Chromatographic System Research Articles

Different Correlation Patterns Between Circulating Amino Acids and Body Temperature in Fibromyalgia Syndrome: A Cross-Sectional Study

The aim of this study was to analyze the association between circulating amino acids and central and peripheral body temperature in subjects with and without fibromyalgia syndrome (FMS). A total of 47 patients with FMS and 59 healthy subjects were included in the study. The concentration of amino acids was determined in serum samples using a fluorimeter coupled with a high-performance liquid chromatography system. An infrared thermography camera was used to estimate peripheral hand temperatures. The core temperature of the body was estimated using an infrared thermometer, which was applied to the axillary and tympanic areas. Correlations between several thermographic variables of the hands and tryptophan, methionine, 3-methylhistidine, histidine, glutamic acid, and tyrosine were identified exclusively within the FMS group. In contrast, correlations between aminoadipic acid and serine and thermographic variables were observed only in the healthy control group. The concentrations of asparagine and lysine correlated with thermographic variables in both groups. The essential amino acid leucine was found to correlate with axillary temperature in FMS patients. However, it should be noted that the observed associations between aminoadipic acid and tryptophan blood concentrations and axillary temperature were limited to the control group. Several correlations were identified between circulating amino acids and different body temperatures in both healthy controls and patients with FMS. However, the correlation pattern differs significantly between FMS patients and healthy controls. These findings suggest the possibility of a change in the function of several amino acids in the thermoregulatory process in patients with FMS.

Membrane chromatographic test system for the determination of bisphenol A in drinking water based on the use of an aptamer

A membrane test system has been developed for the rapid determination of bisphenol A in drinking water, utilizing a conjugate of gold nanoparticles with an aptamer that specifically binds the target analyte, and a conjugate of mercaptosuccinic acid with a carrier protein impregnated in the test zone of the strip. The working principle of the test system is based on the binding of free gold nanoparticles in the test zone, which are formed as a result of the competitive interaction of the aptamer with bisphenol A and its release from the surface of the gold nanoparticles. Conjugates of gold nanoparticles with aptamers of different compositions were obtained and tested. Optimal conditions were selected to achieve a low detection limit for bisphenol A. The developed test system allows for the detection of bisphenol A within 15 minutes with a detection limit of 13.5 ng/mL. The suitability of the test system was confirmed by testing drinking water; the detection rate of bisphenol A ranged from 88.2 to 101.3%.

A Review of Liquid Chromatography-Mass Spectrometry and its Applications in Chemical Analysis

Liquid Chromatography-Mass Spectrometry (LC-MS) has emerged as a powerful analytical technique combining the separation capabilities of liquid chromatography with the high sensitivity and selectivity of mass spectrometry. This sophisticated instrumentation enables precise identification and quantification of complex chemical mixtures across diverse fields including pharmaceuticals, environmental monitoring, food safety, and biological research. Modern LC-MS systems offer enhanced resolution, improved ionization methods, and sophisticated mass analyzers that facilitate accurate molecular weight determination and structural elucidation of compounds. Recent technological advancements have led to the development of ultra-high-performance liquid chromatography (UHPLC) systems coupled with high-resolution mass spectrometers, enabling faster analysis times and improved detection limits. The versatility of LC-MS is demonstrated through its applications in proteomics, metabolomics, drug development, and quality control processes. Integration of artificial intelligence and machine learning algorithms has further enhanced data processing capabilities, allowing for automated peak identification and complex mixture analysis. The continuous evolution of LC-MS technology has addressed previous limitations in ionization efficiency, matrix effects, and quantification accuracy. This analytical technique has revolutionized chemical analysis by providing comprehensive molecular information, making it an indispensable tool in modern analytical laboratories.

Chitosan extracted from the feather of Dosidicus gigas crosslinked with glutaraldehyde for use as a 99Mo adsorbent

The production of Technetium-99 m (99mTc) is important for cancer diagnosis. The solvent extraction is the most widely used method to obtain 99mTc; however, due to the evaporation of the polluting solvent into the environment, a radiochemical separation using a chromatographic column system based on adsorbent materials would be cleaner and more efficient.In this study, squid feather (Dosidicus gigas) chitosan (SFC) polymers have been synthesized and crosslinked with glutaraldehyde solutions at concentrations of 50, 40 and 25 % to study the adsorption of 99Mo with the aim of test a promising material to produce 99mTc in the chromatographic column of the 99Mo/99mTc generator. Likewise, the same methodology of synthesis was performed using a commercial chitosan (CC) as a control. Deacetylation degree was measured obtaining 74.15 % and 77.65 % for the SFC and CC polymers, respectively. The molecular weight obtained was 990.72 kDa for SFC and 722.72 kDa for CC and the characterization of the biopolymers was performed with FTIR, SEM, XRD, TGA y FT Raman techniques. Equilibrium time, adsorbent mass and pH effect were evaluated as factors that influence the adsorption process of 99MoO4-2. The zero load point (pHPZC) analysis confirmed the positive charge of the crosslinked polymers, improving the adsorption capacity of 99MoO4-2 at low pH values between 2 and 4, following a pseudo-first order model. With respect to the adsorption isotherms, they exhibit maximum values of 99MoO42- of 482 mg g−1 SFCG40 and 502 mg g−1 CCG25 with a better fit to the Langmuir theoretical model.

Continuous chromatography system with 6-zone and 18-column dynamic tandem connection technique for the enrichment of total flavonoids from Epimedium koreanum Nakai.

Natural flavonoids have been shown to have many pharmacological activities. Efficient and continuous enrichment of total flavonoids with high content and low mobile phase usage from complex natural products is greatly needed at the moment. In this study, a new continuous chromatography system (CCS) with 6 zones and the18-column dynamic tandem connection technique was developed and used to enrich total flavonoids from Epimedium koreanum Nakai (EKN). The 18 columns were divided into 6 zones, and the principle of adynamic series of three columns was adopted for each zone to achieve continuous automatic separation and enrichment of total flavonoids under the control of alogic control valve. The CCS separation conditions were established based on single-column chromatography anda theoretical calculation model of the CCS. By means of the self-designed device and method, 485.11±3.16 g of total flavonoids were isolated from 16.2 kg of EKN. It is worth noting that the total content of 18 types of flavonoids in the samples enriched by the CCS was increased from 2.84±0.07% to 88.29±0.22%, the total recovery rate was 92.20±0.38%, and theRSD of each flavonoid was less than 5.0%. Furthermore, compared with single-column chromatography filled with the same volume of chromatography filler, the entire process saved about 2/3 of the mobile phase usage. In summary, the developed device and method could efficiently and continuously enrich total flavonoids from EKN with high-content and low mobile phase usage and would have a wide application prospect in the separation and enrichment of natural products.

Four-Dimensional Lipidomic Analysis Using Comprehensive Online UHPLC × UHPSFC/Tandem Mass Spectrometry.

Multidimensional chromatography offers enhanced chromatographic resolution and peak capacity, which are crucial for analyzing complex samples. This study presents a novel comprehensive online multidimensional chromatography method for the lipidomic analysis of biological samples, combining lipid class and lipid species separation approaches. The method combines optimized reversed-phase ultrahigh-performance liquid chromatography (RP-UHPLC) in the first dimension, utilizing a 150 mm long C18 column, with ultrahigh-performance supercritical fluid chromatography (UHPSFC) in the second dimension, using a 10 mm long silica column, both with sub-2 μm particles. A key advantage of employing UHPSFC in the second dimension is its ability to perform ultrafast analysis using gradient elution with a sampling time of 0.55 min. This approach offers a significant increase in the peak capacity. Compared to our routinely used 1D methods, the peak capacity of the 4D system is 10 times higher than RP-UHPLC and 18 times higher than UHPSFC. The entire chromatographic system is coupled with a high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) in both full-scan and tandem mass spectrometry (MS/MS) and with positive- and negative-ion polarities, enabling the detailed characterization of the lipidome. The confident identification of lipid species is achieved through characteristic ions in both polarity modes, information from MS elevated energy (MSE) and fast data-dependent analysis scans, and mass accuracy below 5 ppm. This analytical method has been used to characterize the lipidomic profile of the total lipid extract from human plasma, which has led to the identification of 298 lipid species from 16 lipid subclasses.

Modeling the extra column volume of a micro simulated moving bed chromatography system: Introducing the equivalent radial flow rate distribution

Recently, the focus in chromatography model development has expanded to include the modeling of extra column volume (ECV), particularly in small- and lab-scale systems where ECV can constitute a significant portion of the total volume. Typically, ECV is modeled with 1D approaches, for example with combinations of dispersed plug flow reactors (DPFRs) and continuously stirred tank reactors (CSTRs). However, radial inhomogeneities in the ECV concentration profile necessitate higher-dimensional models for more accurate predictions. Searching for a suitable modeling approach for a micro simulated moving bed chromatography (μSMB) system, we investigated whether the 2D laminar flow model can be extended to account for additional dispersion effects, such as Dean vortices, through an equivalent radial flow rate distribution (eqFRD). For this purpose, we conducted 3D CFD simulations of the respective ECV and adapted the radial flow rate profile of a 2D simulation to match the residence time distribution observed in the CFD results. Applying the eqFRD model led to a significant improvement in prediction accuracy for isolated ECV segments, increasing from 90% to 97% compared to traditional ECV models. However, when these models were applied to the full μSMB system, the choice of ECV model had minimal impact on overall results as long as retention time within the ECV was accurately predicted. This suggests that, in the studied system, the column has a greater influence on peak shape than the ECV, allowing simpler ECV models to suffice in certain contexts. Despite these advances, significant deviations between predicted and experimental results were observed, indicating that factors such as the transition between the column and ECV, as well as detector effects, should be considered in future research. The results underscore the importance of selecting an ECV model in the context of the entire system, balancing accuracy with computational efficiency.

Modeling indirectly detected analyte peaks in ion-pair reversed-phase chromatography

In indirect detection, sample components lacking detectable properties are detected by adding a detectable component to the eluent, a so-called probe that interacts with the analytes to be detected. This study focuses on modeling indirect detection in two principally different cases. In case (1), the analyte component has the same charge as the probe component, so the probe acts as a co-ion of the analyte. In case (2), the analyte component has the opposite charge to the probe, so the probe acts as a counter-ion of the analyte.In the co-ion case (1), the analytes are alkyl sulfonates, and a competitive bi-Langmuir isotherm model was used. In the counter-ion case (2), the analytes are amines, and a modified bi-Langmuir isotherm model, incorporating ion-pairing on the stationary phase surface, was derived and applied for simulating the elution profiles. The chromatographic system comprised an XBridge Phenyl column as the stationary phase and an acetonitrile/phosphate buffer mixture with varying concentrations of sodium 2-naphthalenesulfonate as the eluent. In both cases, the detectable probe component was sodium 2-naphthalenesulfonate.The applied isotherm models successfully predicted system peaks with high agreement in both model cases, with calculated relative errors in retention times typically below 4.72 % and often below 1 %. The models were employed to predict the sensitivity of analytical methods, demonstrating excellent agreement between experimental and calculated sensitivities. These findings confirm the validity of the new adsorption isotherm model under these experimental conditions.

Capillary electrophoresis separations with Betaine:Urea, a deep eutectic solvent as the separation medium

BackgroundCapillary electrophoresis (CE) is a highly versatile separation technique widely used in analytical chemistry. Traditionally, CE can be categorized as either aqueous or non-aqueous systems based on the buffer solvents employed. For decades, non-aqueous CE has been predominantly associated with the use of organic solvents, a perception deeply ingrained in the scientific community. However, growing concerns about the health and environmental impacts of these solvents, driven by the principles of green chemistry, have prompted a reevaluation of their use. In response to these concerns, our group recently introduced a deep eutectic solvent (DES), specifically Proline:Urea, as an innovative and eco-friendly separation medium for CE. This approach not only enhances the sustainability of CE separations but also offers a new perspective for the development of innovative CE separation media. ResultsBuilding on our previous work, here we report the use of the second DES, Betaine:Urea (BU), as a new separation medium that offers further improved performance for CE applications. The DES was systematically characterized, with key physical properties relevant to CE separations, such as thermal properties, viscosity, dielectric constant, Joule heating effect, and UV transmittance, being thoroughly examined. Using a complex sample of 10 structurally similar naphthalene derivatives, we demonstrated the efficiency of BU in capillary zone electrophoresis (CZE) for separating analytes with varying charges (including cations, neutrals, and anions) and sizes. Additionally, we established the first micellar electrokinetic chromatography (MEKC) system in this DES using sodium dodecyl sulfate (SDS) as the surfactant. This system successfully resolved 6 structurally similar neutrals that could not be separated by conventional aqueous SDS-MEKC, highlighting the versatility of this DES-type separation medium. Furthermore, BU showed several advantages over the previously reported DES, Proline:Urea, particularly in terms of stability, viscosity, and Joule heating effects. SignificanceThis study holds the potential to challenge the traditional notion that “CE separation media are merely categorized into aqueous and organic solvents”. Given that DESs are “designer” solvents with highly tunable properties and environmentally friendly characteristics, the introduction of BU as a viable alternative to traditional solvents not only expands the media available for CE separations, but also offers a more efficient and potentially more sustainable option for specific analyses.

Aggregation-Dispersion Chromatography: Application of Elastin-like Polypeptides

Protein purification is a crucial step for various downstream applications like drug development, antibody preparation, and structure determination. The constant pursuit is for methods that are more efficient and cost-effective. We propose a novel approach using an elastin-like polypeptide (ELP) as an aggregation core that serves as an anchor between the beads in a chromatography column. In this method, a chilled sample containing a [target protein type] fusion protein is loaded onto a pre-equilibrated IMAC (immobilized metal affinity chromatography) column with a low-salt buffer. The column is then washed with a warm buffer containing high salt to remove impurities. Here, the key step involves warming the column above the ELP’s transition temperature (Tt), which triggers its aggregation. This aggregation is expected to trap the target protein tightly between the beads. Subsequently, a harsh wash with high salt and high imidazole can be applied to remove even persistent contaminants, achieving high protein purity. Finally, the temperature is lowered, and a cold, low-salt buffer is introduced to reverse the aggregation and elute the purified target protein. This method has the potential to eliminate the need for sophisticated chromatography systems while still achieving high protein purity.

Cost-effective low-volume sample introduction system for open tubular capillary ion chromatography via a hybrid negative pressure approach

Open tubular capillary ion chromatography (OTIC) exhibits a minute sample/eluent volume separation with a low-pressure operation (<30 psi) and potentially develops for a portable analytical device. In this study, a novel and cost-effective low-volume sample-introduction system has been developed for the OTIC and applied for separating water-soluble small inorganic ions. This innovative design uses a hybrid negative pressure (HNP) system, considerably reducing sample consumption to ∼7 μL per injection while maintaining excellent repeatability (<4 % relative standard deviation) on a 50 μm bore capillary tube. Various sample-introduction parameters, such as moving time of the sample zone, injection pressure, and sample viscosity, were optimized for the separation of model anions (Cl−, Br−, NO2−, and NO3−) and cations (Na+, K+, Ca2+, and Mg2+) in a non-suppressed OTIC mode. The performance of this HNP-OTIC system was demonstrated through the analysis of water-soluble small inorganic ion determination in low-volume particulate matter extracted samples, yielding recovery percentages of 78–119 % for cation determination in PM10 samples. In addition, the determination of K+ in PM2.5 samples exhibited no significant difference from the results obtained using a commercial packed IC. The cost-effective and efficient OTIC system has the potential to become a promising alternative for atmospheric analysis, especially in tracking K+ as an ion tracer for PM2.5 source identification.

A sustainable HPTLC approach for green assessment of Tyrosine to phenylalanine ratio in chronic kidney disease

The kidney is considered the main site for the net release of Tyrosine (Tyr) to the circulation through hydroxylation of Phenylalanine (Phe) using phenylalanine hydroxylase enzyme. In chronic kidney disease (CKD) patients the enzyme activity is impaired, therefore the serum concentration ratio of Tyr/Phe is reduced compared to healthy individuals. This motivates us to develop a cost effective, green, simple, sensitive, and selective HPTLC method to measure the levels of Tyr and Phe in serum samples. The analysis was carried out using acetonitrile: ethanol: 25% ammonia solution: ethyl acetate (6.5: 1.5: 1: 0.5) as a mobile phase. Rf values were 0.55 ± 0.05 for Phe and 0.39 ± 0.05 for Tyr. Densitometry scanning was performed using UV detector and dual wavelengths of 210 and 225 nm were obtained. A linear correlation was observed between the levels of Phe and Tyr, ranging from 50 to 700 ng band− 1 and 50 to 600 ng band− 1, respectively, under the optimum conditions. The method selectivity, linearity, precision, accuracy, and robustness were all confirmed in accordance with ICH recommendations. Calculations of the separation and resolution factors, number of theoretical plates, and height equivalent to theoretical plates prove to the chromatographic system accuracy and high separation efficiency. The developed method exhibits an acceptable eco-scale when measuring the method greenness using AGREE and GAPI softwares. It was applied for the determination of Phe and Tyr concentrations in human serum samples.

OpenPrep: Sensor Event-driven Open Column Chromatographic Sample Preparation System.

We propose a sensor event-driven, open-column chromatographic sample preparation system─OpenPrep. This system replaces the problematic flow selector valve with a noncontact built-in sensor, eliminating carryover and clogging issues, which are commonly encountered in pump-driven chromatographic systems. The innovative valveless column-flow design, combined with a compact gantry dispenser and motion stage-based fraction collector, reduces the sample flow path to a disposable component with a postcolumn dead volume of only 0.5 mL. This configuration is superior to conventional dynamic flow systems owing to its ability to minimize sample consumption, eluent loss, and cleaning waste generation. Thus, OpenPrep can be ideal for preventing cross-contamination. Owing to the rapid flow-transfer and stop approach, OpenPrep achieves higher reagent delivery accuracy and precision compared to ISO 8655-2 piston-operated pipettes, by minimizing liquid residue on the dispensing tip. Sensor-driven flow control ensures precise eluent collection and automatic regulation of the mobile-phase flow rate through an online calibration algorithm, meeting all the requirements for accurate and reproducible column flow control. OpenPrep features an open architecture for both hardware and software, offering high flexibility for analyzing samples with considerable variations in radioactivity and matrix compositions, such as radioactive waste. The proof-of-concept implementation of a multistage sequential separation strategy, using five resin columns to separate Re, Sr, Fe, and Ni from a single sample, demonstrates that OpenPrep provides enhanced flexibility and reconfigurability, improved operational efficiency, and cost-effective customization. Excellent chemical yields were obtained for Re, Sr, and Ni in the range of 83-97%, with less than 2.6% relative standard deviation at a flow rate three times faster than the gravity method. Additionally, improved recovery performance of Fe was achieved by applying a slower flow rate compared to gravity flow.

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.

Advancements in mass spectrometry-based proteomics: anew era in pathology research and diagnostics.

Mass spectrometry (MS)-based proteomics is rapidly transforming pathology research and diagnostics by enabling comprehensive studies of protein expression and post-translational modifications (PTMs). This article discusses recent advancements in MS-based proteomics, focusing on emerging technologies in sample preparation, MS instrumentation, and data analysis. These developments are scrutinized for their applications in clinical cohort studies and molecular pathology diagnostics. The article reviews innovations in automated sample preparation, chromatography systems, advanced MS technologies, and proteomic data analysis in the context of pathology. Specific applications such as liquid biopsy, spike-in heavy peptide panels, immunopeptidomics, and PTM screening are highlighted alongside opportunities for data integration. Recent technological improvements have significantly increased the throughput, precision, and scope of proteomic studies, enabling the analysis of large clinical cohorts and small specimens with unprecedented sensitivity. Advanced MS techniques have broadened applications, opening new avenues for discovery and diagnosis of marker proteins and therapeutic targets. Advancements in MS-based proteomics have created new opportunities in clinical research and diagnostics. By facilitating more comprehensive and integrated analyses of proteomes, these technologies are set to play apivotal role in the future of personalized medicine and pathology research.

Ultra-high pressure slalom chromatography: Application to the characterization of large DNA and RNA samples relevant in cell and gene therapy

Slalom chromatography (SC), initially co-discovered by Boyes and Kasai in the late 1980s, has recently re-emerged as a breakthrough technique to rapidly analyze DNA samples. With the development of cutting-edge ultra-high pressure liquid chromatography (UHPLC) systems and columns, SC now offers enhanced resolution and sensitivity for analyzing large DNA samples. By revisiting the fundamentals of the SC retention mechanism (non-equilibrium separation mode) and considering the physicochemical properties of DNA biopolymers (contour length, extension under shear flow, relaxation time), we provide analytical chemists with a general strategy and framework for selecting the most relevant applications in the expanding field of cell and gene therapy. We then present proof-of-concept data demonstrating the rapid separation (under 2 min) of plasmid digest samples containing linear double-stranded (ds) DNA macromolecules starting from 2 kbp to 25 kbp, as well as the accurate size determination (±6%) of linear dsDNAs. Additionally, we show rapid baseline separation and quantification of extensible linear dsDNAs, along with the more rigid plasmid dsDNA (supercoiled/circular/nicked circular). We also quantify dsRNA impurities present in vitro transcription (IVT) media used for producing new mRNA therapeutics and assess dsRNA structural heterogeneity (conformational isomers). These findings aim to support in a near future application chemists in addressing emerging bioanalytical challenges in cell and gene therapy by offering advanced SC columns and methods.

Sustainable and efficient monitoring of tryptophan and tyrosine serum levels: a green HPTLC method as a biomarker for type 2 diabetes

In recent years, there has been considerable interest in using amino acids like tryptophan (Trp) and tyrosine (Tyr) as biomarkers for various diseases, including type 2 diabetes mellitus (T2D). In diseases like T2D, the metabolism of Trp and Tyr is altered. The activity of enzymes involved in Trp metabolism increases, leading to a decrease in its serum level. On the other hand, the serum level of Tyr increases due to the suppressed activity of its metabolizing enzymes. These observations suggest that Trp and Tyr metabolism may play a crucial role in the pathophysiology of type 2 diabetes. Our study highlights the potential utility of Trp and Tyr as biomarkers for the early detection, prognosis, and monitoring of this metabolic disorder. Given these observations, we aimed to develop a high-performance thin-layer chromatographic (HPTLC) method that is sensitive, selective, rapid, and environmentally friendly for estimating the concentrations of Trp and Tyr in biological fluids, particularly serum samples. To evaluate the method, we performed analysis using serum samples from controlled and streptozotocin-induced diabetic rats. Our main objective was to develop a method that is sensitive and selective for precisely determining Trp and Tyr serum levels, which could serve as potential biomarkers for T2D. Fluorescence and absorption modes were employed for densitometry scanning. We assessed the precision and high separation efficiency of the chromatographic system by calculating parameters such as separation and resolution factors, number of theoretical plates, and height equivalent to theoretical plates. To evaluate the environmental impact of our proposed method, we employed the AGREE (Analytical GREEnness metric) and GAPI (Green Analytical Procedure Index) greenness assessment tools. The results confirmed that our method is environmentally friendly and exhibits superior eco-friendliness and greenness compared to other reported methods.Graphical

Geochemical Parameters of Oil and Rock Samples from Murzuq Basin, Libya: An Application for Paleoenvironment Description

Seven sedimentary rocks and seven crude oils undertook a geochemical analysis to describe their organic matter type and then tag the Paleoenvironments based on the organic matter. The samples were collected from B1-NC151, A1-NC58, P1- NC1, E1-NC174, D1-NC151, H29-NC115, and D1-NC174 boreholes, located in Murzuq Basin. The study was established to understand life forms in the ecosystem based on biomarkers that biochemical techniques recognized. Microscopy was used for kerogen typing and Spore Color Index reference. Organic matter (EOM) was extracted from cutting samples. Furthermore, hydrocarbons were saturated and aromatic fractionated from the samples to investigate using Gas chromatograph system. Spore color index (SCI), range between 1.5-3.5, Amorphous organic matter (AOM) from 42 to 95 percentage, Phytoclasts % were 4-15.5, and Palynomorphs % range 0.5 to 7.5. The percentage of Carbon 85.66-86.29, Hydrogen 13.16-13.73, Nitrogen 0.06 -0.27, and Sulfur 0.31-0.70, besides wax were measured, ranging among 1.3 to 6.0 %. Pristine/n-C17, Phytane/n-C18, Pristine/Phytane, carbon preference index measurements (CPI) and DBT/P as saturated hydrocarbon ratios from whole oil chromatographs were established. Tricyclic terpanes, hopanes and n-alkanes were distinguishing as the most important oil biomarkers that reserved the characteristics in the Paleoenvironments.

A rapid protocol for distinguishing the quality of Sanshengyin and identifying potential markers by the “three-in-one” fingerprint profiles with antioxidant activity

It is imperative to establish a standardized model for the quality control of traditional prescriptions, exemplified by Sanshengyin (SSY). This study introduces a multi-component and multi-level "three-in-one" fingerprint quality evaluation system designed to predict the comprehensive quality of traditional Chinese medicine (TCM). Initially, a five-wavelength high-performance liquid chromatography (HPLC) fusion spectrum was developed to enhance the stability of SSY within the chromatographic system. Additionally, a quantification method was employed to streamline the calculation and visualization processes of ultraviolet (UV) and infrared (IR) spectral systems. Through this approach, chromatographic and spectral fingerprints were amalgamated into a holistic analytical strategy. The average linear quantitative fingerprint method (ALQFM) was employed to swiftly differentiate the grades of 17 sample batches. Additionally, six quality markers were identified for content determination. Correlation analysis between the total content of these quality markers and macroscopic quantitative similarity was conducted to assess the accuracy of ALQFM in evaluating the quality of TCM represented by SSY. Finally, the binary correlation analysis model was employed to investigate the correlation between antioxidant activity and fingerprint profiles, facilitating the prediction of potential bioactive compounds in SSY. This research offers a straightforward, cost-effective, and holistic approach for assessing the quality consistency of TCM. The method is recommended for adoption by medical institutions and enterprises to enhance the standardization of TCM quality control.

Green RP-HPLC method for the estimation of carfilzomib in bulk, protein nanocarriers and human plasma: Application of chemometrics and Monte-Carlo simulations

Carfilzomib is a tetrapeptide epoxyketone that has shown potential clinical outcomes in the treatment of multiple myeloma. However, inaccuracies in quantifying such peptide drug products have arisen due to poor stability, low solubility, time-consuming techniques, complex physicochemical properties, and use of non-green solvents with less recyclability. This provides a substantial urge to develop an ecological and sensitive analytical method for quantifying peptide drugs from matrix formulation and biological samples in early as well as lateral stages of product development in pharma industries. As a result, the study aimed to develop a robust ecological method for estimation of carfilzomib via Green RP-HPLC using analytical quality by design (AQbD) paradigms with specific application in protein nanoparticles and biological matrix. Initially, an appropriate wavelength for quantification of carfilzomib was chosen using principal component analysis (PCA) as a chemometric tool.Risk assessment followed by factor screening studies using 8-factor Placket-Burman Design aided in earmarking critical method parameters (CMPs) affecting critical analytical attributes (CAAs). Further, Central Composite Design (CCD) was employed for design space optimisation to demarcate optimum chromatographic conditions, which were corroborated for robustness using Monte-Carlo simulations. The method was validated as per ICH Q2 (R1), followed by quantifying the greenness of the method using Green Assessment tools. The method optimisation resulted in the optimal chromatographic conditions using Green RP-HPLC. The chromatographic system was equipped with a Phenomenex Aeris Peptide-XC C18 column (150 × 4.6 mm × 5 µm), and the mobile phase was composed of isopropanol:methanol:0.1 M PBS (pH 5.5 adjusted using 0.1 % formic acid) (35:45:20v/v), with a 1 ml/min flow rate at a 210 nm ʎmax. The optimised chromatographic conditions resulted in a short retention time (RT) of 4.95 mins, 0.87 tailing factor (TF), 4,875,122 peak area (PA), and 8995 theoretical plate count (TPC). The method demonstrated linearity in a wide range of concentrations (0.1–20 µg/ml) with a correlational coefficient of 0.997 and < 2 % RSD. The method unearthed a high precision rate with more than 95 % of drug recovery in protein nanoparticles and human plasma, thereby confirming the accuracy and sensitivity of the developed method. Chemometrics and Monte-Carlo simulations ratified the robustness and sensitivity of the developed analytical method of Carfilzomib with established greenness and a high degree of practical utility in protein-based nano formulations and human plasma matrix for life cycle product development.