RP-C18 Column Research Articles

Development of a simple HPLC method for the determination of urinary O-aminohippuric acid (OAH) and an establishment of OAH reference interval

Background: O-aminohippuric acid (OAH) is considered a low-abundance urinary fluorescent metabolite with the potential to be an innovative lung cancer biomarker. There is a lack of simple methods for measuring urinary OAH metabolite, and the measurement needs to be normalized by urinary creatinine, which is produced at a constant rate. Thus, the newly developed method must be able to determine urinary creatinine and OAH simultaneously in the same run. Objective: This work aimed to develop and validate a simple high-performance liquid chromatography (HPLC) method for measuring urinary OAH and to establish the reference intervals of OAH in healthy individuals. Materials and methods: We synthesized OAH standard in a simple route and optimized simple HPLC method for simultaneous measurement of creatinine and OAH in a single run. Analysis was performed on a RP-C18 column with a gradient elution system of acetonitrile - ammonium acetate buffer (pH 6.5, 100 mM). After implementing optimal conditions, the procedure was compiled according to the International Council for Harmonisation (ICH) validation parameters. The developed method was used for the establishment of reference intervals of a total of 120 random urine samples of healthy individuals. Results: The linear range of the calibration curve for creatinine and OAH were 1-1000 µg/mL and 0.1-100 µg/mL, respectively. The recoveries ranged for both metabolites were between 91.35 % and 109.12%. The relative standard deviations (RSDs) for the intra-day and inter-day results ranged from 0.11-0.66 % to 0.16-1.73 %, respectively. The limits of detection (LOD) and quantification (LOQ) were 0.258 µg/mL and 0.783 µg/mL for creatinine, while OAH was 0.045 µg/mL and 0.137 µg/mL, respectively. The method was successfully applied to establish reference intervals of OAH in healthy individuals and was defined as 0.420-2.287 mmol/mol creatinine. Conclusion: According to various validated parameters, the proposed method was proven to quantify urinary OAH and creatinine in a single run. It can also be analyzed noninvasively without additional sample processing. Reported herein is the first establishment of OAH reference intervals in healthy individuals, which may benefit the utilization of OAH as a noninvasive biomarker for lung cancer detection in the future.

An enrichment strategy based on hydrophobic tagging and reversed-phase chromatographic separation for the analysis of lysine-containing peptides

Lysine (K) is widely used in the design of lysine-targeted crosslinkers, structural elucidation of protein complexes, and analysis of protein-protein interactions. In "shotgun" proteomics, which is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), proteins from complex samples are enzymatically digested, generating thousands of peptides and presenting significant challenges for the direct analysis of K-containing peptides. In view of the lack of effective methods for the enrichment of K-containing peptides, this work developed a method which based on a hydrophobic-tag-labeling reagent C10-S-S-NHS and reversed-phase chromatography (termed as HYTARP) to achieve the efficient enrichment and identification of K-containing peptides from complex samples. The C10-S-S-NHS synthesized in this work successfully labeled standard peptides containing various numbers of K and the labeling efficiency achieved up to 96% for HeLa cell protein tryptic digests. By investigating the retention behavior of these labeled peptides in C18 RP column, we found that most K-labeled peptides were eluted once when acetonitrile percentage reached 57.6% (v/v). Further optimization of the elution gradient enabled the efficient separation and enrichment of the K-labeled peptides in HeLa digests via a stepwise elution gradient. The K-labeled peptides accounted for 90% in the enriched peptides, representing an improvement of 35% compared with the number of peptides without the enrichment. The dynamic range of proteins quantified from the enriched K-containing peptides spans 5-6 orders of magnitude, and realized the detection of low-abundance proteins in the complex sample. In summary, the HYTARP strategy offers a straightforward and effective approach for reducing sample complexity and improving the identification coverage of K-containing peptides and low-abundance proteins.

Fast and Easy Simultaneous Determination of Riboflavin, Folic Acid, All-Trans-Retinol and α-Tocopherol in Human Serum by LC/MS/MS for Bariatric Patients.

The aim of this study was to develop and validate methods for the determination of vitamins B2, B9, E and A in serum using liquid chromatography with mass spectrometry (MS) detection. Vitamin analysis was performed using an ultra performance liquid chromatography combined with tandem MS. The compounds were separated on a BEH C18 RP column (2.1 × 100mm, 1.7μm) using a gradient elution with an analysis time of 10min. Sample preparation included protein precipitation with ethanol. The concentration range in human serum was as follows: riboflavin 5-1000nmol/L, folic acid 2.5-250nmol/L, α-tocopherol 0.5-100μmol/L and all-trans-retinol 25-2500nmol/L. Accuracy and precision were validated according to Food and Drug Administration guidelines, with coefficients of variation ranging from 3.1-11.7% and recoveries from 94.4-107.5%. Routine monitoring of the complex range of vitamins in bariatric medicine is still not common. This is despite the fact that patients are at risk for glitch deficits, especially of a neurological nature. An analytical method that allows for the complex measurement of both water-soluble and fat-soluble vitamins is important and necessary for the clinical monitoring of bariatric patients. The method we have described could benefit both clinical practice and nutritional research.

Simultaneous assay of pyrimethamine and methylparaben in extemporaneous pediatric suspensions by an isocratic stability-indicating UPLC-DAD method

Pyrimethamine (PYM) is an antiparasitic drug used in the treatment of congenital toxoplasmosis, available only as tablets. Therefore, preparing extemporaneous formulations and their quality control is essential for safe pediatric treatment. This study aimed to develop and validate a stability-indicating method by ultra-performance liquid chromatography (UPLC) to quantify PYM and methylparaben (MP) in extemporaneous oral suspension and evaluate its applicability to the tablets assay. The analytical conditions included RP-C18 column, mobile phase composed of 0.05 M acetate buffer pH 4.5 and organic mixture (acetonitrile:methanol), at 60:40 (v/v), flow rate of 0.2 mL/min, injection volume of 2 µL and detection at 272 nm. PYM was detected at 3.9 min and MP at 4.7 min, without interference of the degradation products or other excipients. Linearity was observed in the range of 0.5 to 25 μg/mL. The method showed accuracy (mean recovery between 98.0 and 102.0%) and precision (RSD < 2%) for suspension and tablets; the method was robust regarding the variations in the analytical conditions. The developed method met the requirements of current guidelines, was indicative of stability, and was suitable for PYM determination in extemporaneous preparations and tablets.

Validation of HPLC-DAD Based Stability Indicating Assay Method for Ornidazole in Periodontal Polymeric Hydrogel: Robustness Study using Quality by Design (QbD) Approach

In the present study, a gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method has been designed and validated to quantify Ornidazole (OZ) in the marketed formulation (oral gel) with the application of QbD. The proposed HPLC method utilizes an RP C18 column (4.6 x 250 mm, 5 μm). The mobile phase contained HPLC grade Solvent A (water) and Solvent B (acetonitrile) and ran at a flow rate of 1 mL/min in a gradient mode. The linearity was plotted in an OZ range of 1–12 μg/mL and the correlation coefficient was found to be 0.9998. The limit of quantification, as well as the limit of detection, has been recorded as 0.70 μg/mL and 0.23 μg/mL. Inter- and intra-day precision (RSD) percentages were reported respectively within a range of 0.262–0.589 and 0.179–0.879. At 80, 100, and 120 percent different OZ levels, the average accuracy was 99.55, 99.58, and 99.92, respectively. The result of the study indicates that the OZ gel estimate HPLC method has good sensitivity, reproducibility, accuracy, and precision. This method may be used for the estimation of OZ in a gel formulation in quality control laboratories.

AQbD Assisted Method Optimization for Simultaneous Quantification of Potential Phytosterols in Novel Polyherbal Formulation by LC-PDA Method

Background: Herbal medications are preferred by individuals with cardiovascular disorders (CVDs) worldwide for their distinct benefits in preventing and treating illnesses and lesser side effects. A novel polyherbal formulation compiled of ten plants (Allium sativum, Rubia cordifolia, Garcinia cambogia, Terminalia arjuna, Acacia catechu, Helianthus annus, Vitis vinifera, Commiphora mukul, Linum usitatissimum, and Piper nigrum) was developed for treating cardiovascular diseases. As a part of standardization, marker analysis using high-performance liquid chromatography (HPLC) will be highly encouraged. Materials and Methods: In the present study, we selected lupeol, stigmasterol, and β-sitosterol and went for simultaneous quantification using design of experiments (DoE) software (AQbD-Analytical quality by design) to optimize the experimental parameters. The phenomenex RP C18 column, with 250 mm length, 4.6 mm internal diameter, 5 μm particle size, was preferred for the study. Mobile phase with methanol and 0.01% formic acid solution in water surged with a flow rate of 1.5 mL/min. using a maximum detection wavelength of 208 nm. Results and Discussion: Under the specified circumstances, we determined the retention times of lupeol, stigmasterol, and β-sitosterol to be 14.056, 15.544, and 17.431 minutes, respectively, with the desirability of 0.975. The concentrations of lupeol, stigmasterol, and β-sitosterol in the extract were determined to be 30.72, 370.6, and 212.64 μg/10 mg, respectively. The linear range of 31.25 to 500 μg/mL was found to have an R2 value of 0.9972, 0.9976 and 0.9968, respectively. 0.2534, 0.754, 0.421 μg/mL and 0.7681, 2.29, 1.28 μg/mL were the limit of detection (LoD) and limit of quantitation (LoQ) values, respectively. The RSD for precision was determined to be 0.92, 0.98, and 0.96%, respectively. Recovery experiments were conducted, and the accuracy of the suggested approach was between 99 and 102%. Conclusion: Using the modernized software tool- AQbD (analytical quality by design), the study was first scientifically validated for the quantification of three phytomarkers in the novel polyherbal formulation.

False-positive MDA findings in HRMS-based screening of putrefied postmortem blood samples-Identification of the interference as N-acetyltyramine.

An unidentified compound in putrefied postmortem blood samples showed identical accurate mass and chromatographic behavior as 3,4-methylenedioxyamphetamine (MDA) and led to false-positive preliminary screening results. The aim of the study was to identify this unknown interference. Postmortem blood samples were analyzed after protein precipitation on a QExactive Focus high-resolution mass spectrometer (Thermo Fisher, Germany) coupled to a RP C18 column (Macherey-Nagel, Germany). Based on the analysis of mass spectrometry (MS) adducts and isotope ratios using fullscan (m/z 134-330) information, the empiric formula of the protonated molecule [M + H]+ of the unknown compound was found to be C10H14O2N (+ 0.6 ppm). Product ion spectra recorded using normalized collision energy 22% showed a base peak of C8H9O1 (+ 1.5 ppm) and a low-abundant water loss to C7H9 (+ 1.9 ppm), neutral losses of C2H2O and NH3 were found. Based on fullscan and MS-MS information and under consideration of the observed order of neutral losses, the compound was presumptively identified as N-acetyltyramine. This assumption was supported by SIRIUS software showing a SIRIUS score of 99.43% for N-acetyltyramine. Finally, the putative structure annotation was confirmed by a reference compound. The described false-positive MDA findings could be attributed to the presence of N-acetyltyramine in putrefied blood samples. Being an isomer of MDA, N-acetyltyramine could not be distinguished by high-resolution data of the protonated molecules. The presented results once again highlight that false-positive findings may occur even in hyphenatedhigh-resolution mass spectrometry (HRMS) when using full-scan information only.

Profiling and evaluation of phenolic compounds in olive mill wastewater in Jordan

Abstract Phenolic compounds (PCs) are prevalent in olive mill wastewater (OMW), offering numerous health benefits but concurrently posing environmental challenges. This study aims to comprehensively characterize PCs in OMW, both qualitatively and quantitatively. An accurate, sensitive, and cost-effective method was successfully developed for extracting and quantifying seven PCs in OMW. The extraction procedure was optimized to achieve the maximum recovery using a liquid-liquid extraction method. Concentrations of the identified PCs were determined using a high-performance liquid chromatography-diode array detector (HPLC-DAD). The seven PCs peaks were successfully separated on an RP-C8 column within a 23-minute runtime using a gradient mobile phase. The method underwent thorough validation, producing satisfactory results. Subsequently, the developed method was applied to analyze OMW from four olive mills in Jordan, revealing PC concentrations ranging from 139 to 430 mg/L. Tyrosol and hydroxytyrosol were identified as the most abundant compounds. Additionally, gas chromatography-mass spectrometry (GC-MS) separated and identified forty PCs. The total phenolic content was quantified, reaching a value of 1839 mg/L. Moreover, the antioxidant activity was assessed, yielding a maximum value of 95.8%. These results underscore the substantial levels of PCs in OMW, highlighting the importance of economically utilizing this water.

Limonoids from the Branches and Leaves of Aglaia lawii and Their Cytotoxic Activities.

Three undescribed limonoids (1-3), named aglaians G-I, and one new natural product azedaralide (4), together with nine known analogues (5-13) were isolated from the branches and leaves of Aglaia lawii by RP C18 column, silica gel column, Sephadex LH-20 column chromatography and preparative HPLC. The structures of the new compounds were elucidated by IR, HRESIMS, 1D, 2D NMR, electronic circular dichroism (ECD) calculations and X-ray crystallography diffraction analysis. The results of bioassay showed that the compound 12 exhibited potential inhibitory activity against six human tumor cell lines (MDA-MB-231, MCF-7, Ln-cap, A549, HeLa and HepG-2) with IC50 values as 8.0-18.6 μM.

Ecofriendly single-step HPLC and TLC methods for concurrent analysis of ternary antifungal mixture in their pharmaceutical products

Two accurate, sensitive, and selective methods for simultaneous determination of miconazole nitrate (MIC), nystatin (NYS), and metronidazole (MET) in pure state or drug product were established and verified. First, RP-HPLC-DAD was designed. Separation was accomplished using a ZOBRAX Eclipse Plus RP-C8 column that was running under an isocratic elution of methanol: 0.05% aqueous solution of sodium dodecyl sulphate (40: 60 v/v), with a flow rate that was regulated at 0.8 mL/min. The column temperature was adjusted at 25 °C and diode array detector was monitored at 220 nm. The linearity range of the proposed method was achieved at the concentration of 5–50, 4–50, and 4–40 µg/mL and the attained retention time for the studied drugs was 2.52, 3.52 and 4.99 min for MIC, NYS, and MET, correspondingly. Second, a TLC-densitometric approach was used to resolve the three compounds. Resolution of the three cited drugs was carried out using TLC aluminum plates pre-coated with 0.25 mm silica gel 60 F254. A developing solvent comprised ethyl acetate: toluene: methanol: triethyl amine: formic acid (3: 1: 7: 0.3: 0.1 by volume) (pH = 5.5) was utilized and scanning of the resolved bands at 215 nm. Linearity of the developed TLC method was evaluated and evident to be 0.4–2, 0.4–2.2, and 0.4–2 μg/band for MIC, NYS, and MET, in that order. The suggested chromatographic methods were verified according to ICH directives. The findings of the developed chromatographic procedures were statistically compared with the results of the reported ones using student’s t-test and F-test. Furthermore, two green assessment tools evaluated the indicated methods' level of greenness (GAPI and AGREE).

In Vitro Permeability Study of Homotaurine Using a High-Performance Liquid Chromatography with Fluorescence Detection Pre-Column Derivatization Method.

Homotaurine (HOM) is considered a promising drug for the treatment of Alzheimer's and other neurodegenerative diseases. In the present work, a new high-performance liquid chromatography with fluorescence detection (HPLC-FLD) (λex. = 340 nm and λem. = 455 nm) method was developed and validated for the study of substance permeability in the central nervous system (CNS). Analysis was performed on a RP-C18 column with a binary gradient elution system consisting of methanol-potassium phosphate buffer solution (pH = 7.0, 0.02 M) as mobile phase. Samples of homotaurine and histidine (internal standard) were initially derivatized with ortho-phthalaldehyde (OPA) (0.01 M), N-acetylcysteine (0.01 M) and borate buffer (pH = 10.5; 0.05 M). To ensure the stability and efficiency of the reaction, the presence of different nucleophilic reagents, namely (a) 2-mercaptoethanol (2-ME), (b) N-acetylcysteine (NAC), (c) tiopronin (Thiola), (d) 3-mercaptopropionic acid (3-MPA) and (e) captopril, was investigated. The method was validated (R2 = 0.9999, intra-day repeatability %RSD < 3.22%, inter-day precision %RSD = 1.83%, limits of detection 5.75 ng/mL and limits of quantification 17.43 ng/mL, recovery of five different concentrations 99.75-101.58%) and successfully applied to investigate the in vitro permeability of homotaurine using Franz diffusion cells. The apparent permeability (Papp) of HOM was compared with that of memantine, which is considered a potential therapeutic drug for various CNSs. Our study demonstrates that homotaurine exhibits superior permeability through the simulated blood-brain barrier compared to memantine, offering promising insights for enhanced drug delivery strategies targeting neurological conditions.

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.

Competing ligand exchange-solid phase extraction method of polyphenols from wine

A simple and efficient solid phase extraction technique by a ligand-exchange scheme using functionalized silica nanocomposites has been developed and optimized for the extraction of phenolic compounds from wine. Five polyphenolic compounds - naringin, rutin, p-coumaric acid, quercetin, genistein from untreated wine samples were pre-concentrated and cleaned on adsorbent with covalently immobilized fragments of phosphonic acid – APA covered with Fe3+ ions – Fe/APA The cartridge was conditioned with 5 mL of methanol and equilibrated with 5 mL of 10% (v/v) aqueous MeOH. Co-extracted ballast substances were rinsed from the sorbent with 5 mL of 10% (v/v) aqueous MeOH. Retained phenolic compounds were selectively eluted with 1% formic acid in MeOH. A mobile phase gradient containing 0.1% formic acid and acetonitrile was used for the final baseline chromatographic separation on an RP C18 column. Limits of detection are in the range of 2.2 to 3.9 mg L−1. The method was applied to quantify five phenolic compounds in red and white wine samples.

Determination of Pinoresinol 4-O-beta-D-glucopyranoside and Vladinol F as Markers in Pandanus tonkinensis Fruits by High Performance Liquid Chromatography

According to the traditional know-how, the Northern wild pineapple (Pandanus tonkinensis) has been used efficiently as a major component of oriental remedies for liver protection. Based on the experimentally found chemical composition of Pandanus tonkinensis and their obtained biological activities in terms of the hepatoprotection purpose, two structure-elucidated compounds pinoresinol 4-O-beta-D-glucopyranoside and vladinol F were selected as potential markers for this medical plant. This paper is aiming to present the validation of proposed HPLC method for simultaneous quantification of the both pinoresinol 4-O-beta-D-glucopyranoside and vladinol F in the fruits of Pandanus tonkinensis with the following conditions: using RP-C18 (250 mm × 4.6 mm; 5 μm) column, detection wavelength measured at 228 nm, the mobile phases consisting of acetonitrile and 0.1% acetic acid in gradient mode with a flow rate of 1mL/min and injection volume was 10 mL. Method validation showed a high specificity, linear calibration ranging from 25.5 to 76.4 mg/ml (r= 0.9991) and from 26.0 to 77.9 mg/ml (r= 0.9987) for two markers, respectively. Besides, a good repeatability and intermediate precisions (RSD &lt; 2%), a reliable accuracy (recovery efficiency between 99.4 and 101.5%, RSD &lt; 1.73%) as well as the obtained limits of quantification in values of 2.55 m/g and 2.60 mg/g for the two mentioned markers were received. Using the above validated method, the content of two markers - pinoresinol 4-O-beta-D-glucopyranoside and vladinol F in Pandanus tonkinensis fruits collected from different locations (Thanh Hoa, Hoa Binh, Thai Nguyen) had been determined falling in the range of 0.0250 - 0.0435 mg/g and 0.0243 - 0.0371 mg/g, respectively.&#x0D; Keywords: pinoresinol 4-O-beta-D-glucopyranoside, vladinol F, markers, Pandanus tonkinensis, HPLC

Development and Validation of a modified QuEChERS coupled to Liquid Chromatography-Tandem Mass Spectrometry for the Rapid and Accurate Determination of Acrylamide in Cereal-Based Baby Foods.

With the widespread consumption of cereal-based baby food by children, acrylamide contamination is a prevalent risk that may have carcinogenic consequences. This study aims to develop and validate a modified QuEChERS protocol without solvent exchange, followed by rapid separation and accurate determination of acrylamide in cereal-based baby foods using RP-LC-MS/MS. Samples were extracted using a modified AOAC QuEChERS version and cleaned up with basic alumina. Separation was performed on the Phenomenex® Kinetex C18 column (100 Å × 3.5µm × 4.6 mm × 150 mm) using a gradient elution program with a mobile phase of 10-mM ammonium formate/methanol. Determinations were conducted by ESI-MS/MS in positive ion mode. Basic alumina yielded clean extracts, resulting in acceptable recovery percentages and a tolerable ME < 5%. This has allowed extraction without a solvent exchange step. Efficient separation was achieved at a tR of 3.39 ± 0.05 employing an RP-C18 column with core-shell properties in a relatively short analysis run time of only 5 min. Trueness, precision, LOD, LOQ, linearity range, and R2 results were 92.5-104.6%, RSD≤12.2%, 5 µg/kg, 20 µg/kg, 4.0-1000.0 µg/kg, and > 0.9999, respectively. The test method applicability was demonstrated by proficiency testing and 50 real samples of cereal-based baby foods. Most of the tested samples were in violation of acrylamide's established EU benchmark (40 µg/kg). Acetate-buffered QuEChERS in conjunction with optimized amounts of basic alumina was confirmed as a superior driving approach for optimal method performances. Successful selection of the RP-C18 column is the proper means toward selective separation for acrylamide at a relatively short analysis run time. The modified AOAC QuEChERS with a d-SPE of basic alumina assisted in reducing the ME to tolerable levels while maintaining acceptable method performance. The RP-C18 column of core-shell properties enabled a rapid and accurate acrylamide determination.

Screening Of Oxytetracycline And Tetracycline Residues In Pork Marketed In Guwahati City And Its Adjoining Areas And Confirmation By Ultra-Fast Liquid Chromatography-UV/Vis

The antimicrobial residues in the animal-based food products that exceeds the maximum residue limits (MRLs) beyond permissible daily consumption attracted considerable attention due to their public health significance. The present study aimed to detect Oxytetracycline and Tetracycline residues in pork marketed in and around Guwahati City (Kamrup Metropolitan) and its adjoining areas. The preliminary screening of 261 pork samples by microbial inhibition assay using endospores of Bacillus subtilis MTCC 441 as test organism revealed that none of the samples were positive for antimicrobial residues except 3 (three) which were doubtful to have traces of antimicrobial residues. A total of 80 pork samples including the doubtful samples were further screened by Ultra-Fast Liquid Chromatography system (Model: Shimadzu Prominence LC-20AD, Detector- SPD-20A-UV/Vis; RP C18 Column: BDS Premium, 250 mm x 4.6 mm, 5 µm) to detect Oxytetracycline and Tetracycline residues. Oxytetracycline residues were detected in 2.5% of the samples, while none of the samples detected Tetracycline residue. The concentrations of the residues were 0.471 µg/g and 0.610 µg/g, respectively, which is well above the MRL value recommended by Codex Alimentarius. However, considering the detection of 2.5% Oxytetracycline residues and the absence of Tetracycline residue indicates that these antimicrobial compounds are not frequently misused in pig husbandry practices in these areas under study.

Establishment of HLB-UPLC-MS/MS Method to Determine Hainanmycin Sodium in Chicken Liver

Objective: Controlling coccidiosis disease in poultry is significant for healthy breeding, but antibiotic residues can pose a great threat to food safety, the ecological environment, and human health. A special anticoccidial drug called Hainanmycin has been widely used in Asian countries for many years, while a few studies focus on its detection. In this study, we established a hydrophiliclipophilic balance (HLB) purification and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method to determine the hainanmycin in chicken liver. Methods: It was extracted from the sample with 0.1% formic acid-methanol (10+90, v/v) and then it was purified on a hydrophilic-lipophilic balance (HLB) cartridge. Chromatography separation was performed on an RP-C18 column, and detection of the hainanmycin was done by positive electrospray ionization (ESI＋) under the multiple reaction monitoring (MRM) modes. Results: The quantification ion pair was m/z 907.5 and m/z 846.4. The calibration curve showed excellent linearity in the range of 10 to 1000 μg·L-1, with a correction coefficient of 0.99. When spiked at 5, 100, and 500 μg·kg-1, the recoveries ranged from 80.82% to 88.09%, with the intra-day and interday coefficients of variation from 10.76% to 13.48% and from 10.80% to 12.71%, respectively. The limit of quantification (LOQ) and detection(LOD) were 5.05 μg·mL-1 and 1.55 μg·mL-1, respectively. Conclusion: This method showed high sensitivity and accuracy for determining hainanmycin in chicken liver.

Intrinsic Permeation and Anti-Inflammatory Evaluation of Curcumin, Bisdemethoxycurcumin and Bisdemethylcurcumin by a Validated HPLC-UV Method

Curcumin shows anti-inflammatory activity, and it has been widely investigated for neurodegenerative diseases, adjuvant treatment in AIDS and antitumor activity against different tumors, among other activities. The goal of this work was to evaluate the capacity of curcumin and its derivatives (bisdemethoxycurcumin and bisdemethylcurcumin) in preventing the irritant effects of topically applied xylol and to assess the intrinsic capacity of curcuminoids in permeating human skin by ex vivo permeation tests. Its secondary goal was to validate an HPLC method to simultaneously determine the curcuminoids in the samples from the ex vivo permeation studies and drug extraction from the skin. Curcuminoid quantification was performed using an RP-C18 column, at isocratic conditions of elution and a detection wavelength of 265 nm. The method was specific with a suitable peak resolution, as well as linear, precise, and accurate in the range of 0.195-3.125 μg/mL for the three curcuminoids. Bisdemethylcurcumin showed the greatest permeation through the human skin, and it was the curcuminoid that was most retained within the human skin. The anti-inflammatory activity of the curcuminoids was evaluated in vivo using a xylol-induced inflammation model in rats. Histological studies were performed to observe any changes in morphology at the microscopic level, and these three curcuminoids were found to be respectful within the skin structure. These results show that these three curcuminoids are suitable for anti-inflammatory formulations for dermal applications, and they can be properly quantified using HPLC-UV.

RP-HPLC Method for Quantification of Bilastine and Monteleukast Sodium in Pharmaceutical Dosage form

A simple, accurate, precise and robust reverse phase high performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous estimation of Bilastine and Monteleukast Sodium in bulk and pharmaceutical dosage form. Chromatographic separation was performed on an Zodiac sil RP C18 column (100mm × 4.6mm, 3µm) with mobile phase consist a mixture of 75:25% v/v acetonitrile: phosphate Buffer pH adjusted to 5.2 with ortho-phosphoric acid in an isocratic elution mode, at a flow rate of 1 ml/min. The detection was monitored at 272nm. The retention time of Monteleukast Sodium and Bilastine were found to be 3.668 min and 2.746 min respectively. Linearity range was found between 20-100µg/ml for Bilastine with correlation coefficient of 0.999 and 5-25µg/ml for Monteleukast Sodium with correlation coefficient of 0.999. This method was validated with respect to linearity, precision, accuracy, ruggedness, limit of detection, limit of quantification and robustness. This method was successfully applied for the simultaneous estimation of Bilastine and Monteleukast Sodium in bulk and pharmaceutical formulation.

Determination of Pterostilbene in Pharmaceutical Products Using a New HPLC Method and Its Application to Solubility and Stability Samples

The quantification of a natural bioactive compound, pterostilbene (PTT), in commercial capsule dosage form, solubility, and stability samples was carried out using a rapid and sensitive high-performance liquid chromatography (HPLC) approach. PTT was quantified on a Nucleodur (150 mm × 4.6 mm) RP C18 column with a particle size of 5 µm. Acetonitrile and water (90:10 v/v) made up the mobile phase, which was pumped at a flow speed of 1.0 mL/min. At a wavelength of 254 nm, PTT was detected. The developed HPLC approach was linear in 1–75 µg/g range, with a determination coefficient of 0.9995. The developed HPLC approach for PTT estimation was also rapid (Rt = 2.54 min), accurate (%recoveries = 98.10–101.93), precise (%CV = 0.59–1.25), and sensitive (LOD = 2.65 ng/g and LOQ = 7.95 ng/g). The applicability of developed HPLC approach was revealed by determining PTT in commercial capsule dosage form, solubility, and stability samples. The % assay of PTT in marketed capsules was determined to be 99.31%. The solubility of PTT in five different green solvents, including water, propylene glycol, ethanol, polyethylene glycol-400, and Carbitol was found to be 0.0180 mg/g, 1127 mg/g, 710.0 mg/g, 340.0 mg/g, and 571.0 mg/g, respectively. In addition, the precision and accuracy of stability samples were within the acceptable limit, hence PTT was found to be stable in solution. These results suggested that PTT in commercial products, solubility, and stability samples may be routinely determined using the established HPLC method.