UPLC System Research Articles

Rapid Identification of Chemical Constituents in New Compound Aloe Capsule Based on UPLC-Q-Orbitrap-MS.

The chemical composition of the new compound aloe capsule (NCAC) has not been fully elucidated to date, which poses challenges for pharmacology, scientific, and rational drug use. In this study, a rapid and sensitive method was established to comprehensively study the constituents in the NCAC. The contents of the NCAC were analyzed by UPLC-Q-Orbitrap-MS and data postprocessing technology. Firstly, the extract was separated by UPLC system, and the fragment information was obtained under positive and negative ions and then compared with the characteristic fragments described in the literature, so as to achieve the purpose of rapid identification of the compound of NCAC. A total of 56 compounds including 21 chromones, 6 pyranones, 7 anthraquinones, 7 anthrone, 5 alkaloids, 4 amino acids, and 6 other components were detected in NCAC by comparing the retention time and mass spectrometry information and retrieving the reference literature. The UPLC-Q-Orbitrap-MS method was developed and utilized successfully to identify the major constituents in NCAC and would be helpful for improving the quality control standard of NCAC.

Caffeine Content Prediction in Coffee Beans Using Hyperspectral Reflectance and Machine Learning

The application of hyperspectral data in machine learning models can contribute to the rapid and accurate determination of caffeine content in coffee beans. This study aimed to identify the machine learning algorithm with the best performance for predicting caffeine content and to find input data for these models that can improve the accuracy of these algorithms. The coffee beans were harvested one year after the seedlings were planted. The fresh beans were taken to the spectroscopy laboratory (Laspec) at the Federal University of Mato Grosso do Sul, Chapadão do Sul campus, for spectral evaluation using a spectroradiometer. For the analysis, the dried coffee beans were ground and sieved for the quantification of caffeine, which was carried out using a liquid chromatograph on the Waters Acquity 1100 series UPLC system, with an automatic sample injector. The spectral data of the beans, as well as the spectral data of the roasted and ground coffee, were analyzed using machine learning (ML) algorithms to predict caffeine content. Four databases were used as input: the spectral information of the bean (CG), the spectral information of the bean with additional clone information (CG+C), the spectral information of the bean after roasting and grinding (CGRG) and the spectral information of the bean after roasting and grinding with additional clone information (CGRG+C). The caffeine content was used as an output to be predicted. Each database was subjected to different machine learning models: artificial neural networks (ANNs), decision tree (DT), linear regression (LR), M5P, and random forest (RF) algorithms. Pearson’s correlation coefficient, mean absolute error, and root mean square error were tested as model accuracy metrics. The support vector machine algorithm showed the best accuracy in predicting caffeine content when using hyperspectral data from roasted and ground coffee beans. This performance was significantly improved when clone information was included, allowing for an even more accurate analysis.

Chromatographic Comparison of Commercially Available Columns for Liquid Chromatography in Polar Pesticide Detection and Quantification Using a Score-Based Methodology.

The detection and quantification of polar pesticides in liquid chromatography coupled with mass spectrometry present significant analytical challenges. This study compares the performance of three LC columns (Hypercarb™, Raptor Polar X™, and Anionic Polar Pesticide™) in separating and quantifying eleven polar pesticides in chicken eggs using a score-based methodology. Analytes include glyphosate, its metabolites, and other high-polarity pesticides like Ethephon, Glufosinate, and Fosetyl aluminum, included in the EU's official control plan. Polar pesticides, characterized by high polarity and hydrophilicity, lead to analytical issues such as poor retention and unconventional peak shapes with traditional reversed-phase methods. Their weak interaction with hydrophobic stationary phases complicates separation, necessitating specific stationary phases to enhance retention and selectivity. This study evaluates these columns' efficacy in complex matrices like chicken eggs and other food samples. Chromatographic separation was performed using a UPLC system coupled with a Q-TOF mass spectrometer; extraction and purification involved freeze-out, centrifugation, and filtration steps. The study highlights the critical role of column selection in achieving accurate and reliable separation and quantification of highly polar analytes in matrices of animal origin, offering in the meantime an easy-to-apply methodology of selection for the right determination of the best chromatographic column for different purposes.

Abstract P209: The novel angiotensin Alamandine-(1-5) is increased in plasma of pediatric patients with primary nephrotic syndrome

Background and Aim: The Renin Angiotensin System (RAS) has an important role in the pathophysiology of primary nephrotic syndrome (NS). It was previously found that urinary levels of angiotensin converting enzyme 2 (ACE2) are reduced in patients with NS in comparison to health controls. The aim of this study was to measure the novel angiotensin peptide, Alamandine-(1-5), in plasma samples of patients with primary NS and to compare with sex and age-matched controls. Methods: Blood samples of pediatric patients with primary NS (n=11) and of sex and age-matched health controls (n=11) were collected in sterile tubes with 7.5% (w/v) EDTA. After plasma separation and purification by solid phase extraction, the circulating levels of Alamandine and of Alamandine-(1-5) were measured by Mass Spectrometry using multiple reaction monitoring on an ACQUITY I-Class UPLC system coupled to electrospray ionization (ESI) tandem mass spectrometry (LC-ESI-MS/MS Xevo TQ-S). Data were compared between patients and controls and associated with laboratory findings. Continuous variables were presented as mean ± standard error of the mean. Results: A total of 11 patients (7 males) with NS with mean age of 10.65±3.66 were compared to 11 controls (6 males) and with 10.86±4.35 years. In terms of disease response, 5 patients were in partial remission, 5 patients in total remission and only 1 patient was in disease relapse. All patients have normal renal function. Plasma levels of Alamandine-(1-5) were significantly higher in patients when compared to controls (28.03±7.54 vs. 10.68±3.02 pg.mL-1 in controls). Despite a mild decrease in patients, plasma levels of Alamandine did not differ between groups (19.54±6.33 vs 26.09±6.12 pg.mL-1 in controls). No correlations were found between RAS molecules and proteinuria and with the medications in use. However, Alamandine-(1-5) negatively correlated with plasma levels of albumin (r= -0.695; p=0.038). In addition, Alamandine negatively correlated with total cholesterol (r=-0.700; p = 0.036), LDL (r=-0.800; p=0.004), and HDL (r = -0.762; p = 0.038). Conclusion: The novel RAS peptide Alamandine-(1-5) is significantly increased in primary NS and inversely correlated with plasma albumin levels. These findings suggest a potential role of Alamandine-(1-5) in NS

Development and Validation of a Novel Isotope Dilution-Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Serum C-Peptide.

Mass spectrometry (MS) methods exhibit higher accuracy and comparability in measuring serum C-peptide concentrations than immunoassays. We developed and validated a novel isotope dilution-ultraperformance liquid chromatography-tandem MS (ID-UPLC-MS/MS) assay to measure serum C-peptide concentrations. Sample pretreatment involved solid-phase extraction, ion-exchange solid-phase extraction, and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (Cayman Chemical, Ann Arbor, Michigan, USA). We used an ExionLC UPLC system (Sciex, Framingham, MA, USA) and a Sciex Triple Quad 6500+ MS/MS system (Sciex) for electrospray ionization in positive-ion mode with multiple charge states of [M+3H]3+ and multiple reaction monitoring transitions. The total run time was 50 mins, and the flow rate was 0.20 mL/min. We evaluated the precision, trueness, linearity, lower limit of quantitation (LLOQ), carryover, and matrix effects. Method comparison with electrochemiluminescence immunoassay (ECLIA) was performed in 138 clinical specimens. The intra- and inter-run precision coefficients of variation were <5% and the bias values for trueness were <4%, which were all acceptable. The verified linear interval was 0.050-15 ng/mL, and the LLOQ was 0.050 ng/mL. No significant carryover or matrix effects were observed. The correlation between this ID-UPLC-MS/MS method and ECLIA was good (R=0.995, slope=1.564); however, the ECLIA showed a positive bias (51.8%). The developed ID-UPLC-MS/MS assay shows acceptable performance in measuring serum C-peptide concentrations. This will be useful in situations requiring accurate measurement of serum C-peptide in clinical laboratories.

Differential proteomic expression in non-functional pituitary neuroendocrine tumours and pituitary glands.

Pituitary neuroendocrine tumours (PitNETs) are common accounting for 10 to 25 % of all intracranial tumours. This project describes the feasibility of developing a novel membrane-based biomarker that could be used for fluorescent guided surgery. The aim was to catalogue the differential expression of membrane proteins between non-functional PitNETs and pituitary glands. Ten pituitary gland tissue specimens were obtained from the National Institute of Health (NIH) NeuroBio-Bank and twenty non-functional PitNETs were obtained from the Northwestern University Nervous System Tumour Bank. Mass spectrometry analysis using an Orbitrap Fusion Lumos Tribrid Mass Spectrometer linked to a Dionex Ultimate 3000 UPLC system was undertaken. Data Dependent Acquisition Mass Spectrometry and Data Independent Acquisition Mass Spectrometry was then completed. Pathway enrichment analysis was performed using clusterProfiler v4.6.0. Functional enrichment analysis was conducted using Gene Ontology terms and Reactome pathways. Differential expression analysis between the two groups revealed a total of 2110 significant differently expressed proteins (DEPs), with 1387 of these also having a Log2 fold change either greater than 1, or less than -1. Of the 2110 DEPs, 925 were upregulated in tumours compared to control, while 1185 were downregulated. We have demonstrated a proteomic comparison between non-functional PitNETs and normal pituitary glands. These results demonstrate differences consistent with contemporary literature but shows that NOTCH3 and PTPRJ are up-regulated in non-functional PitNETs compared to pituitary glands.

Modified atmosphere affects glucosinolate metabolism during postharvest storage of broccoli

Broccoli is a vegetable with a growing consumer demand worldwide due to its important nutritional properties, including high amounts of glucosinolates. However, this vegetable has a short postharvest life and quickly loses its organoleptic and nutritional quality. Modified atmosphere is one of the numerous methodologies used to delay deterioration during postharvest storage of broccoli heads. In this study, the effect of a modified atmosphere during postharvest storage of broccoli on glucosinolate metabolism was evaluated. Five glucosinolates were identified by using a UPLC system coupled to a mass spectrometer. We detected one aliphatic glucosinolate and four indolic glucosinolates, and their concentration was found to decrease during storage. The decrease in content was less marked, and in some cases, an increased level was observed in the treated samples. Moreover, the treatment made it possible to maintain higher expression (analyzed by real-time quantitative PCR) of genes linked to glucosinolate biosynthesis. We also detected an increased expression of some genes related to indolic glucosinolate biosynthesis. Overall, storage of broccoli in modified atmospheres allowed for maintaining better visual quality and higher levels of glucosinolates.

Tick-borne encephalitis virus transmitted singly and in duo with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum bacteria by ticks as pathogens modifying lipid metabolism in human blood

BackgroundTicks are vectors of various pathogens, including tick-borne encephalitis virus causing TBE and bacteria such as Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum causing e.g. viral-bacterial co-infections (TBE + LB/HGA), which pose diagnostic and therapeutic problems. Since these infections are usually accompanied by inflammation and oxidative stress causing metabolic modifications, including phospholipids, the aim of the study was to assess the level of polyunsaturated fatty acids and their metabolism (ROS- and enzyme-dependent) products in the blood plasma of patients with TBE and TBE + LB/HGA before and after pharmacotherapy.MethodsThe total antioxidant status was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid. The phospholipid and free fatty acids were analysed by gas chromatography. Lipid peroxidation was estimated by measuring small molecular weight reactive aldehyde, malondialdehyde and neuroprostanes. The reactive aldehyde was determined using gas chromatography coupled with mass spectrometry. The activity of enzymes was examined spectrophotometrically. An analysis of endocannabinoids and eicosanoids was performed using a Shimadzu UPLC system coupled with an electrospray ionization source to a Shimadzu 8060 Triple Quadrupole system. Receptor expression was measured using an enzyme-linked immunosorbent assay (ELISA).ResultsThe reduced antioxidant status as a result of infection was accompanied by a decrease in the level of phospholipid arachidonic acid (AA) and docosahexaenoic acid (DHA) in TBE, an increase in DHA in co-infection and in free DHA in TBE with an increase in the level of lipid peroxidation products. The enhanced activity of enzymes metabolizing phospholipids and free PUFAs increased the level of endocannabinoids and eicosanoids, while decreased 15-PGJ2 and PGE2 was accompanied by activation of granulocyte receptors before pharmacotherapy and only tending to normalize after treatment.ConclusionSince classical pharmacotherapy does not prevent disorders of phospholipid metabolism, the need to support treatment with antioxidants may be suggested.

Comprehensive phytochemical profiles and antioxidant activity of Korean local cultivars of red chili pepper (Capsicum annuum L.).

Red chili pepper (Capsicum annuum L.), which belongs to the Solanaceae family, contains a variety of phytochemicals with health-promoting properties including capsaicinoids, phenolics and fatty acids. Red chili pepper is one of the most consumed vegetables in Korea and occupies the largest cultivated area among spices. In this study, the ethanolic extracts from two Korean local cultivars, namely Subicho and Eumseong, were analyzed using a hybrid trapped ion mobility Q-TOF mass spectrometer equipped with a UPLC system, and their phytochemical profiles were then compared with those of a common phytophthora disease-resistant cultivar called Dokbulwang, which is extensively used for red chili pepper powder in public spaces across Korea. Utilizing high-resolution ion-mobility Q-TOF MS analysis, 458 and 192 compounds were identified from the three different red chili peppers in positive and negative ion modes, respectively, by matching with a reference spectral library. Principal component analysis revealed clear distinctions among the three cultivars, allowing us to identify key phytochemical components responsible for discriminating the local cultivars from the public cultivar. Furthermore, the assessment of total flavonoid, phenolic, and antioxidant activity in the red pepper extracts, highlighted their diverse molecular and chemical profiles. Despite the higher total flavonoid and phenolic content values observed in the public cultivar, the radical scavenging rate was higher in the local cultivars, particularly in Subicho. This suggest the presence of stronger antioxidant compounds in the local cultivar, indicating their potential health benefits due to their rich content of bioactive compounds. Notably, the local cultivars exhibited significantly higher proportions of organic compounds (more than four times) and terpenoids (more than two times) compared to the public cultivar. Specifically, higher levels of five major capsaicinoid compounds were found in the local cultivars when compared to the public cultivar. The observed disparities in phytochemical composition and antioxidant activities indicate the molecular diversity present among these cultivars. Further exploration of the bioactive compounds in these local cultivars could prove invaluable for the development of native crops, potentially leading to the discovery of novel sources of bioactive molecules for various applications in health and agriculture.

The development and validation of a stability indicating Rp-UPLC method for the simultaneous estimation of clarithromycin, amoxicillin, and vonoprazan in a physical mixture

A novel technique was developed for simultaneous quantification of clarithromycin (CLA) amoxicillin (AMO), and vonoprazan (VON) in a mixture using the reverse phase ultra-performance liquid chromatography (RP-UPLC) technique and validated as per International Council for Harmonization (ICH) guidelines as there was no literature published for its estimation by UPLC. The method was developed using an acquity UPLC system from waters corporation with Hibar Bis phosphonate C18 column (100 × 2.1 mm, 2 μm) at 35°C and tunable ultra-violet detector (TUV) with detection wavelength at 210 nm, has a run time of below 3 minutes. The mobile phase proportion of 60:40 of 0.1 N monobasic potassium phosphate buffer (pH 3.8) and acetonitrile at a flow velocity of 0.2 ml/ minute was utilized. Linearity was observed for CLA, AMO, and VON between the concentration ranges of 25–150, 25–150, and 1–6 μg/ml, respectively, and R2 was 0.999 for CLA, AMO, and VON. Accuracy and precision were within 2% of the coefficient of variation (RSD) for all drugs. The observed mean percentage recoveries for the CLA, AMO, and VON were determined to be 99.74%, 99.07%, and 99.8%, respectively. The stability of the approach was assessed using degradation studies by exposing it to acid, alkali, oxidizing agent, heat, Ultra Violet (UV) light, and water as per ICH guidelines.

Determination of cytotoxic, antioxidant activities and LC/MS-MS profiles of three endemic Verbascum L. species

The Verbascum genus includes many species used in folk medicine or the treatment of various diseases. In this study, the cytotoxic, antioxidant activity, and LC/MS-MS profiles of three Verbascum species, which are endemic in Eskişehir and its surroundings, were investigated. The cytotoxic effects of methanol extract of V.detersile, V. eskisehirensis, and V.gypsicola species on the cervical (HeLa) and ovarian cancer (SKOV-3) cells were investigated using a colorimetric assay. The results indicated that cytotoxic effect was not observed after treatment of SKOV-3 cells with Verbascum. On the other hand, the cytotoxic activity of V. detersile was found to be 0.1910 mg/dL and 1.057 mg/dL for HeLa cells after 24 or 48 hours incubation with V.detersile, respectively. The antioxidant activity was determined as 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging activity, trolox equivalent antioxidant capacity (TEAC assay), and also the total phenolic content of the samples was found. Total phenols were estimated as Gallic acid equivalents (GAE), expressed as mg Gallic acid/in 1g extract previously described by Singletton. LC/MS profiles separation and detection of phytochemicals of the extract were performed on a Shimadzu UPLC system consisting of a vacuum degasser, an autosampler (SIL20A Shimadzu Autosampler), a binary pump (LC20AD Shimadzu), an oven (CTO20A Shimadzu Column Oven) and DAD dedector (SPD M20A Shimadzu DAD Detector). In terms of antioxidant activity, V. gypsicola was found to have the least antioxidant activity among the three extracts, which is also correlated with the total amount of phenolic content in its content. In this way, it differs from other species. V. detersile exhibits a different chemical profile from the other two species with the iridoid catalpol derivatives it contains. Apigenin pentoside is the only flavonoid molecule detected in V. detersille.

Exometabolites of streptomycetes isolated from the Odesa bay exhibit a toxic effect against human cancer cell lines

The aim of the work was to determine the cytotoxic activity of marine actinobacteria exometabolites on the human larynx adenocarcinoma Hep-2 and rhabdomyosarcoma RD cell lines. Exometabolites of 6 strains of Streptomyces sp. were extracted with ethyl acetate and their composition was determined with UltiMate™ 3000 BioRS UPLC System coupled to mass spectrometer. Cytotoxic activity of exometabolites against cancer cells was determined after 24 hours of incubation by microscopic examination of cell morphological changes and the degree of cell monolayer degeneration. Exometabolites from Streptomyces sp. Myt7b strain showed the highest cytotoxic activity and at concentration of 500.0 μg/ml caused up to 90.0% death of RD and Hep-2 cells. The presence of 53 identified compounds with potential cytotoxic activity, including enterocin, 6-prenyltryptophol, medelamine A and turbinaric acid was detected in Streptomyces sp. Myt7b exometabolome. It is concluded that this strain is a promising producer of compounds with anticancer activity. Keywords: cytotoxic activity, exometabolites, marine streptomycetes, RD and Hep-2 cancer cells

Determination of quinclorac and quinclorac methyl ester in honey by online SPE-UPLC-MS/MS

A method employing online solid phase extraction (SPE) coupled to UPLC-MS/MS was developed for the determination of residues of the acid herbicide quinclorac plus its transformation product, quinclorac methyl ester, in honey. The analytical method involved dissolving the honey in a mixture of methanol:water followed by direct injection into a two-dimensional UPLC system which is used to perform an automated SPE cleanup on a reusable phenyl cartridge prior to the target analytes being transferred onto an analytical UPLC column for subsequent chromatographic separation followed by MS/MS detection. The limits of quantitation for quinclorac and quinclorac methyl ester in honey were both set at 0.5 µg kg−1 and the method detection limit was estimated to be 0.012 µg kg−1 for each compound. The working analytical range (0.5–100 µg kg−1) was validated by analysing a series of spiked replicate honey samples. The method was applied to the analysis of various honeys obtained from numerous different commercial sources. Quinclorac was detected in 9 out of 30 samples at concentrations ranging from 0.6 to 31.5 µg kg−1. Quinclorac methyl ester, which is estimated to be significantly more toxic than the parent herbicide itself, was not detected in any honey sample.

B-206 The Determination of Vanillylmandelic Acid (VMA), Homovanillic Acid (HVA) and 5-Hydroxyindolacetic Acid (5-HIAA) by LC-Ms/MS Assay in Urine Samples for Diagnosis of Neuroendocrynes Tumors

Abstract Background The catecholamines dopamine, norepinephrine and epinephrine, as well the neurotransmitter serotonin are biogenic amines that were formed by enzymatic descarboxylation of aminoacids in the body. They realize an important function in central nervous system. Vanillylmandelic acid (VMA) and Homovanillic Acid (HVA) are degradation products of the catecholamines, and 5-Hydroxyindolacetic Acid (5-HIAA) is a serotonine degradation product. They are important to the diagnosis of neuroendocrynes tumors, that produce catecholamines and/or serotonine in excess, resulting in elevated levels of VMA, HVA, and/or 5-HIAA in 24 h collection urine. Objective This study aimed to validate a simple LC-MS/MS method for quantification of VMA, HVA and 5-HIAA in isolated and 24 h urine samples for the diagnosis of neuroendocrines tumors. Methods Using a deep-well microtiter plate, 20 µL of isolated or 24 h urine was diluted in 540 µL of 0.05% of formic acid solution. Then, 20 µL of an internal standard solution containing stable isotopes of the analytes were added (VMA-D3, HVA-13C6,18O and 5-HIAA-13C6). The plate was sealed, homogenized for 30 s at 2500 rpm, and centrifuged for 15 min at 4000 rpm and ambient temperature. Then, 5.0 µL of the supernatant was injected into the LC-MS/MS system. Chromatographic separation was performed on the Waters ACQUITY UPLC system equipped with Kinetex XB-C18 1.7 µm X 50 mm X 2.1 mm column (Phenomenex), and methanol and formic acid 0.2% solution as mobile phase in 3.5 min of gradient mode separation. The detection was performed on a Waters XEVO TQ-S Micro mass spectrometer with electrospray ionization (ESI+). The current method was validated for carryover, linearity, precision, recovery, and limit of quantification, and it was evaluated by comparing the analysis in the developed method and a commercial kit by HCPL-ECD, of 15 real samples from volunteers in the laboratory. Results The method was successfully validated for all the parameters. The linearity was between 0.5–100.0 mg/L. The precision was less than 6.3%, the recovery was between 85.4%–103.4% and the quantification limit was 0.5 mg/L. The comparison with the reference method showed good coefficient correlations (R2 &amp;gt; 0.92) for the three analytes. Conclusion We validate a simple LC-MS/MS method to quantify three metabolites to help the diagnosis of neuroendocrynes tumors. The method represents a simplification of the extraction compared with the commercial kit employing HPLC-ECD, and an important reduction of the per sample production cost, without loss of quality.

B-183 The Impact of 3-EPI-25-Hydroxyvitamin D on 25-Hydroxyvitamin D Measurement by Liquid Chromatography/Mass Spectrometry

Abstract Background 25-hydroxyvitamin D is the recommended target to evaluate vitamin D status. The accurate measurement of 25-hydroxyvitamin D by liquid chromatography/mass spectrometry (LC/MS/MS) is impacted by the presence of the biologically inactive epimer of 25-hydroxyvitamin D2 and D3, 3-EPI-25-hydroxyvitamin D2 and D3. The epimers are isomers and share the same mass and fragment transitions as 25-hydroxyvitamin D2 and D3. For an accurate measurement the epimer should be chromatographically separated from 25-hydroxyvitamin D. Herein we assess the significance of 3-EPI-25-hydroxyvitamin D in our patient population. Methods A LC/MS/MS method was developed to chromatographically separate 25-hydroxyvitamin D2 and D3 from 3-EPI-25-hydroxyvitamin D2 and D3 on a Waters Xevo TQ-S Micro with an Acquity UPLC System using a 2.6 μm Pentafluorophenyl (PFP) column (Phenomenex). There were 231 residual serum samples analyzed. Results The cohort tested consisted of 16 infant samples (&amp;lt;1 years), 62 pediatric samples (1–17 years), adult samples (≥18 years), and 20 pregnant samples. 3-EPI-25-hydroxyvitamin D2 was not detected in any sample analyzed (&amp;lt;1 ng/mL). The prevalence and concentration of 3-EPI-25-hydroxyvitamin D3 was variable (Fig. 1). Conclusion The specificity of 25-hydroxyvitamin D measurement by LC/MS/MS is not affected by 3-EPI-25-hydroxyvitamin D2 as it is not present in our patient population. However, 3-EPI-25-hydroxyvitamin D3 is a significant interference. The presence of 3-EPI-25-hydroxyvitamin D3 is variable with the highest prevalence and concentration in infants. The prevalence and concentration decrease with age and is minimal in pregnancy. For infant and pediatric samples, an assay that chromatographically separates 3-EPI-25-hydroxyvitamin D3 from 25-hydroxyvitamin D3 is needed. For adult samples, it may be appropriate not to separate 25-hydroxyvitamin D3 from the epimer. For this patient population a balance between accuracy and throughput must be established.

A-103 Stability Study of the Biogenic Amines 5-Hydroxyindoleacetic Acid (5-HIAA), Vanillylmandelic Acid (VMA), and Homovanillic Acid (HVA) in Acidified Vs Non-acidified Human Urine Samples

Abstract Background Carcinoid tumors and neuroblastomas are disorders in which some neurotransmitters are produced. The first one is characterized by the overproduction of serotonin, which the main metabolite is 5-Hydroxyindoleacetic acid (5-HIAA). In neuroblastomas, approximately 90% of the tumors produce catecholamines, wherein Vanillylmandelic acid (VMA) and Homovanillic acid (HVA) are the main final products of catecholamine metabolism. Due to the great clinical importance of these disorders, our group previously developed a very sensitive method to identify and quantify VMA, HVA, and 5-HIAA by LC-MS/MS. Thus, was performed a study of sample stability to identify the impact of no-acidification of the samples and the optimal storage temperature to perform a high accuracy analysis. The study aimed to evaluate stability of acidified and non-acidified urine samples under refrigerated and frozen conditions to identify and quantify VMA, HVA and 5-HIAA compounds. Methods Isolated urine samples were collected from 24 volunteers. For each compound, the stability of non-acidified samples was compared with HCl acidified samples, aiming for a final pH among 1 and 4. Independent aliquots of the samples were made to obtain the following cohort: Baseline (day of collection acidification), 24 h post-collection acidification, 4 days, 8 days and 15 days acidification. Those samples were divided into 4 groups: A—refrigerated storage and non-acidified; B—frozen and non-acidified; C—refrigerated and acidified (default) and D—frozen and acidified. The quantification of the analytes was performed by a previously validated method that employ a simple dilution of the samples followed by the LC-MS/MS analysis. Chromatographic separation and detection were performed on the Waters ACQUITY UPLC system equipped with RP-C18 column and gradient separation and on a Waters XEVO TQ-S Micro mass spectrometer and electrospray ionization (ESI+), respectively. Results The comparison between the basal groups (collection day) was identical for the 3 compounds, in the non-acidified groups (A and B) and also in the acidified group (C and D). Pearson’s Correlation was 0.995, 0.993 and 0.995 (P &amp;lt; 0.0001) for VMA,HVA and 5 -HIAA, respectively. Stability for refrigerated samples has been confirmed for up to 7 days and 15 days for compound 5-HIAA. For 15-day samples, one of the differences exceeded the total allowed error (31.3%-Westgard). Frozen samples (non-acidified or acidified) reverse percentage differences when compared to the standard group. In addition, for group B (frozen and non-acidified) the differences exceeded the total allowed error. However, comparing those days after collection and basal groups, it was calculated that the maximum acceptable period to maintain the stability of the samples was up to 7 days in refrigerated condition. Conclusion The stability for the urinary measurement of VMA, ,HVA and 5-HIAA was considered acceptable in this study. The maximum period allowed for acceptable stability is up to 7 days in the refrigerated condition.

Whiteness and greenness assessment with efficacy evaluation of two UPLC systems applied for the quantification of cinnarizine and dimenhydrinate along with their toxic impurities

Fixed-dose combination of cinnarizine (CIN) and dimenhydrinate (DMH) is an effective therapy for patients diagnosed for having central and/or peripheral vestibular disorder. Comparison and evaluation of two UPLC systems for analysis of CIN and DMH along with their toxic impurities namely 1- (diphenylmethyl)piperazine (DPP) and benzophenone (BZP), respectively in pharmaceutical dosage form matrix using diode array detector (DAD) and fluorescence detector (FLD) was applied. The chromatographic conditions of UPLC systems were carried out on poroshell UPLC column C18 (3.0 × 150 mm, 1.9 μm) as a stationary phase. An isocratic elution was established using a mobile phase made of methanol and triethylamine (pH = 4.0 ± 0.02) in the ratio of (94:6, v/v). The flow rate was 0.3 mL/min using injection volume 0.5 μL through application of DAD at 256.0 nm while was done at 251.0 nm as excitation wavelength and 310.0 nm and 330.0 nm as emission wavelength for DMH and CIN in case of FLD with total run time less than 5 min. The detected linearity range for CIN was 0.5–40.0 μg/mL and that for DMH was 1.0–50.0 μg/mL using DAD while the linearity range used in FLD was 0.1–6.0 μg/mL for CIN and was 0.25–8.0 μg/mL for DMH. Different whiteness and greenness assessment tools were demonstrated to evaluate the health and safety hazard of the proposed methods such as GAPI, AGREE and RGB12 tools, as well as the chemical components risk for the mobile phase were assessed by a green specific tool named D-CHEMS-1. The two proposed UPLC systems were sustainable, green, simple, fast, and effective and were successfully used for determination of the studied drugs in their combined dosage form excluding any contribution from excipients thus, will be useful in quality control department in pharmaceutical companies.

GC-MS and LC-MS/MS Analysis of Secondary Metabolites in the Methanolic Extract of Uncaria callophylla Blume ex Korth. Stems

Uncaria callophylla Blume ex Korth. is a wild plant species belonging to the Rubiaceae family. These plants thrive in the peat swamp forest of Kalimantan, Indonesia. The stems of Uncaria callophylla have been used by the Dayak Ngaju tribe to treat various diseases and to restore stamina while they work in the forest. The present study was conducted to identify the secondary metabolites in the methanolic extract of Uncaria callohylla stems to increase the knowledge about this plant and its potential in medicinal applications and drug discovery. The analysis was conducted using GC-MS and LC-MS/MS. GC-MS analysis was performed with the Agilent Technologies 7890 Gas Chromatography equipped with an Auto Sampler and 5975 Mass Selective Detector. The LC-MS analysis was carried out in the C18 column of the Waters Acquity UPLC system. MS analysis was performed with an electrospray ionization source (ESI) in positive and negative ion modes, and the compounds were identified using a UNIFI data processor with a mass spectrum library of natural active substances from the Waters Traditional Medical Scientific Library database based on UPLC/QTof MSE Data Acquisition. GC-MS analysis revealed the presence of 24 peaks, and nine of them showed quality 90% or higher, namely n-Hexadecanoic acid (16.63%), 1,3,7-Trimethyl-3,7-Dihydro-1H-Purine-2,6-Dione (13.07%), Hexadecanoic Acid, Methyl Ester; 1,2-Benzenedicarboxylic Acid, Diethyl Ester; 9,12-Octadecadienoic Acid, Methyl Ester; Methyl 14-Methylheptadecanoate; (9E,12E)-9,12-Octadecadienoic Acid; Z, E-3,13-Octadecadien-1-o1; and 2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-hexanethyl-, (all-E)-. LC-MS/MS analysis revealed the presence of five compounds, namely Arecatannin A1, Sweroside_2, Uncarine A, Epianhydrobelachinal, and Betulonic acid. All the secondary metabolites identified from GC-MS and LC-MS/MS analysis have biological activity, which indicates their medicinal potencies. From the result, it can be concluded that Uncaria callophylla stem methanolic extract contains various bioactive compounds justifies its traditional use, and therefore it can be developed further for new drug discovery.

Validated UPLC-MS/MS method for the determination of ivosidenib in rat plasma: Application to a pharmacokinetic study

AbstractIvosidenib (AG-120) is an unlisted, but estimated to be valid, oral inhibitor for isocitrate dehydrogenase 1 (IDH1) in the phase Ⅰ study of IDH1-mutated acute myeloid leukemia (AML) patients. This paper presents the investigation and validation of a rapid, effective, qualitative and quantitative determination method of ivosidenib in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The samples were treated using acetonitrile precipitation to remove protein influence. Then, the supernatant was extracted to analyze plasma concentration traits. In the UPLC system, acetonitrile and water containing 0.1% formic acid were selected as a cosolvent mobile phase, applying a gradient elution to isolate compounds in a C18 column. Mass detections were performed on a triple quadruple mass spectrometer in positive ion mode. Electroshock characteristic fragment ionization was used for m/z 583.95→214.53 for ivosidenib for quantitative determination, m/z 583.95→186.6 for qualitative determination, and m/z 492.06→354.55 for IS. The selectivity, linearity, stability, accuracy and precision were verified by reaching the guideline criteria from European Medicine Agency (EMA) and the Food and Drug Administration (FDA). The calibration curve was linear over the concentration range of 2–2,000 ng mL−1 for ivosidenib in rat plasma with a lower limit of quantification (LLOQ) of at least 2 ng mL−1. Additionally, there was no distinct matrix effect or carry-over phenomenon. The method was successfully established and applied to separate ivosidenib from plasma, with the entire analytical process being performed within 3 min for each sample, which shows high-efficiency and convenience for further studies of ivosidenib.

Hypoglycemic activities of flowers of Xanthoceras sorbifolia and identification of anti-oxidant components by off-line UPLC-QTOF-MS/MS-free radical scavenging detection

ObjectiveTo identify phytochemical constituents present in the extract of flowers of Xanthoceras sorbifolia and evaluate their anti-oxidant and anti-hyperglycemic capacities. MethodsThe AlCl3 colorimetric method and Prussian Blue assay were used to determine the contents of total flavonoids and total phenolic acids in extraction layers, and the bioactive layers was screened through anti − oxidative activity in vitro. The Waters ACQUITY UPLC system and a Waters ACQUITY UPLC BEH C18 column (2.0 mm × 150 mm, 5 μm) were used to identify the ingredients. And anti-oxidative ingredients were screened by off-line UPLC-QTOF-MS/MS-free radical scavenging. The ameliorative role of it was further evaluated in a high-fat, streptozotocin-induced type 2 diabetic rat model and the study was carried out on NADPH oxidase (PDB ID: 2CDU) by molecular docking. ResultsCombined with the results of activity screening in vitro, the anti − oxidative part was identified as the ethyl acetate layer. A total of 24 chemical constituents were identified by liquid chromatography-mass spectrometry in the ethyl acetate layer and 13 main anti-oxidative active constituents were preliminarily screened out through off-line UPLC-QTOF-MS/MS-free radical scavenging. In vivo experiments showed that flowers of X. sorbifolia could significantly reduce the blood glucose level of diabetic mice and alleviate liver cell damage. Based on the results of docking analysis related to the identified phytocompounds and oxidase which involved in type 2 diabetes, quercetin 3-O-rutinoside, kaempferol-3-O-rhamnoside, isorhamnetin-3-O-glucoside, and isoquercitrin showed a better inhibitory profile. ConclusionThe ethyl acetate layer was rich in flavonoids and phenolic acids and had significant anti-oxidant activity, which could prevent hyperglycemia. This observed activity profile suggested X. sorbifolia flowers as a promising new source of tea to develop alternative natural anti-diabetic products with a high safety margin.