray light. Therefore, this detector indicated a low stray light level (0.011 %) and noise level (2.5 × 10−4 AU). This detector was applied for the commercial HPLC system to detect HbA1c level, and showed a low limit of detection (0.5 μg/mL) and excellent repeatability (≤ 2.03 %). The sensitivity was enhanced by 3.4 times when the optical path length was increased from 0.5 mm to 2 mm and the stray light was blocked by optical slits. The miniaturized microchip-based absorbance detector developed shows a great potential for application in portable and compact HPLC.

Validation of an Isocratic HPLC Method for Simultaneous Estimation of Major Phytosterols in Prunus spinosa L. extracts.

This study aimed to develop a rapid method for separation of stigmasterol, campesterol and β-sitosterol in Prunus spinosa L. (sloe) fruit extracts by High Performance Liquid Chromatography system. Samples were prepared by Soxhlet extraction method and separated on a high strength silica C18 column using acetonitrile-methanol mobile phase and Photodiode Array Detector. The optimized method resulted in a linear calibration curve ranging from 1.7 ng mL-1 to 130 ng mL-1 for all three phytosterols. Analyses of internal and external phytosterol standards showed good linearity (R2 of 0.998 to 0.999); LOD and LOQ were determined to be 2.33×10-7-2.18×10-4 and 7.07×10-7-6.60×10-4 mg mL-1, respectively. Repeatability and reproducibility precision analyses showed acceptable values of RSD %. β-sitosterol was the predominant phytosterol (51.53-81.03 % of total) among all samples. Method validation parameters indicated that this analytical method can be applied for accurate and precise determination of campesterol, stigmasterol and β-sitosterol, in selected extracts.