Ultra-high Performance Liquid Chromatography Method Research Articles

Validation of a UPLC-MS/MS Method for Multi-Matrix Biomonitoring of Alternaria Toxins in Humans.

Mycotoxins, natural toxins produced by fungi, contaminate nearly 80% of global food crops. Alternaria mycotoxins, including alternariol (AOH), alternariol monomethylether (AME), and tenuazonic acid (TeA), present a health concern due to their prevalence in various plants and fruits. Exposure to these toxins exceeds the threshold of toxicological concern in some European populations, especially infants and toddlers. Despite this, regulatory standards for Alternaria toxins remain absent. The lack of toxicokinetic parameters, reference levels, and sensitive detection methods complicates risk assessment and highlights the necessity for advanced biomonitoring (HBM) techniques. This study addresses these challenges by developing and validating ultra-high performance liquid chromatography method coupled with tandem mass spectrometry to quantify AOH, AME, TeA, and their conjugates in multiple biological matrices. The validated method demonstrates robust linearity, precision, recovery (94-111%), and sensitivity across urine (LOD < 0.053 ng/mL), capillary blood (LOD < 0.029 ng/mL), and feces (LOD < 0.424 ng/g), with significantly lower LOD for TeA compared to existing methodologies. The application of minimally invasive microsampling techniques for the blood collection enhances the potential for large-scale HBM studies. These advancements represent a step toward comprehensive HBM and exposure risk assessments for Alternaria toxins, facilitating the generation of data for regulatory authorities.

QbD green analytical procedure for the quantification of tolvaptan by utilizing stability indicating UHPLC method

For the first time a new QbD-assisted green stability indicating ultra-high-performance liquid chromatography (UHPLC) method was developed and validated for quantifying Tolvaptan. The method is simple, quick, cost-effective, and stable, and it was used to formulate a quality target product profile (QTPP) with strategically defined critical analytical attributes (CAAs) to meet specific criteria. Chromatographic separation was undertaken using a 10 cm long column of ACE excel super C18 with an interior diameter of 2.1 mm and particle size of 1.7 µm. The analysis was performed under controlled conditions at 25 ℃ with the mobile phase flowing at a rate of 0.2 mL/min and detection occurring at 220 nm. Injected 3 µL of standard by using an isocratic mobile phase system consisting of acetonitrile and water in a 95:5 v/v ratio. The diluents, prepared by mixing acetonitrile with water at a 90:10 volumetric ratio, were utilized. The analyte’s retention time was determined to be 1.63 min. The developed method provided reliable results with accuracy exceeding 99% and a correlation coefficient exceeding 0.999 ranged between 10 and 150 µg/mL across the range for LOQ—150% levels. Notably, during forced degradation testing, Tolvaptan exhibited susceptibility to acidic hydrolysis. The method effectively separated degradation products during stress testing, demonstrating its stability-indicating status. Environmental sustainability assessment of the developed method was conducted through the investigation of various indicators of Complex GAPI, Analytical Eco scale and Analytical GREEness and it was concluded the optimized method aligns with environmentally friendly practices.

Development and validation of stability indicating ultra‐high‐performance liquid chromatographic method for assay and impurities of mexiletine hydrochloride and mexiletine hydrochloride capsules

AbstractMexiletine (MEX) is an antiarrhythmic drug used to treat acute and chronic ventricular arrhythmias. A simple isocratic, rapid, specific, stability‐indicating, and sensitive reverse‐phase ultra‐high‐performance liquid chromatography (UHPLC) method was developed and successfully validated to quantify the assay and impurities of MEX hydrochloride. The new UHPLC method showed the optimum separation of MEX and its seven impurities in 20 min by using sodium acetate buffer (0.14 mM) with 0.3% of glacial acetic acid (pH 4.8) and methanol in the ratio of 40:60 (v/v) as mobile phase. The method used an Acquity HSS T3 (100 × 3.0 mm, 1.8 µm) chromatographic column at a 0.2 mL/min flow rate. The injection volume is 2 µL, and 254 nm is the detection wavelength. Note that, 25°C is the column oven temperature. The method is linear over the concentration range of the quantification limit (0.03%) to 150% (0.3%) of the specification level (0.2%) for the impurities and 50%–150% for the assay method. The quality control lab of bulk drugs and finished dosage form successfully applied the new UHPLC method for quantification of assay and impurities of MEX hydrochloride.

A comparative metabolomic investigation of different sections of Sicilian Citrus x limon (L.) Osbeck, characterization of bioactive metabolites, and evaluation of in vivo toxicity on zebrafish embryo.

Citrus fruits are a diverse and economically important group of fruit crops known for their distinctive flavors and high nutritional value. Their cultivation and consumption contribute significantly to the global agricultural economy and offer a wide range of health benefits. Among the genetic diversity of citrus species, Citrus x limon (L.) Osbeck is particularly relevant due to its chemical composition and potential health benefits. Two cultivars from the Sicily region (southern Italy) were compared for their phenolic content and preliminary antioxidant activity to select the distinctive extract with potential biological activity. A detailed characterization revealed the occurrence of phenolics, coumarins, and flavonoids. The quantification of metabolites contained in the selected extract was performed by an ultrahigh-performance liquid chromatographic method coupled with an ultraviolet detector. Different concentrations were tested in vivo through the fish embryo acute toxicity test, and the 50% lethal dose of 107,833µgmL-1 was calculated. Finally, the effect of the extract on hatching was evaluated, and a dose-dependent relationship with the accelerated hatching rate was reported, suggesting a Femminello Zagara Bianca green peel upregulating effect on the hatching enzymes. PRACTICAL APPLICATION: Citrus fruits and their products continue to be one of the natural food sources with the highest waste output. In this study, we demonstrate how food industry waste, particularly lemon peel, is rich in bioactive compounds with anti-inflammatory and antioxidant properties that may be used in the nutraceuticals industry.

Innovative Reversed-Phase Chromatography Platform Approach for the Fast and Accurate Characterization of Membrane Vesicles' Protein Patterns.

Outer membrane vesicles (OMVs) have been widely explored to develop vaccine candidates for bacterial pathogens due to their ability to combine adjuvant properties with immunogenic activity. OMV expresses a variety of proteins and carbohydrate antigens on their surfaces. For this reason, there is an analytical need to thoroughly characterize the species expressed at their surface: we here present a simple and accurate reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC) method developed according to quality by design principles. This work provides an analytical alternative to the classical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) characterization. The higher selectivity and sensitivity of the RP-UHPLC assay allow for the identification of additional protein species with respect to SDS-PAGE and facilitate its precise relative abundance quantification. According to validation results, the assay showed high accuracy, linearity, precision, repeatability, and a limit of quantification of 1% for less abundant proteins. This performance paves the way for improved production campaign consistency while also being analytically simple (no sample pretreatment required), making it suitable for routine quality control testing. In addition, the applicability of the assay to a wider range of vesicle classes (GMMA) was demonstrated.

Validation, measurement uncertainty estimation and evaluation of UHPLC greenness for simultaneous determination of metoprolol tartrate and hydrochlorothiazide in binary tablet

A novel green ultra high performance liquid chromatography (UHPLC) method was validated and estimated the measurement uncertainty for simultaneous determination of metoprolol tartrate (MET) and hydrochlorothiazide (HCT) in binary tablet. A Zorbax® SB-C18 column with isocratic elution (flow rate 0.9 mL min−1) at 25°C was used. Analytes and HCT degradation product were separated in approximately 1 min using acetonitrile:water:triethylamine (17:83:0.2, v/v) as mobile phase. Analytical curves were linear with (R 2 > 0.99 for MET and HCT) with detection limits of 2.42 and 1.05 µg mL−1 for MET and HCT, respectively. Precision measurements showed RSD% from 0.39 to 1.2% and method accuracy by recovery tests was 100 ± 2%. Measurement uncertainties using Eurachem procedure were 100.1 ± 2.8% and 100.3 ± 2.4% for MET and HCT, respectively. This UHPLC method was accurate, precise, linear and selective, making it suitable for pharmaceutical quality control. It also proved eco-friendly compared to other reported methods.

The association between plasma IgG N-glycosylation and neonatal hypoxic-ischemic encephalopathy: a case-control study.

Hypoxic-ischemic encephalopathy (HIE) is one of severe neonatal brain injuries, resulting from inflammation and the immune response after perinatal hypoxia and ischemia. IgG N-glycosylation plays a crucial role in various inflammatory diseases through mediating the balance between anti-inflammatory and pro-inflammatory responses. This study aimed to explore the effect of IgG N-glycosylation on the development of HIE. This case-control study included 53 HIE patients and 57 control neonates. An ultrahigh-performance liquid chromatography (UPLC) method was used to determine the features of the plasma IgG N-glycans, by which 24 initial glycan peaks (GPs) were quantified. Multivariate logistic regression was used to examine the association between initial glycans and HIE, by which the significant parameters were used to develop a diagnostic model. Though receiver operating characteristic (ROC) curves, area under the curve (AUC) and 95% confidence interval (CI) were calculated to assess the performance of the diagnostic model. There were significant differences in 11 initial glycans between the patient and control groups. The levels of fucosylated and galactosylated glycans were significantly lower in HIE patients than in control individuals, while sialylated glycans were higher in HIE patients (p < 0.05). A prediction model was developed using three initial IgG N-glycans and fetal distress, low birth weight, and globulin. The ROC analysis showed that this model was able to discriminate between HIE patients and healthy individuals [AUC = 0.798, 95% CI: (0.716-0.880)]. IgG N-glycosylation may play a role in the pathogenesis of HIE. Plasma IgG N-glycans are potential noninvasive biomarkers for screening individuals at high risk of HIE.

Analysis of vitamin D and its metabolites in biological samples – Part I: Optimization and comparison of UHPSFC-MS/MS and UHPLC-MS/MS methods

Fat-soluble vitamin D is an essential bioactive compound important for human health. Insufficient vitamin D levels can result not only in bone disease but also in other disorders, such as cancer, metabolic disorders, and diseases related to poor immune function. The current methods commonly used for vitamin D analysis are often applied to determine the levels of the most abundant metabolite in plasma, i.e., 25-OH-D2/D3. These methods do not consider the presence of other hydroxylated and esterified metabolites, including isomers and epimers, which are typically found in low concentrations.In this study, we developed a fast and selective ultra-high performance supercritical fluid chromatography (UHPSFC) method using a 150 mm long 1-amino anthracene (1-AA) column and a mobile phase consisting of carbon dioxide and methanol/isopropanol (1/1, v/v) mixed with 8 % water. After thorough optimization of column temperature and back pressure, the separation of four vitamin D3 esters, vitamin D3 and D2, and eight mono- and di-hydroxylated metabolites, including three groups of isomers, was achieved in 10 min. Two ion sources, atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization optimized within this study, were compared in tandem mass spectrometry (MS/MS) detection. No significant sensitivity differences were observed. Subsequently, the same 1-AA column chemistry was examined in ultra-high performance liquid chromatography (UHPLC) as the stationary phase that could hypothetically bring different selectivity in the separation of vitamin D and its metabolites. However, this hypothesis was rejected, and C18 was used as a stationary phase in the final optimized UHPLC-MS/MS method. Despite detailed optimization, the final 15 min UHPLC method was not able to separate di-hydroxylated isomers of vitamin D3, while it enabled better resolution of esterified forms compared to UHPSFC.Optimized methods provided similar repeatability of retention times and peak areas, with RSD < 2 % and 10 %, respectively. The lowest limits of quantification were in the range of 1.2 – 4.9 ng/mL for UHPSFC-APCI-MS/MS, while for UHPLC-APCI-MS/MS, they were typically in the range of 2.6 – 9.6 ng/mL. Based on the obtained results, the UHPSFC-APCI-MS/MS method was the most promising approach for fast, selective, and sensitive analysis that could be applied in the analysis of biological samples with emphasis on the separation of both hydroxylated and esterified metabolites, including isomeric forms.

Analytical quality by design based on knowledge organization: A case study of developing an ultrahigh-performance liquid chromatography method for the detection of phenolic compounds.

Despite numerous successful cases, there are still some challenges in using analytical quality by design (AQbD) for the development of analytical methods. Knowledge organization helps to enhance the objectivity of risk assessment, reduce the number of preliminary exploratory experiments, identify potential critical method parameters (CMPs) and their scope. In the present study, we aimed to develop a simple, rapid, and robust analytical method for detecting phenolic compounds in Xiaochaihu capsule intermediates utilizing knowledge organization. Knowledge organization and AQbD were combined to obtain the initial analytical conditions through knowledge collection, extraction, reorganization, and analysis. The quantitative relationship between critical method attributes (CMAs) and CMPs was then established by a definitive screening design. The method operable design region was calculated using an exhaustive Monte Carlo approach based on the probability of reaching the standard. Robustness investigation and methodological validation were finally performed. Analytical target profiles, CMAs, potential CMPs, and initial analytical conditions were initially identified, and the optimized ranges of operating parameters were obtained. A UHPLC method was successfully established for the analysis of phenolic compounds in ginger-ginger pinellia percolate, and the method validation outcomes were also satisfactory. The developed method can be a reliable means to detect the phenolic compounds of Xiaochaihu capsule intermediates. Knowledge organization provides a new approach for making better use of prior knowledge, significantly enhancing the efficiency of analytical method development. The approach is versatile and can be similarly applied to the development of other methods.

Ultra‐high‐performance liquid chromatography method development for the quantification of Molnupiravir and its process‐related impurities using Box‐Behnken experimental design

AbstractMolnupiravir, a promising antiviral agent, has gained significant attention for its potential in treating viral infections, particularly in the context of the coronavirus disease 2019 pandemic. This study aimed to develop and validate an ultra‐high‐performance liquid chromatography (UHPLC) method for the quantification of Molnupiravir and its associated impurities (Imp‐I, Imp‐II, Imp‐III, and Imp‐IV). The method involved a systematic investigation of critical method parameters and their optimization using Quality by Design principles. The percentage of organic modifiers, column temperature, and flow rate were systematically optimized using the Box‐Behnken design. The developed method was assessed for specificity, system suitability, accuracy, precision, linearity, and detection limits. The results demonstrate strong specificity, with the analysis remaining accurate even in the presence of impurities. The developed method exhibits excellent precision, with repeatability and intermediate precision showing relative standard deviation values ranging from 0.78% to 1.14% for impurities and 0.82% to 1.91% for Molnupiravir. Furthermore, the method displays exceptional linearity, covering a wide range of concentrations. The linear regression analysis yields high coefficients of determination (r2, 9993–0.9997), confirming the linearity. The developed UHPLC method is well‐suited for the accurate and reliable analysis of Molnupiravir and its impurities, making it a valuable tool for quality control and pharmaceutical research applications.

Development, validation and clinical implementation of a UPLC-MS/MS bioanalytical method for simultaneous quantification of cabotegravir and rilpivirine E-isomer in human plasma

A reversed phase ultra-high performance liquid chromatography method was developed for the simultaneous quantification of cabotegravir (CAB) and the E-isomer of rilpivirine (RPV) in human EDTA plasma, also considering RPV E-isomer instability. Because of the instability of RPV (and CAB) in all light conditions, the (RPV Z-isomer/total RPV)-isomer ratio of RPV was determined for each stock, calibration curve standard, quality control sample and patient sample. [2H3]-CAB and [13C6]-RPV were used as internal standard. Sample preparation involved protein precipitation of plasma using methanol. An HSS T3 column with a guard column (set at 40 °C) was used for analyte separation. The mobile phase components were 65 % 0.1 % formic acid in water (A) and 35 % 0.1 % formic acid in acetonitrile (B) and the flow rate was 0.5 mL/min. Detection was performed with tandem mass spectrometry (MS/MS) in a total runtime of 3.0 min. The assay was validated over the concentration range of 0.0500 – 10.0 mg/L for CAB and 0.00300 – 3.00 mg/L for RPV. The average within-day and between-day accuracies for the assay in plasma were 101 % and 101 % for CAB and 97.6 % and 98.5 % voor RPV, respectively. Within-day and between-day precision in coefficients of variations (CV) were 5.0 %. Extraction recovery was 99 % and 102 % for CAB and its internal standard and 95 % and 97 % for RPV and its internal standard. As our aim was that the (Z-isomer RPV/total RPV) response ratio in patient samples had to be less than 10 % to give reliable results, the (Z-isomer RPV/total RPV) response ratio in stocks, calibration curve standards and internal quality control samples were also taken into account being maximal 0.9 % and 2.3 % respectively. This assay has been successfully used in our Therapeutic Drug Monitoring (TDM) service for people living with HIV on long-acting injectable therapy with CAB/RPV and will also be used in future pharmacokinetic studies.

Rapid and validated UHPLC method for simultaneous determination of sofosbuvir, ledipasvir and paracetamol as commonly repurposed drugs for COVID-19 treatment: application in spiked human plasma

BackgroundSofosbuvir/ledipasvir (SOF/LDV), a combination of antiviral drugs, has been recently repurposed for COVID-19 management, according to Food and Drug Administration approval. Paracetamol (PAR) identified as a first-line antipyretic for COVID-19 symptoms' management. The use of these three drugs together has significantly influenced the management of COVID-19 by providing symptomatic relief via inhibiting viral activity. A validated ultra-high performance liquid chromatographic (UHPLC) method has been introduced for the quantification of these repurposed drugs in COVID-19 treatment. This novel chromatographic method allows the simultaneous detection of SOF, LDV, and PAR in bulk. Additionally, the method has been applied to determine the levels of SOF and LDV in human plasma samples with PAR used as an internal standard.ResultsA new UHPLC method was developed, using a mobile phase with a combination of acetonitrile and 0.1% orthophosphoric acid in a proportion of 42:58 (v/v).Flow rate was set at 0.4 ml/min, and UV detection was adjusted at 254 nm. The concentration of SOF, LDV, and PAR were measured by their corresponding peak areas, and showed linear relationships between concentration and peak area within the ranges of (5–60) µg/ml for SOF, (2–22) µg/ml for LDV, and (1–22) µg/ml for PAR. The presented UHPLC method was used to quantify the amounts of SOF, LDV, and PAR in both bulk samples and human plasma samples being spiked with the mentioned analytes. The elution process was completed within 4 min, with retention times of 3.28 min for SOF, 2.28 min for LDV, and 1.70 min for PAR. The method showed high separation selectivity, with an injection volume of 1µl. The precision, accuracy and repeatability of the method were found to be within acceptable limits.ConclusionThe recently developed method has been successfully validated in accordance with the guidelines set by the International Council for Harmonization (ICH). This validation process ensures that the method is suitable for routine quality control analysis, making it convenient for regular use.

Validation and Greenness assessment of UPLC Method for bioanalysis of Bilastine in mice PK study

Bilastine, is a second-generation antihistamine drug which is used in the treatment of allergic rhinoconjunctivitis and urticaria. It exerts its effect as a selective histamine H1 receptor antagonist. In this study, an ultra-high-performance liquid chromatography (UPLC) method was developed to measure the concentrations of Bilastine in mouse blood, and the method was applied in measuring the pharmacokinetics of the analyte after oral and intravenous administration. The analyte was extracted by solid phase extraction method. A UPLC BEH C18 column (2.1 mm × 100 mm, 1.8 ?m particle size) was used for chromatographic separation by gradient elution using acetonitrile-water (0.1% formic acid) as the mobile phase at a flow rate of 0.4 mL/min. Bilastine was administered to the mice orally at 2 mg/kg and intravenously at 0.05 mg/kg. Blood was collected at various time intervals, and the blood samples were processed after collection and analyzed by UPLC. The intra-day and inter-day accuracy of Bilastine were 91%-103% and 85%-107%, respectively, and the precision (RSD, %) was less than 15% for both intra-day and inter-day measurements. The matrix effect ranged from 95% to 108%, and the recovery was higher than 70%. The half-life of bilastine in plasma was found to be 7.02 ± 5.21h. Bilastine has a good linear relationship in the range of 10-500 ng/mL, and the lower limit of quantification was 10 ng/mL. The precision, accuracy, extraction recovery, matrix effect, and stability meet the requirements of the guiding principles. A robust and reliable UPLC method was fully optimized and developed to detect the blood concentration of bilastine in mice and the samples were analyzed by Empower software.

Stable isotope labeling differential glycans discovery in the serum of acute myocardial infarction by ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high resolution mass spectrometry

Acute myocardial infarction (AMI) poses a grave threat to human life. However, most clinical biomarkers have limitations of low sensitivity and specificity. Therefore, screening novel glycan biomarkers with high sensitivity and specificity is crucial for the prevention and treatment of AMI. The novel method of ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) with d0/d5-BOTC probe labeling for relative quantification of glycans based on Pronase E digestion was established to screen novel glycan biomarkers in the serum of 34 AMI patients relative to healthy volunteers. The monosaccharide model D-glucosamine was used to investigate the effectiveness of the derivatization; the limit of detection (S/N = 3) was 10 amol. The accuracy was verified based on the consistency of different theoretical molar ratios (d0/d5 = 1:2, 2:1) and intensity ratios following digestion of glycoprotein ribonuclease B. Expressions of H4N4F3SA, H4N6F2, H4N6SA, H4N6F3 and H5N4FSA in the serum were significantly different (p < 0.0005) between AMI patients and healthy volunteers. The area under the receiver operating characteristic curve (AUC) for H4N6SA, H5N4FSA, and H4N6F2 was greater than 0.9039. Based on the proposed method, H4N6SA, H5N4FSA, and H4N6F2 in human serum showed high accuracy and specificity and may serve as potential glycan biomarkers, crucial for the diagnosis and treatment monitoring of AMI.

Development of validated HPTLC and UHPLC methods for quantification of Withanolide in extract and formulation

High performance thin layer chromatography (HPTLC) and Ultra-High-Performance Liquid Chromatography (UHPLC) techniques were developed and validated to quantify Withanolide in extract and formulation. On Al-backed silica gel 60 F254 TLC plates (10 cm × 10 cm, layer thickness 0.2 mm), which had been prewashed with methanol, HPTLC separation was carried out. Dichloromethane: Methanol: Toluene: Acetone in various ratios produced good separation in mobile phase (5:1:1:0.5 v/v). Camag TLC scanner densitometric scanning at 365 nm determined and quantified. This approach produced compact Withanolide spots at Rf 0.48. ICH guidelines verified HPTLC's precision, reproducibility, and accuracy. Withanolide linearity was 500-3000 ng/spot with R2= 0.9994. LOD &amp; LOQ were found to be 9.48 &amp; 28.73 ng respectively. For UHPLC, Cosmosil C18 was used with acetonitrile: water (0.2 % OPA) (70:30, v/v) mobile phase. Flow rate was 1.5 mL/min. Under optimal chromatographic conditions, Withanolide was retained for 5.9 min and detected at 254 nm. ICH guidelines verified UHPLC's precision, repeatability, and accuracy. Withanolide linearity was 10-60 μg/mL with R2= 0.9994. LOD along with LOQ were 0.411 and 1.245 μg. HPTLC and UHPLC procedures utilized for regular quality control and quick screening of active components from plant extracts.

Simultaneous quantification of (E) and (Z) isomers of rilpivirine and four degradation products in bulk and tablets by reversed-phase ultra-high-performance liquid chromatography and confirmation of all by molecular weight.

The (E)-isomer of rilpivirine is an approved antiretroviral drug used to treat human immunodeficiency virus. A simple, fast, accurate, and precise analytical method is required to confirm the quality, purity, efficacy, and safety of drug substances and drug products containing rilpivirine. This research article offers a comprehensive ultra-high performance liquid chromatography method for the simultaneous separation and quantification of (E) and (Z) isomers of rilpivirine, including two amide impurities, one nitrile impurity, and one dimer impurity, in both bulk and tablet forms. After complete validation, the proposed reversed-phase ultra-high-performance liquid chromatography method has proven to be simple, fast, linear, accurate, and precise, with a lower limit of quantification and detection of 0.05 and 0.03μg/ml, respectively, for all six analytes. Separation was achieved on a Waters Acquity ethylene bridged hybrid Shield RP18 (150×2.1mm, 1.7μm) column maintained at 35.0°C using a gradient elution of acetonitrile and 0.05% formic acid in 10mM ammonium formate at a flow rate of 0.30ml/min. A systematic forced degradation study on the undissolved rilpivirine revealed the formation of acid-base hydrolyzed amide impurities (Impurity-A and Impurity-B), oxidative nitrile impurities (Impurity-C), and Z-isomer and dimer impurities of rilpivirine (Impurity-D and Impurity-E) due to alkaline hydrolysis and photodegradation. The proposed method is primarily appropriate for applications requiring the precise determination of desired and undesired isomers of rilpivirine and its degradation products, such as those involving the safety, efficacy, and quality roles of rilpivirine in bulk and tablet forms. Additionally, the proposed ultra-high-performance liquid chromatography method in combination with a mass spectrometer and photo-diode array detector is helpful for the confirmation and correct identification of all analytes.

Analysis of a Broad Range of Carbonyl Metabolites in Exhaled Breath by UHPLC-MS.

Analysis of volatile organic compounds (VOCs) in exhaled breath (EB) has shown great potential for disease detection including lung cancer, infectious respiratory diseases, and chronic obstructive pulmonary disease. Although many breath sample collection and analytical methods have been developed for breath analysis, analysis of metabolic VOCs in exhaled breath is still a challenge for clinical application. Many carbonyl compounds in exhaled breath are related to the metabolic processes of diseases. This work reports a method of ultrahigh-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-MS) for the analysis of a broad range of carbonyl metabolites in exhaled breath. Carbonyl compounds in the exhaled breath were captured by a fabricated silicon microreactor with a micropillar array coated with 2-(aminooxy)ethyl-N,N,N-trimethylammonium (ATM) triflate. A total of six subgroups consisting of saturated aldehydes and ketones, hydroxy-aldehydes, and hydroxy-ketones, unsaturated 2-alkenals, and 4-hydroxy-2-alkenals were identified in the exhaled breath. The combination of a silicon microreactor for the selective capture of carbonyl compounds with UHPLC-MS analysis may provide a quantitative method for the analysis of carbonyls to identify disease markers in exhaled breath.

99mTc]Technetium-Labeled Niosomes: Radiolabeling, Quality Control, and In Vitro Evaluation.

The aim of this research was to develop technetium-99m ([99mTc]Tc)-radiolabeled niosomes and evaluate the cancer cell incorporation capacity of radiolabeled niosomes. For this purpose, niosome formulations were developed by film hydration method, and prepared niosomes were characterized to particle size, polydispersity index (PdI), ζ-potential value, and image profile. Then, niosomes were radiolabeled with [99mTc]Tc using stannous salts (chloride) as a reducing agent. The radiochemical purity (RP) and stability in different mediums of the niosomes were assessed by ascending radioactive thin-layer chromatography (RTLC) and radioactive ultrahigh-performance liquid chromatography (R-UPLC) methods. Also, the partition coefficient value of radiolabeled niosomes was determined. The cell incorporation of [99mTc]Tc-labeled niosome formulations, as well as reduced/hydrolyzed (R/H)-[99mTc]NaTcO4 in the HT-29 (human colorectal adenocarcinoma) cells, was then assessed. According to the obtained results, the spherical niosomes had a particle size of 130.5 ± 1.364 nm, a PdI value of 0.250 ± 0.023, and a negative charge of -35.4 ± 1.06 mV. The niosome formulations were effectively radiolabeled with [99mTc]Tc using 500 μg mL-1 stannous chloride for 15 min, and RP was found to be over 95%. [99mTc]Tc-niosomes showed good in vitro stability in every system for up to 6 h. The log P value of radiolabeled niosomes was found as -0.66 ± 0.02. Compared to R/H-[99mTc]NaTcO4 (34.18 ± 1.56%), the incorporation percentages of [99mTc]Tc-niosomes (88.45 ± 2.54%) were shown to be higher in cancer cells. In conclusion, the newly developed [99mTc]Tc-niosomes showed good prototype for potential use in nuclear medicine imaging in the near future. However, further investigations, such as drug encapsulation and biodistribution studies, should be performed, and our studies are continuing.

STABILITY INDICATING ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF RUXOLITINIB IN BULK AND PHARMACEUTICAL DOSAGE FORM USING UPLC

Objective: To establish a simple and selective UPLC (Ultra-high Performance Liquid Chromatography) method for the determination of Ruxolitinib in tablet and bulk dosage forms. Methods: Chromatographic separation was achieved on a C8 column with the dimensions of (250×4.6m ID) 5 µm length; the mobile phase composition was a mixture of pH 6.2 with glacial acetic acid: Methanol: acetonitrile in the ratio of 40:30:30 was passed through the designated column with a flow rate of 1 ml per minute and the UV (Ultra Violet) detection was witnessed at 254 nm. Results: Linearity was observed in the range 50-150 µg/ml for Ruxolitinib (r² =0.9998) for drugs estimated by the proposed methods was in good agreement with the label claim. The % recovery of the drug was found to be between 98 and 102%. The drug was used for determining stability studies for acid, alkali, thermal, photolytic, and peroxide degradation. Conclusion: The method for determining Ruxolitinib was discovered to be simple, precise, accurate, and high resolution, with a shorter retention time, making it more acceptable and cost-effective for routine analysis.

Green Solid-Phase (Micro)Extraction of Andrographolides’ from Human Plasma Samples Followed by UHPLC-DAD-QqQ-MS/MS Analysis

Since inflammatory bowel disease is a progressive disease, optimal treatment early in the disease course is paramount. Still, the use of conventional drugs is accompanied by side effects. The herbal therapies for gastrointestinal disorders are traditionally widespread in Asia, while in Western countries have been adopted in recent years. Andrographis paniculata is a herbaceous plant used in anti-inflammatory treatment. To improve its clinical application, an analytical method with the potential to define a relationship between dose and plasma concentration of active ingredients is prerequisite. Considering the increasing demand for eco-friendly bioanalytical methods, our research aimed to implement new sample preparation technique microextraction by packed sorbent for extraction of marker compounds of A. paniculata from plasma. This green procedure using polymeric sorbent based on the poly (styrene-divinylbenzene) allowed miniaturization of sample volume (100 µL), decrease in consumption of solvents, and high recovery (92–98%). A highly selective and sensitive reversed-phase ultrahigh-performance liquid chromatography method has been developed and validated. The newest greenness assessment software AGREE calculator was used for evaluation of the method’s environmental performance. It suggests that our method conforms to the eco-friendly conditions (score 0.8). The sample preparation was identified as weak point of the bioanalytical methods which highlights the importance of microextraction.