C18 Column Research Articles

Targeted ultra-high performance liquid chromatography-tandem mass spectrometry assay for the quantification of medium-chain phosphatidylcholines in platelets of coronary artery disease patients

In a recent untargeted clinical lipidomics study of platelets of coronary artery disease (CAD) patients, medium-chain phosphatidylcholines (MCPCs) with C8 and C10 fatty acyl residues were found significantly upregulated in the patient group with acute coronary syndrome (ACS) as compared to chronic coronary syndrome (CCS) and healthy controls. To support this finding, this work presents the development and optimization of a targeted UHPLC-QTrap-MS/MS method with multiple reaction monitoring acquisition for the quantitative analysis of MCPCs (PC 10:0/8:0, PC 16:0/8:0, PC 10:0/20:4 and PC 10:0/10:0) in platelets for biomarker validation. A systematic optimization of chromatographic and mass spectrometric parameters was performed. A charged surface hybrid CSH C18 (1.7 µm, 130 Å) column and fine-tuned gradient elution with 2-propanol/acetonitrile and ammonium acetate as additive to the mobile phase was employed in the final method in ESI negative mode. Four selected PC standards (PC 6:0/6:0, PC 8:0/8:0, PC 10:0/10:0 and PC 12:0/12:0), which cover well the carbon and retention rime range of the target analytes, were used for the optimization process and calibration. Quantification was based on matrix-matched calibration with these four selected commercially available MCPC standards as surrogate calibrants and PC 6:0/6:0 (d22) as internal standard. Furthermore, an organic solvent and fatty acyl carbon number-corrected response factor approach gave also accuracies within acceptance limits of bioanalytical validation guidelines and has more generic applicability. Compared to the previous untargeted RPLC-ESI-QTOF-MS/MS method, the optimized targeted UHPLC-QTrap-MS/MS assay showed increased sensitivity and selectivity for the detection of medium-chain PCs in platelet samples of CAD (LOQs in the range of 0.5-5 nmol/L). The method performance parameters indicated its suitability for a future biomarker validation study of MCPCs in platelets.

Deep eutectic supramolecular polymers based HPLC stationary phase: Green synthesis strategy and promising application prospects

BackgroundDeep eutectic solvents (DESs) have been widely and significantly applied in various fields due to their outstanding features such as low cost, easy preparation and good biodegradability. As novel derivatives of DESs, deep eutectic supramolecular polymers (DESPs) combine the macroscopic state of DESs with the covalent interactions of supramolecular polymers, which also possess the properties of DESs as multifunctional materials. Therefore, DESPs are believed to be promising candidates for separation science. However, there are no studies on the application of DESPs as stationary phases for HPLC analysis. ResultsIn this work, a novel DESP based HPLC stationary phase (Poly(DES)@SiO2) was developed for the first time through a green synthesis method by using DES as the polymerization monomer as well as the reaction medium. The results manifest that this novel Poly(DES)@SiO2 column can well interact with analytes through various mechanisms, and realize selective separation of a wide range of structurally similar hydrophilic/hydrophobic substances. More importantly, the separation of hydrophobic analytes on the Poly(DES)@SiO2 column is less time-consuming with fewer organic eluent, although the column efficiency is slightly lower than that of commercial C18 column. Furthermore, the Poly(DES)@SiO2 column exhibits excellent mechanical stability and satisfactory separation repeatability for steroid hormones. Therefore, a reliable method was established for detecting steroid hormones in actual samples with the recoveries ranging from 94.56 % to 103.84 %, which can meet the detection needs of commonly seen steroid hormones in food and the environment. SignificanceIn summary, this work provides some valuable theoretical references for the synthesis of new DESP based stationary phases through a green and facile strategy, and meanwhile, verifies the feasibility of DESP for effective HPLC separations. In addition, the promising application prospect of DESP based stationary phases in the analysis of complex samples has also been demonstrated, expanding the potential application of DES in separation science.

Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry.

In this study, a simple and sensitive liquid chromatography tandem mass spectrometric method was developed and validated for the determination of iptacopan and two acyl glucuronidation metabolites in monkey plasma. The plasma sample was precipitated with acetonitrile and then separated on an Acquity UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm) using 0.1% formic acid and 5 mM ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometry (MS) detection was performed in positive multiple reactions monitoring (MRM) mode with precursor-to-production transitions. The developed assay was validated over the range of 1-2000 ng/mL for three analytes with correlation coefficient (r) more than 0.99. The validation parameters including accuracy, precision, carryover effect, matrix effect, recovery, and stability were all within the acceptable limits. The validated method has been applied to investigate the pharmacokinetics of iptacopan and its two acyl glucuronidation metabolites in monkey plasma. After intravenous administration, iptacopan showed low clearance (2.75 mL/min/kg) in monkey plasma. After oral administration, the bioavailability was 55.43%. The exposure (AUC0-t) of direct acyl glucuronide (AG) of iptacopan accounts for 9.73% of the iptacopan plasma exposure. The AUC0-t of AG of dealkylated metabolite of iptacopan was present at a lower level, accounting for 0.5% of the iptacopan plasma exposure.

Simultaneous determination of 17 perfluoroalkyl substances in beef by EMR-Lipid dispersive solid-phase extraction and ultra performance liquid chromatography-tandem mass spectrometry

The ability to accurately analyze perfluoroalkyl substance (PFAS) levels in beef is imperative in order to effectively assess food-safety risks and ensure consumer safety because PFASs are harmful and prevalent in beef. In this study, we developed a rapid and accurate method for the simultaneously determination of the 17 PFASs in beef using dispersive solid-phase extraction (d-SPE) and ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and optimized the mobile phase system, extraction solvent, and d-SPE materials. Samples were finally extracted using 0.1% (v/v) formic acid in acetonitrile, cleaned using d-SPE with PSA, C18, GCB, and EMR-Lipid, separated using an Acquity Premier BEH C18 column (100 mm×2.1 mm, 1.7 μm) with 0.5 mmol/L ammonium fluoride aqueous solution and methanol as the mobile phases at a flow rate of 0.3 mL/min. Analytes were detected in negative ion switching mode (ESI-) with multiple reaction monitoring (MRM) scanning, and quantitatively analyzed using the internal standard method. The 17 PFASs exhibited linearity in the 0.2-20.0 μg/L range under the optimal experimental conditions, with correlation coefficients of 0.9915-0.9999. The method delivered limits of detection (LODs) of 0.003-0.007 μg/kg and limits of quantification (LOQs) of 0.01-0.02 μg/kg. The 17 PFASs exhibited recoveries of 71.1%-127.4% with RSDs of 0.6%-14.4% when spiked at three levels (0.05, 0.5, and 1.8 μg/kg). We optimized the mobile phase system, which revealed that, compared with 2.0, 5.0, and 10.0 mmol/L ammonium formate or ammonium acetate in aqueous methanol, 0.5 mmol/L ammonium fluoride in aqueous methanol exhibited higher sensitivities for all the 17 PFASs, with PFASs bearing long-chain carboxylic acids (C10-C18) showing 1-2 fold increases in sensitivity. PFASs do not dissociate in acidic environments, favoring their entry into the organic phase. Therefore, we investigated the effect of extractant acidity, which revealed that the 17 PFASs were better extracted using 0.1% (v/v) formic acid in acetonitrile. The beef matrix has a complex composition; consequently, d-SPE adsorbents were required to purify samples and reduce matrix effects. The purification effects of four adsorbents (PSA, C18, GCB, and EMR-Lipid) toward the 17 PFASs and the amount of EMR-Lipid used were investigated, which revealed that 100 mg PSA+80 mg C18+40 mg GCB+150 mg EMR-Lipid exhibited superior matrix-purification behavior. We also investigated the effects of various injection solutions and types of syringe filter, with pure methanol selected for reconstitution and high-speed supernatant centrifugation applied prior to injection. The developed method is simple, rapid, sensitive, and reproducible, and can be used to simultaneously, rapidly, and accurately determine various perfluoroalkyl compounds in beef.

A novel RP-HPLC method for the simultaneous quantification of azithromycin and dexamethasone in marketed ophthalmic drops

A straightforward, precise, accurate, specific, and sensitive RP-HPLC technique has been established for the quantification of Azithromycin Dihydrate and Dexamethasone Sodium Phosphate in their commercial formulation. The wavelength maxima for Azithromycin and Dexamethasone were considered to be 215 nm for estimation. In RP-HPLC separations were carried out on a Zorbax SB C18 column (250mm X 4.6mm, 5 μm) and mobile phase consisting of Acetonitrile: Monobasic Potassium Phosphate (KH2PO4) buffer in the proportion of 75:25 v/v and pH of the buffer was adjusted to 5.5 with OPA. The estimation was carried out at 215 nm using UV detector keeping the flow rate of 1.2 ml/min and injection volume of 50 μl. The current method demonstrates good linearity over the range of 20 - 120 μg/ml and 2 - 12 μg/ml for Azithromycin and Dexamethasone respectively. No interference of excipients was found during estimation. The recommended techniques were verified and determined to be specific, accurate, and exact. The approaches were effectively used for the concurrent quantification of both medicines in pharmaceutical formulations, making them suitable for regular quality control examination.

RGB Trichromatic Whiteness Assessment of Bio Analytical Chromatographic Tool Using Fluorescence for Quantitation of Semaglutide: Application to Pharmaceutical Preparations and Spiked Plasma.

Semaglutide (SEMG) is one of the most widely used and trending medications to treat type II diabetes and obesity.This work aimed to develop a liquid chromatography with spectroflourimetric detection (HPLC-flourimetry) analysis of SEMG in both its tablet dosage form and plasma. The power of fluorescence detection coupled with HPLC proved its capability as a bioanalytical tool to assay SEMG in plasma samples owing to its simplicity and sensitivity which reached below the Cmax of SEMG. Separation was done using a C18 column with mobile phase of acetonitrile and water acidified with orthophosphoric acid (pH 3.5) (1.41 × 10-5M) in isocratic mode in ratio 57:43 and 1mL/min flow rate after extraction using protein precipitation. Detection was carried out at λ excitation of 238nm and λ emission of 416 and 307nm for SEMG and the internal standard, respectively. Evaluation of greenness of the proposed method was done using AGREE (Analytical GREEnness Metric Approach), ComplexGAPI (Complementary Green Analytical Procedure Index) & the new algorithm RGB 12 model (Red-Green-Blue). They showed that these methods can be a greener alternative with acceptable sensitivity for analysis of SEMG. The developed seven min-assay was validated per ICH as well as FDA bio analytical methods' guidelines to prove its applicability for routine sample analysis and future pharmacokinetic studies.

Development of a gradient method for sulfamethoxazole, trimethoprim, isoniazid, and pyridoxine hydrochloride in rabbit plasma through QbD-driven investigation

The current study developed a method for quantifying four drugs—Sulfamethoxazole, Trimethoprim, Isoniazid, and Pyridoxine—in rabbit plasma. The method uses gradient liquid chromatography based on analytical quality by design. To achieve separation, a Eclip Plus C18 (250 mm × 5 mm, 4.6 µm) column with L1 packing was used, and analytes were detected at 254 nm at ambient temperature. The optimized mobile phase consisted of 50 mM potassium dihydrogen phosphate buffer (pH 6.5) and Methanol. The concentration of Methanol was 3% (0–5 min), 15% (5–15 min), 55% (15–27 min), and 3% Methanol until the end of the 30-min runtime, and the flow rate was set at 0.95 mL/min. Control Noise Experimentation was used to screen studies, revealing that flow rate, pH, and Methanol concentration significantly affected the analytical attributes. The study identified critical attributes (resolution and asymmetric factor) and developed a quality target method profile. A central composition design was used to optimize the essential parameters. The method developed for the drugs showed peaks at retention times of 6.990 min for Isoniazid, 7.880 min for Pyridoxine, 15.530 min for Sulfamethoxazole, and 26.890 min for Trimethoprim, respectively. The method was validated with linearity in the range of 10–640 ng ml−1, with R2 of 0.9993, 0.9987, 0.9993, and 0.9992 for Sulfamethoxazole, Trimethoprim, Isoniazid, and Pyridoxine, respectively.

Insights into type 2 diabetes mellitus in atrial fibrillation patients: a proteomics approach

Abstract Introduction Atrial fibrillation (AF) and Type 2 Diabetes Mellitus (T2DM) are two closely interconnected conditions, sharing a complex relationship within cardiac pathophysiology. Common risk factors such as obesity, hypertension, inflammation, and endothelial dysfunction contribute to their coexistence. T2DM is known to drive cardiac fibrosis and promote electrical and structural remodeling of the heart, creating a substrate conducive to AF onset, while AF in T2DM patients increases the risk of adverse cardiovascular outcomes, such as stroke. Understanding this complex relationship is pivotal for improving the therapeutic and preventive strategies for these patients. This study aims to examine the proteomic differences between AF patients with and without T2DM, providing novel insights into pathophysiological mechanisms and identifying novel therapeutic targets and biomarkers. Methods In total, 14 AF patients undergoing open-heart surgery, comprising of 6 T2DM individuals and 8 non-T2DM individuals have been included. The mean age of the T2DM patients was 69.5 ± 10 years, while the non-T2DM was 69.3 ± 8.2 and BMI was 29.2 ± 2.9 and 29.5 ± 3 respectively. Right atrial appendages (RAA) from these patients were obtained in an RNA stabilization solution during open-heart surgery and processed using the phenol-based method for protein extraction. The proteins were digested with trypsin to generate peptides, followed by an additional clean-up step using C18 pipette tips. The prepared peptide samples were analyzed by employing a label-free-based quantitative (LFQ) approach, using a C18 (75cmx75cm) column. Proteomic analysis was performed using a Nano liquid chromatography system coupled to tandem mass spectrometer (nano LC-MS/MS), followed by a comprehensive bioinformatics analysis, using appropriate software. Results A total of 1548 proteins were identified, of which 899 could be quantified using LFQ analysis. Applying strict significance criteria, including FDR <0.1 and Abundance ratio (AR)>2 or <0.5, we identified 100 differential expressed proteins, with 21 upregulated and 79 downregulated in T2DM (Fig.1). Most notably, DPP9 and ATP1A2 were overexpressed in hearts of AF patients with T2DM (AR=100), while INPPL1 and ARID5A were underexpressed (AR=0.01), thereby representing attractive therapeutic targets. In pathway enrichment analysis (Fig.2), the dysregulated proteins participate in noteworthy pathways, including Non-alcoholic fatty liver disease (NAFLD), Type II diabetes mellitus, and Insulin signaling pathway. Conclusion Given the increased morbidity associated with the coexistence of AF and T2DM, there is a pressing need for novel therapeutic strategies tailored to this patient population. While AF and T2DM share certain molecular characteristics in the heart, this study emphasizes the necessity for further evaluation and validation of the distinct proteomic signatures identified.Fig.1:Volcano PlotFig.2:Protein Enrichment Network

Simultaneous qualitative and quantitative analysis for evaluating constituents of Atractylodis macrocephalae rhizome by UPLC-QTOF-MS

AbstractAtractylodis macrocephalae rhizome (AMR) belongs to medicine food homology. Its' clinical application of invigorating the spleen-stomach of AMR was applied to various diseases. In this research, a UPLC-QTOF-MS method was developed for qualitative and quantitative analysis of AMR, simultaneously. A Waters Acquity BEH C18 column (2.1 mm × 100 mm, 1.7 μm particle size) was used for separation of AMR multi-components. The column was eluted with a mobile phase of 0.1% formic acid-water and 0.1% formic acid-acetonitrile. Electron spray ionization with positive-ion mode and external standard method was utilized for quantifying the nine analytes in AMR. Constituents of AMR were scanned by UPLC-QTOF-MS and then identified by mass fragments and chromatographic information compared with the published literature and reference standards. Under positive mode, a total of 61 chemical compositions including 16 terpenoids, 8 polyacetylenes, 6 aromatics, 5 flavonoids, 5 coumarins, 5 organic acids, 4 amino acids, 3 fatty acids, 3 aliphatics, 2 steroids, and 2 alkenes, a nucleoside and an aldehyde were identified. Simultaneously, the contents of three amino acids (L-tyrosine, L-phenylalanine, and L-tryptophan), three sesquiterpenoids (atractylenolide Ⅲ, atractylenolide Ⅱ, and atractylenolide Ⅰ), a flavonoid (rutin), an organic acid (ferulic acid), and a pentacyclic triterpenoid (oleanolic acid) were determined in seventeen AMR batches. Amino acids and triterpenoid were quantified for the first time in AMR. The UPLC-QTOF-MS method developed in this article was reliable, practical, and useful for qualitative and quantitative evaluation of AMR multi-components.

A bioanalytically validated RP-HPLC method for simultaneous quantification of rivaroxaban, paracetamol, and ceftriaxone in human plasma: a combination used for COVID-19 management

Rivaroxaban is a direct oral anticoagulant medication that has been found to be beneficial for the management of thromboembolic events linked to the Corona virus disease 2019 (COVID-19) pandemic, which has resulted in more than 6 million deaths worldwide. Hence, a sensitive, selective, and green bioanalytically validated method was developed using RP-HPLC coupled with DAD for the simultaneous determination of rivaroxaban in human plasma, and two co-administered drugs, namely, paracetamol, an analgesic, and ceftriaxone, an antibiotic, that are used in the management of COVID-19. An Exsil 100 ODS C18 column (250 × 4.6 mm, 5 μm) was used as the stationary phase, and acetonitrile: water: methanol at a ratio of 60:30:10 (v/v/v) was used in isocratic mode as the mobile phase with a flow rate of 0.7 ml/min. The method was validated over a concentration range of 0.1–10.0 µg/mL for rivaroxaban, and 1.0–15.0 µg/mL for paracetamol and ceftriaxone. The lower limits of detection (LLODs) were found to be 0.03, 0.32, and 0.32 µg/ml for rivaroxaban, paracetamol, and ceftriaxone, respectively. Moreover, the lower limits of quantitation (LLOQs) were found to be 0.1, 0.96, and 0.98 µg/ml. The developed method showed excellent accuracy and precision for the determination of the aforementioned drugs. Four metrics were used to evaluate the greenness of the developed method. The results revealed that the suggested method is green, with values of 81 and 0.6 for the analytical eco-scale and analytical greenness assessment (AGREE), respectively.

Development and Validation of a Method for Detecting and Quantifying Mitragynine in Kratom Samples Using HPLC-PDA

Kratom (Mitragyna speciosa Korth) has been identified as a New Psychoactive Substance (NPS) by the United Nations Office on Drugs and Crime (UNODC) and included in the list of prohibited ingredients in food supplements and traditional medicine by Indonesian FDA. Therefore, a rapid, easy, and reliable analytical method is necessary to detect and quantify this plant and its products. This study developed a method for the detection and quantification of kratom products based on a unique compound, mitragynine, as a biomarker. A previous survey of determining mitragynine in Kratom using GC-MS, LC-MS/MS, UPLC, and HPLC-PDA. Previously, the HPLC-PDA method used a C8 column. Yet, for efficiency, it is also necessary to develop a test method using a C18 column. Analysis was performed using an HPLC - PDA system with Waters Atlantis T3-C18 (250 x 4.6 mm, 5 µm) column. The mobile phase comprises acetonitrile and formic acid 0.05%, pH 5.0 (75:25 v/v), delivered at a 1.0 mL/min flow rate. Detection was carried out at a wavelength of 225 nm. The analytical method was validated with test parameters of selectivity, system suitability, accuracy, precision, linearity, detection limit, and quantification limit. The validation study demonstrated an excellent linear concentration range of 1.96 - 6.01 µg/mL with a correlation coefficient of 0.9996; the detection limit is 0.14 µg/mL, while the limit of quantification is 0.45 µg/mL—accuracy method of 98.88 - 101.44% and a bias of 0.27%. The percent relative standard deviation for six independent assay determinations was 0.67%, and the intermediate precision was 1.56% on two days. The mitragynine amounts in these materials ranged from 6.01 to 6.31 mg/g of dried leaf material. Based on the research results, it can be concluded that the method developed provides quick, simple, reliable, accurate, and valid, and has an advantage over existing methods in terms of simplicity of sample preparation, short analysis time, and cost-effectiveness compared to GCMS and LCMS/MS and can be applied for future analysis in Kratom samples.

Fast Determination of Propofol in Human Plasma Using C18-Functionalized Magnetic Nanomaterials Followed by Supercritical Fluid Chromatography.

Novel C18-functionalized magnetic nanomaterials; i.e., C18@poly-styrene-divinylbenzene-glycidyl methacrylate-Fe3O4 (C18@PS-DVB-GMA-Fe3O4) have been synthesized by using N, N-dimethyloctadecylamine as modifying agent, which could be beneficial to remove the blood phospholipids. The C18@PS-DVB-GMA-Fe3O4 nanoparticles have been used and evaluated in the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure for human plasma prior to the analysis of propofol by supercritical fluid chromatography (SFC). In the QuEChERS procedure, human plasma samples are directly mixed with extraction solvent and C18@PS-DVB-GMA-Fe3O4 nanoparticles, and the extraction and cleanup procedures have been accomplished simultaneously. The SFC separation was performed with a C18 column (Thermo Scientific™ Acclaim™ 120, 250 × 4. 6mm, 5μm) within 5min, using thymol as the internal standard. Supercritical carbon dioxide was used as the mobile phase with methanol as the cosolvent at the flow rate of 1.0mL/min. The column temperature was 38°C, and detection wavelength was 275nm. A good linearity was obtained among the propofol concentration range of 0.5-10mg/L (R2 = 0.9997) with the limit of detection of 0.17mg/L. Recoveries were in the range of 76.5-91.9%, with RSD less than 7.9%. These results suggested that method is convenient, rapid with high accuracy and little matrix effect, and suitable for rapid determination of propofol plasma concentration.

Online Comprehensive Two-Dimensional Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry-Based Metabolic Profiling and Comparison Enabling the Characterization of 1146 Ginsenosides and More Explicit Differentiation of Ginseng.

This work was designed for the in-depth characterization and holistic comparison of up to 12 ginseng varieties, which can benefit the development of functional foods and ensure their authenticity in the food industry. An online comprehensive two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry (2D-LC/QTOF-MS) approach was established by configurating the XCharge C18 and HSS Cyano columns. Under the optimal conditions, we characterized a total of 1146 ginsenosides (including 876 potentially new compounds) from 12 ginseng varieties by reference to an in-house library of 573 known ginsenosides and 70 reference compounds. The online 2D-LC/QTOF-MS-based untargeted metabolomics workflows were developed, by which 126 potential ginsenoside markers were unveiled and utilized to establish the key identification points for each ginseng species. Compared with the conventional liquid chromatography/mass spectrometry metabolomics, our multidimensional chromatography approach performed better in discriminating multiple ginseng varieties. This work demonstrates a potent and practical methodology to identify easily confused functional plants.

Retention Mechanisms of Basic Compounds in Liquid Chromatography with Sodium Dodecyl Sulfate and 1-Hexyl-3-Methylimidazolium Chloride as Mobile Phase Reagents in Two C18 Columns

Reversed-phase liquid chromatography (RPLC) relies on a non-polar stationary phase and a more polar hydro-organic mobile phase, where compound retention is primarily governed by hydrophobicity, with more hydrophobic compounds being retained longer. The introduction of secondary equilibria in the chromatographic system through additives, such as anionic surfactants and ionic liquids (ILs), was proposed to mitigate ionic interactions between positively charged analytes and the anionic free silanol groups in non-endcapped stationary phases, thereby preventing increased retention and peak tailing. Additionally, the combined hydrophobic and ionic interactions between cationic analytes and the ions in these additives was demonstrated to create mixed retention mechanisms that influence retention and selectivity. In this regard, this study investigates aqueous chromatographic systems incorporating both the anionic surfactant sodium dodecyl sulfate (SDS) and the IL 1-hexyl-3-methylimidazolium chloride as mobile phase reagents. This combination of reagents modulates the retention, eliminating the need for organic solvents and resulting in highly sustainable HPLC procedures. The chromatographic behavior was assessed using two different C18 columns (Zorbax Eclipse and XTerra-MS). The strength of solute interactions was estimated by calculating equilibrium parameters and the contributions of hydrophobic and ionic interactions through simple mathematical models. Focusing on the retention of six basic drugs (β-adrenoceptor antagonists), the study highlighted the significant role of ionic interactions. The results demonstrate the feasibility of using aqueous systems combining SDS and an IL for the efficient separation of moderately polar basic compounds without the use of organic solvents.

Method for determination of gamma oryzanol in dietary supplement by high performance liquid chromatography

Gamma oryzanol (GO), a phytosterol found most abundantly in rice bran oil as well as corn, barley, rye, vegetable oils and wheat bran. Thanks to its ability to slow down the development of melanin pigments, prevent the effects and spread of ultraviolet rays on the skin's surface, limiting freckles and darkening of the skin, in recent years, GO has been added to dietary supplements to help reduce melasma, brighten and smooth the skin. For the purpose of evaluating and controlling product quality, the GO analysis method was developed and validated. GO was analyzed by HPLC using a PDA detector with a C18 chromatography column (250 mm × 4.6 mm, 5 µm) and a mobile phase consisting of methanol and acetonitrile in isocratic mode 35: 65 (v/v). The test sample was extracted with a mixture of n-hexane and ethanol at a ratio of 2: 5 (v/v). The method was evaluated for system suitability, specificity, calibration curve, precision and accuracy with limit of detection and limit of quantification were 15.0 µg/g and 50.0 µg/g, respectively. The analysis results of 10 real samples including 03 pellet samples and 07 powder samples showed that the method is suitable for analyzing samples with different concentration ranges.

Development and Validation of an Improved HPLC-MS/MS Method for Quantifying Total and Unbound Lenalidomide in Human Plasma.

This study aimed to develop a fully validated HPLC-MS/MS method for quantifying total and unbound lenalidomide concentrations in human plasma. Unbound concentrations were measured using plasma ultrafiltrate prepared with Amicon® Centrifugal Filters. Lenalidomide and lenalidomide-d5 (internal standard) were extracted from 50 μL of human plasma using liquid-liquid extraction. Chromatography was conducted with a Halo® C18 column using 0.1% formic acid and methanol (20:80, v/v) as the mobile phase. The mass spectrometer was operated in a positive ion mode with an electrospray ionization interface and multiple reaction monitoring modes. Calibration curves were linear over the range of 5 to 1000 ng/mL (r2 > 0.996) for both the total and unbound lenalidomide. For total lenalidomide concentrations, between-run precision (coefficients of variation) and accuracy were 1.70-7.65% and 94.45-101.10%, respectively. For unbound concentrations, inter-day precision and accuracy were 1.98-10.55% and 93.95-98.48%, respectively. We developed a highly reproducible, sensitive, and efficient bioanalytical method using a smaller volume of plasma sample (50 μL) with a relatively short run time (2.5 min). The proposed analytical method was successfully applied to measure total and unbound lenalidomide concentrations at various time points in multiple myeloma patients with renal impairment.

Quantification of L-ornithine L-aspartate in health supplement by HPLC in combination with o-phthalaldehyde derivative technique

Dietary supplements containing L-ornithine L-aspartate (LOLA) are on the rise. Thus, a simple, reliable method of analysis for determining LOLA has been developed, which helps to control product quality. In this study, a method for determining LOLA in dietary supplements by HPLC in combination with o-phtalaldehyde derivatives (OPA) was developed and validated. The study optimized conditions for extraction of LOLA from the matrix, then derived with OPA and chromatographic separation on column C18. The detection limits and quantitative limits of the method were conducted on low-content real samples, with LOD and LOQ values achieved at 6.0 and 20.0 μg/g, respectively. The method has good recovery (98 – 106%) and repeatability (1.32 – 3.50%) that meet the method performance requirements according to AOAC. The validated method has been applied to analyze LOLA content in dietary supplements at the National Institute for Food Control.

Enhanced enantioselective separation of racemic menthol via reverse-phase high-performance liquid chromatography: Method development and computational insights for pre-screening

A robust and efficient enantioselective separation of racemic menthol was achieved on a standard C18 column with reverse-phase high-performance liquid chromatography (RP-HPLC) and UV detector. (R)-α-hydroxy-4-methylbenzeneacetic acid was utilized as the pre-column derivatization reagent. The impact of mobile phase composition on diastereomer selectivity was thoroughly investigated, resulting in a high resolution of 2.11 under optimized conditions. The method was rigorously validated for linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). Notably, a separation pre-screening mechanism (SPM) and a prediction model was developed based on density functional theory (DFT) studies. This model elucidated the relationship between molecular polarity differences (△MPI) and chromatographic behavior, facilitating the interpretation and prediction of racemic menthol resolution with various chiral derivatization reagents. The present work not only presents an efficient and economical approach for menthol enantiomeric separation, but also offers valuable insights for the innovative design and advancement of chromatographic methodologies for a wide array of chiral enantiomers.

Quantitation and Abundance of Xylazine and Xylazine Metabolites in Human Urine and Plasma by LC-MS/MS

Abstract The most common cause of drug overdose fatality in the US involves synthetic opioids such as fentanyl. Recently the Director of the White House Office of National Drug Control Policy designated fentanyl adulterated or associated with xylazine as an emerging threat to the US, and a national response plan will include xylazine testing. Xylazine is a clonidine analog and is not FDA-approved for use in humans but has been increasingly detected in unregulated illicit drugs supplies. Our understanding of xylazine detection, metabolism, and clinical impact on patients, however, is limited. The objectives of this study are to develop a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect xylazine and xylazine metabolites in urine and plasma, and to investigate the presence and abundance of xylazine metabolites in remnant urine and plasma samples submitted for clinical testing. This is achieved by first collecting paired urine and serial plasma samples when urine tests positive for xylazine during routine clinical testing. Plasma samples were prepared for LC-MS/MS analysis by protein precipitation with methanol and xylazine-D6 internal standard. Urines were prepared by dilution with xylazine-D6, followed by a hydrolysis with beta-glucuronidase. All experiments were performed using an Aquity HPLC coupled to a Waters TQS-micro triple quadrupole mass spectrometer. LC separation utilized a C18 column with a 4-minute gradient from 2% to 98% mobile phase B (0.1% formic acid and 2mM ammonium acetate in A: water; B: methanol) with a flow rate of 400 μl/min at 50°C. Multiple reaction monitoring for xylazine and 4-OH-xylazine was optimized with commercial standards. Calibration curves for both analytes were prepared from 0.5 – 2,000 ng/mL in plasma, and 5 – 5,000 ng/mL in urine. Product ion scanning was used to detect four additional xylazine metabolites by spectral matching with previously published full scan tandem mass spectra. The highest intensity fragment ions observed in the product ion spectra were used to develop multiple reaction monitoring experiments for four additional xylazine metabolites, previously described as: sulfone-xylazine, oxo-xylazine, OH-sulfone-xylazine, and OH-oxo-xylazine, in 50 remnant urines and 70 individual plasmas. In remnant urines, xylazine concentrations ranged from less than 5 to greater than 5,000 ng/mL. In remnant plasmas, xylazine ranged from less than 0.5 to 158 ng/mL. Previously reported xylazine metabolites were detected in both urine and plasma, and relative peak area ratios were calculated as metabolite-to-xylazine in both matrices. In urine, the highest average peak area ratio (3.1) was observed for sulfone xylazine, after hydrolysis. In plasma, the highest average peak area ratio (1.4) was observed for oxo-xylazine. We were unable to absolutely quantitate either metabolite due to lack of available standards. Future studies include investigation on the association of xylazine exposure and plasma concentrations with clinical presentation and patient outcomes.

A Liquid Chromatography‐Tandem Mass Spectrometry Bioanalytical Method Development and Validation for the Determination of Pacritinib in Plasma

ABSTRACTThe primary objective of the current work is to establish and verify an accurate and linear liquid chromatography‐electrospray ionization‐tandem mass spectrometry procedure for quantifying Pacritinib. An effective chromatographic resolution was obtained using the Hypersil Gold (50 × 4.6 mm, 2.1 µm) C18 column. The mobile phase is a mixture of methanol, 0.1% formic acid, and acetonitrile in the proportion of 20:15:65 (%v/v/v). The procedure involved closely monitoring executed ionic transitions of m/z 473.1/376.2 for Pacritinib and 584.26/101.1 for Brigatinib internal standard in multiple reaction monitoring mode. A strong correlation value (r2) of 0.9997 is associated with the linear plot regression line, which can be represented as y = 0.0001x − 0.0008. The relative standard deviation (RSD) results for the matrix effect were 3.63% and 3.57%, respectively, when the quality control (QC) level was low and high, respectively. Over the course of the study, the percentage average recoveries for Pacritinib were found to be 103.27% in high QC, 97.59% in medium QC, and 94.28% in low QC, respectively. The values that were obtained for the QC samples (0.378, 1.058, 7.56, and 11.34 µg/mL) varied from 2.00% to 4.03%. The developed method was useful for evaluating Pacritinib in biological samples for the purposes of QC, forensics, and bioavailability investigations for individuals.