HPLC System Research Articles

Prescription versus over-the-counter venotonics: HPLC-DAD and static digestive model simulation comparison.

Venotonics are a class of therapeutically active molecules that have vaso-protective effects. They are used to alleviate venous diseases and disorders, particularly venous insufficiency. We compared the composition of prescription versus over-the-counter (OTC) venotonics using high-performance liquid chromatography with UV detection (HPLC-DAD) and simulating their digestion using a static digestive model. From each drug, five tablets were weighed. A homogenate was prepared, and 25 mg of crushed homogenized tablets were weighed into 25 ml volumetric flasks. Dissolved in MeOH and added two drops of saturated NaOH solution. The samples were filtered into vials (Teflon, 0.45 μm) and used for analysis. An Ultimate 3000 HPLC system (Thermo Fisher Scientific, Waltham, MA, USA) consisting of a quaternization pump, autosampler, column thermostat and DAD (UV/VIS detector) was used. The composition of the mobile phase proceeded in a linear gradient from 30% methanol and 70% phosphoric acid (0.15%) in water at time t=0 min. to 80% methanol and 20% phosphoric acid (0.15%) at time t=15 min., at a constant mobile phase flow rate of 1.2 mL/min. Detection was performed using a DAD detector in the 190-450 nm wavelength range. The content of monitored flavonoids was calculated from peaks at a wavelength of 277 nm, in which both flavonoids have their absorption maxima. The static digestive model was used to simulate the digestive phase from the oral cavity to the corresponding intestinal phase. The content of diosmin and hesperidin (mg per table) for a prescription drug: Detralex: 480 mg, 26 mg. The content of diosmin and hesperidin (mg per tablet) for OTC drugs: Venostop: 502 mg, 48 mg, Diosminol: 520 mg, 50 mg, Devenal: 496 mg, 49 mg, Diohes: 493 mg, 46 mg. Digestion did not affect the solubility of all tested drugs. The active substances could not be determined in the non-alkalized sample. After alkalization, part of the insoluble matter was visibly dissolved and converted to a yellow flavonoid complex. Neither diosmin nor hesperidin could be identified afterwards. Our experimental results show that the contents of both listed active substances, diosmin and hesperidin, met the declared amounts in all tested medicaments. Digestion simulation showed identical behaviour in prescription and OTC venotonics. The active substances could not be determined in the non-alkalized sample. Digestion did not affect the solubility of the tested drugs.

Malleatin A and B: New Premyrsine-Type Diterpenes from <i>Euphorbia malleata</i> with Cytotoxic Effects Against A2780 Wild and A2780 R-CIS Ovarian Cancer Cell Lines in Mono or Combination Treatment with Cisplatin

Background: This study focused on macrocyclic diterpenes derived from Euphorbia, particularly myrsinanes, and their potential in cytotoxic and combination treatments for resistant cancer cells. We examine premyrsinanes isolated from Euphorbia malleata and explore their cytotoxic properties. Methods: Euphorbia malleata was collected from Taragh-Roud, Natanz, Iran. The semi-polar chloroform/acetone extract was chromatographed and fractionated using a large silica column. Fractions containing diterpene resonances were selected based on 1H-NMR spectra and were further subjected to smaller silica or Sephadex columns, followed by a recycling HPLC system. The isolated compounds were identified through 1D and 2D-NMR experiments and mass spectrometry. The cytotoxicity of the isolated compounds was assessed using the MTT assay against A2780 wild and A2780 cisplatin-resistant (R-CIS) cells, both in mono and combination treatments with cisplatin. Results: Using a Waters 616 HPLC pump and a YMC prep silica column, we successfully isolated two new premyrsinane diterpenes (Malleatin A and Malleatin B) alongside two known compounds (beta-sitosterol and loliolide). Malleatin A exhibited cytotoxicity against A2780 wild and A2780 R-CIS cells, with an IC50 range of 50 - 65 μM in the MTT assay. While cisplatin demonstrated significant cytotoxic effects on the A2780 wild cell line, it was ineffective against the A2780 R-CIS cells due to their resistance. However, the combination therapy of Malleatin A and cisplatin exhibited a synergistic effect, significantly increasing the mortality rate of the resistant cells compared to monotherapy. The Combination Index (CI) of 0.58 indicates effective synergy, and the Dose Reduction Index (DRI) of 3.65 suggests a favorable reduction in the dosage of cisplatin needed, potentially reducing its associated side effects.

DEVELOPMENT AND OPTIMIZATION OF HPLC METHODS FOR THE CHIRAL SEPARATION OF ARYLPYRAZOLE, CHLOROACETANILIDE AND ORGANOCHLORINE PESTICIDES: CASE STUDIES OF BENZOBICYCLON, ACETOCHLOR AND CHLORDANE.

This work is centered on the development and optimization of High-Performance Liquid Chromatographic methods for the separation of chiral pesticides, particularly benzobicyclon, chlordane and acetochlor employing Agilent Technologies Infinity II chiral HPLC system equipped with a UV – 2000 detector. The stereoisomers were successively separated using a standard analytical column of dimensions 250 mm X 4.6 mm. The Cis– stereoisomers of chlordane exhibited partial baseline separation while the Trans – enantiomers were fully separated. Benzobicyclon and acetochlor enantiomers attained full baseline separation under optimized conditions. Chlordane and benzobicyclon were analyzed using normal phase chromatography [NP]c with a mobile phase of 90% Normal hexane and 10% Isopropanol, while acetochlor was analyzed by reverse phase chromatography [RP] c using 70% methanol and 30% water. Benzobicyclon was later spiked into soil sample and subsequently extracted using Environmental Protection Agency (EPA) methods and the extracts were further analyzed with a shorter analytical column of dimensions 75 mm X 4.6 mm. The HPLC separation revealed that the longer column provided more efficient separation for the benzobicyclon stereoisomers than the shorter column as reflected by high separation factors (?). The high recovery rate of the stereoisomers of benzobicyclon and the internal standard from the soil extracts confirmed the effectiveness of the method of extraction and the HPLC system employed.

B-157 High Resolution Liquid Chromatography - Mass Spectrometry (LC-MS) For Rapid, High-Throughput Clinical Testing of β2-Transferrin in Human Nasal Secretions

Abstract Background Leakage of Cerebrospinal Fluid (CSF) can result from trauma, lacerations, and surgery, and if untreated can result in potentially life-threatening meningitis. CSF leaks can be diagnosed through the detection of β2-Transferrin (β2-Tf) from nasal secretions via gel electrophoresis. β2-Tf is a uniquely glycosylated proteoform of Transferrin found primarily in CSF, which can be distinguished from the most abundant Transferrin proteoform (β1-Tf) by its altered electrophoretic mobility. However, gel-based detection of β2-Tf requires hours of labor and has low specificity due to the numerous alternative glycoforms of Transferrin. Higher-throughput assays are thus desirable for improved patient care. Recently, our group applied High-Resolution Mass Spectrometry (HR-MS) coupled to Microprobe In-Emitter Elution (MPIE) to discriminate Transferrin isoforms. This method successfully resolved β2- from β1-Tf in nasal secretions derived from patient samples with suspected CSF leakage, but has relatively low sample-to-sample throughput. Herein, we describe a novel approach utilizing Liquid Chromatography Mass Spectrometry (LC-MS) to rapidly profile Transferrin glycoforms from patient nasal secretion samples. Furthermore, we explore solutions to address significant blood contamination in nasal secretions, which may otherwise interfere with this approach. Furthermore, we explore solutions to address significant blood contamination in some nasal secretions, which may otherwise interfere with this approach. Methods Our approach consists of two primary steps: 1) Sample processing and high abundance protein depletion via commercial filtration and depletion columns. 2) LC-MS analysis of the resulting extract to resolve Transferrin glycoforms by retention time and m/z. This approach retains the high specificity of the previously demonstrated MPIE-MS method, but takes advantage of existing high-throughput liquid chromatography platforms to reduce the sample-to-sample analysis time. Secretion samples were analyzed using a Vanquish HPLC system coupled to a Q-Exactive Plus Mass Spectrometer (ThermoFisher). Broadband mass spectra were collected (FWHM resolution of 17,500 at 200 m/z), and the deconvoluted average masses were used to identify Transferrin glycoforms. Results Our approach was benchmarked using remnant nasal secretion samples from patients with known CSF leakage. Examining 9 samples, the resulting ESI spectra revealed peaks at 79,554 Da and 78,008 Da, consistent with β1-Tf (Pred. MW (Average): 79,554.71 Da) and β2-Tf (Pred. MW (Average): 78,008.35 Da) within ∼10 ppm mass accuracy. The 79.5 kD peak was observed in all samples, while the 78 kD peak was observed in 7 of 9 samples. Samples with missing β2-Tf were marked by significant blood contamination. Genetic variants (2/9 samples) resulted in mass shifts for both β1- and β2-Tf variants, but Transferrin glycoforms identifiable due to the preserved mass shift between the β1- and β2-Tf peaks (∼1546.4 Da). An optimized LC-MS gradient enables rapid (∼10 minute) sample-to-sample injection times. Samples with blood contamination represent an additional challenge for identification of β2-Tf, owing to ion suppression from blood-derived β1-Tf. We have developed enrichment and depletion methods to increase the sensitivity for β2-Tf in blood contaminated samples, and anticipate that these approaches will reduce or eliminate this challenge. Conclusions Our approach enables high-throughput, MS-based measurement of Transferrin glycoforms (i.e. β1- and β2-Tf) from clinical nasal secretion samples for the first time.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

B-151 Development and Validation of Plasma Catecholamines by Mass Spectrometry

Abstract Background Our established method for measuring plasma catecholamines utilized a Shimadzu HPLC coupled to an ESA Coulochem III electrochemical detector. Chromatographic run times were long, resulting in slow turnaround times. Instrument maintenance was labor intensive, and the assay was prone to interferences. To overcome these limitations, we changed the method to liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurements. Methods After the addition of a labeled internal standard for each analyte, samples are loaded onto 50 mg of activated alumina at pH 8.6, utilizing a Tris-HCl, sodium carbonate and EDTA buffer. After incubation the alumina is then washed with 50% acetonitrile followed by water. Elution off the alumina is performed with 2% acetic acid. Derivatization is then performed on the eluate utilizing acetaldehyde and a cyanoborohydride buffer. Following derivatization, the samples are analyzed for epinephrine, norepinephrine, and dopamine by LC-MS/MS using a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Chromatographic separation was obtained using a Phenomenex Kinetex 2.6 um BiPhenyl 50x3 mm analytical column. Results Runtimes were significantly shorter than with the previous method, and no interferences were observed. Assay validation consisted of: intra/inter assay precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), and interferences. Three levels were assessed for intra assay precision and each of the analytes monitored yielded %CVs of less than 6%. Four levels were assessed for inter assay precision and each of the analytes monitored yielded %CVs of less than 10%. When using linear regression analysis to compare results with the previous clinical assay, the slope, y-intercept and R2 were the following; dopamine: 1.06, 2.74, 0.978; epinephrine: 0.905, 2.20, 0.989; norepinephrine: 0.919, 9.75, 0.953. Spike and recovery from five samples yielded a mean average recovery of 93.4% for dopamine, 100% for epinephrine and 98.1% for norepinephrine. For linearity 4 samples were diluted and the mean % difference from expected was -1.66% for dopamine, -0.25% for epinephrine and 1.78% for norepinephrine. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. Sixty-two common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Conclusions The LC-MS/MS method exceeded the performance of the historical Shimdazu HPLC system with ESA Coluochem III electrochemical detector method. We anticipate that the better analytical performance will improve the testing quality of our patients.

Relationship between 25-OH-D3 levels and cognitive functions in children with obesity

Aims: The inconsistent results about neurocognitive functions in children with obesity may be suggestive of factors like vitamin deficiencies rather than the disorder itself. So we aimed to investigate the 25(OH)D3 levels and cognitive functions in obese children in the present study. Methods: Seventy-two children were included to this study. Forty-one of them were obese children and 31 children were with normal weight. The patients were diagnosed as obese according to body mass index >95 percentile, considering the sex and age-specific growth curves for Turkish children. The participants completed the battery tests of the central nervous system vital signs (CNSVS), a neurocognitive test battery, via computer. The battery calculates seven domain scores (Memory, Psychomotor speed, Processing speed, Reaction time, Complex attention, Executive function, Cognitive flexibility) and a summary score (Neurocognition Index). 25(OH)D3 levels were measured in residual samples using a Shimadzu HPLC system with the aid of a 25(OH)D3 kit. The scores were compared by using commercial software (IBM SPSS Statistics 18). Results: The mean 25-OH-vitamin D levels were 13.41±7.91 µg/L in obese children and 20.31±5.92µg/L in controls. Vitamin D3 levels were significantly lower in obese children than in control group (p

Anion exchange-HPLC method for evaluating the encapsulation efficiency of mRNA-loaded lipid nanoparticles using analytical quality by design

Lipid nanoparticles (LNPs) are emerging nucleic acid delivery systems in the development of mRNA therapeutics such as the severe acute respiratory syndrome coronavirus 2 vaccines. However, a suitable analytical method for evaluating the encapsulation efficiency (EE) of the LNPs is required to ensure drug efficacy, as current analytical methods exhibit throughput issues and require long analysis times. Hence, we developed and validated an anion-exchange HPLC method using Analytical Quality by Design. Three critical method parameters (CMPs) were identified using risk assessment and Design of Experiments: column temperature, flow rate, and sodium perchlorate concentration. The CMPs were optimized using Face-Centered Central Composite Design. The discriminating power of the optimized HPLC method and RiboGreen assay was comparable. The main advantage of this method is that LNPs can be directly injected into the HPLC system without bursting the LNPs loaded with encapsulated poly(A). The optimized HPLC method was validated as robust, high-throughput, and sufficiently sensitive according to the ICH Q2 guidelines. We believe our findings could promote efficient LNPs-based drug development.

Development of an HPLC System Using a Phase-Separation Multiphase Flow as an Eluent— Effect of Pore Size in Column-Packed Particles on the Novel Separation Mode —

Development of an HPLC System Using a Phase-Separation Multiphase Flow as an Eluent— Effect of Pore Size in Column-Packed Particles on the Novel Separation Mode —

Imatinib therapy of chronic myeloid leukemia significantly reduces carnitine cell intake, resulting in adverse events

ObjectiveA prominent, safe and efficient therapy for patients with chronic myeloid leukemia (CML) is inhibiting oncogenic protein BCR::ABL1 in a targeted manner with imatinib, a tyrosine kinase inhibitor. A substantial part of patients treated with imatinib report skeletomuscular adverse events affecting their quality of life. OCTN2 membrane transporter is involved in imatinib transportation into the cells. At the same time, the crucial physiological role of OCTN2 is cellular uptake of carnitine which is an essential co-factor for the mitochondrial β-oxidation pathway. This work investigates the impact of imatinib treatment on carnitine intake and energy metabolism of muscle cells. MethodsHTB-153 (human rhabdomyosarcoma) cell line and KCL-22 (CML cell line) were used to study the impact of imatinib treatment on intracellular levels of carnitine and vice versa. The energy metabolism changes in cells treated by imatinib were quantified and compared to changes in cells exposed to highly specific OCTN2 inhibitor vinorelbine. Mouse models were used to test whether in vitro observations are also achieved in vivo in thigh muscle tissue. The analytes of interest were quantified using a Prominence HPLC system coupled with a tandem mass spectrometer. ResultsThis work showed that through the carnitine-specific transporter OCTN2, imatinib and carnitine intake competed unequally and intracellular carnitine concentrations were significantly reduced. In contrast, carnitine preincubation did not influence imatinib cell intake or interfere with leukemia cell targeting. Blocking the intracellular supply of carnitine with imatinib significantly reduced the production of most Krebs cycle metabolites and ATP. However, subsequent carnitine supplementation rescued mitochondrial energy production. Due to specific inhibition of OCTN2 activity, the influx of carnitine was blocked and mitochondrial energy metabolism was impaired in muscle cells in vitro and in thigh muscle tissue in a mouse model. ConclusionsThis preclinical experimental study revealed detrimental effect of imatinib on carnitine-mediated energy metabolism of muscle cells providing a possible molecular background of the frequently occurred side effects during imatinib therapy such as fatigue, muscle pain and cramps.

Enantiomeric Separation of Triacylglycerols Consisting of Three Different Fatty Acyls and Their Chiral Chromatographic Elution Behavior.

Chromatographic separation of triacylglycerol (TG) enantiomers is a highly challenging task of analytical chemistry because of the similar physicochemical properties. The analysis of chiral TGs is crucial for improving the knowledge of lipid biochemistry and for understanding the nutritional properties of fats and oils. Thus, this study aimed to systematically investigate the chiral resolution of TGs consisting of three different fatty acyls (FAs). Thirty-three asymmetric TG enantiopairs, including 49 synthesized enantiopure TGs and racemic TGs, were analyzed with a recycling chiral HPLC system. Twenty-six enantiopairs were successfully separated. Overall, having both unsaturated and saturated FAs in the outer positions or a difference in carbon chain length between two saturated FAs at the outer positions favored the separation of enantiomers. The retention time at separation correlated negatively with the sn-3 carbon number of the early eluting enantiomer and positively with the carbon number difference between sn-1 and sn-3. When the samples were studied in separate groups based on unsaturation and regioisomers, both the acyl carbon number and the degree of unsaturation of FAs in all three positions influenced the separation and elution behavior of chiral TGs, indicating an active role of both intermolecular interactions and steric hindrances. This is the first systematic study of the chiral separation of TGs consisting of three different FAs using a large number of enantiopairs. The novel findings on the behavior of TG enantiomers in a chiral environment provide important guidance and reference for a stereospecific study of the chemistry and biochemistry of natural lipids.

Gastrointestinal digestion of yerba mate, rosemary and green tea extracts and their subsequent colonic fermentation by human, pig or rat inocula

Polyphenolic compounds are common constituents of human and animal diets and undergo extensive metabolism by the gut microbiota before entering circulation. In order to compare the transformations of polyphenols from yerba mate, rosemary, and green tea extracts in the gastrointestinal tract, simulated gastrointestinal digestion coupled with colonic fermentation were used. For enhancing the comparative character of the investigation, colonic fermentation was performed with human, pig and rat intestinal microbiota. Chemical analysis was performed using a HPLC system coupled to a diode-array detector and mass spectrometer. Gastrointestinal digestion diminished the total amount of phenolics in the rosemary and green tea extracts by 27.5 and 59.2 %, respectively. These reductions occurred mainly at the expense of the major constituents of these extracts, namely rosmarinic acid (−45.7 %) and epigalocatechin gallate (−60.6 %). The yerba mate extract was practically not affected in terms of total phenolics, but several conversions and isomerizations occurred (e.g., 30 % of trans-3-O-caffeoylquinic acid was converted into the cis form). The polyphenolics of the yerba mate extract were also the least decomposed by the microbiota of all three species, especially in the case of the human one (−10.8 %). In contrast, the human microbiota transformed the polyphenolics of the rosemary and green extracts by 95.9 and 88.2 %, respectively. The yerba mate-extract had its contents in cis 3-O-caffeoylquinic acid diminished by 78 % by the human microbiota relative to the gastrointestinal digestion, but the content of 5-O-caffeoylquinic acid (also a chlorogenic acid), was increased by 22.2 %. The latter phenomenon did not occur with the rat and pig microbiota. The pronounced interspecies differences indicate the need for considerable caution when translating the results of experiments on the effects of polyphenolics performed in rats, or even pigs, to humans.

Modern chromatographic method for estimating Loratadine and affections on healthcare

PurposeThe purpose of this study is to establish Loratadine [LRD] quantification in purified and capsule formulations using a precise and specific Reversal Phase with a very high-performance liquid Chromatographic [RP-HPLC] technique. The approach was evaluated in agreement with the principles of the International Conference on Harmonization [ICH]. Arcus EP-C18 Ion Pac column, 5 m, 4.6 mm, 250 mm, mobile phase Methanol: Acetonitrile (60:40) v/v. Dibasic potassium phosphate buffer, pH 7.2, flow rate 1.0 ml/min.Design/methodology/approachThe HPLC system used a 340 nm UV detector for testing. A 10-min run time was used for the analysis. At concentrations ranging from 2 to 10 g/ml, the technique was linear (R2 = 0.9998), exact (intra-day and inter-day relative standard deviation [RSD] values 1.0%), accurate (range recovery = 96%–102%), exclusive and strong.FindingsThe detecting and quantitation limits were 0.92 g/ml and 2.15 g/ml, respectively.Originality/valueThe findings demonstrated that the proposed method could accurately determine LRD in bulk and pill dose formats quickly and accurately.

Versatile injector for inline renewable solid-phase extraction: Application to cyclodextrin-based bioaccessibility assessment in environmental solids

BackgroundSolid phase extraction (SPE) is a standard sample preparation technique in HPLC workflows. Inline cartridges are high-performance alternatives to manual or robotic systems but at long term, they suffer from irreversible sorption of matrix components and sorbent compaction. Bead injection (BI) is a niche fluidic technique that allows renewing a sorbent bed through the manipulation of its suspension. However, there is a need for a versatile and reliable tool in HPLC that can exchange the inline sorbent automatically, resorting to inexpensive and assorted bulk sorbents. ResultsWe present a new flow path for a liquid chromatographic injector to perform inline micro-solid phase extraction. The sample is processed at real time, trapping the analytes and discarding the matrix. Cleaning the matrix and injecting 10 μL of sample takes 70 s, comparable with the injection in commercial HPLC systems. If the aim is to preconcentrate the analytes, average enrichment factors of 250 have been obtained after processing sample volumes of 3200 μL in 16 min (interleavable with the chromatographic step), keeping the peak position and width independent of the injected volume (compared to large volume direct injection).The desired bed mass is automatically and pressure-driven manipulated in the valve, retained by an inline frit, and optionally, after the analysis, removed by forward flow.The chromatographic performance of the new design is compared to the standard 6-port, 2-position HPLC injector. As a case study, we have monitored the extraction kinetics of a cyclodextrin-based bioaccessibility extraction test of persistent organic contaminants in soil, by extracting several fractions in valve, process them with inline SPE with a balanced hydrophilic-hydrophobic reversed-phase sorbent, and inject the bioaccessible compounds into HPLC. Aiming at avoiding carryover, the sorbent bed (ca. 3 mg) is exchanged before every run. It should be noted that this contribution focuses on HPLC, but other non-separative techniques, such as Flow Injection Analysis, can equally benefit from this injection platform. SignificanceThis contribution reports the first use of inline BI-solid phase extraction in HPLC workflows, without heart-cut eluate injection, in which the sorbent can be exchanged automatically by forward flow. This performance is enabled by prototyping a valve that can autonomously swap sorbents in real-time for diverse samples, as a cartridge exchanger, but using cost-effective and environmentally friendlier bulk sorbents (bed masses from sub-mg to 5 mg) without requiring additional hardware.

Development of a HPLC system using a phase-separation multiphase flow as an eluent: an influence of column pressure on phase separation and chromatogram at room temperature.

We have developed a HPLC system where phase-separation multiphase flow works in the separation column as an eluent. We call the novel separation mechanism a phase-separation mode. The ternary mixed solution of water/acetonitrile/ethyl acetate, which is one of the two-phase-separation mixed solutions, caused the phase-separation multiphase flow via phase change from homogeneous to heterogeneous with the temperature effect (from 20 to 0°C). In this study, we tried to perform phase-separation multiphase flow with the pressure effect instead of the temperature one. The fused-silica capillary tube (50cm length and 50µm inner diameter) was allied to the downstream of the column to apply the pressure of 5.5MPa to the system. Model analytes of 2,6-naphthalenedisulfonic acid (2,6-NDS) and 1-naphthol (1-NA) were examined. For example, solutions (the volume% of water/acetonitrile/ethyl acetate; 20:55:25, organic-rich) and (60:30:10, water-rich) were used as eluent. The model analytes were not separated with both solutions at the pressure of 1.5MPa and 20°C. But with the organic-rich solution, 1-NA and 2,6-NDS were separated in this order and with the water-rich solution, they were separated in the reverse order at the pressure of 5.5MPa and 20°C. The phase-separation mode could be performed at the high pressure even at the room temperature.

RP-HPLC Method Development and Validation for Simultaneous Determination of Sofosbuvir and Ledipasvir: A Pharmaceutical Application Study

The objective of the present work was to develop a simple, precise, accurate, reproducible, and specific reversed-phase high performance liquid chromatographic method for the simultaneous determination of sofosbuvir and ledipasvir in pharmaceutical dosage form. The HPLC system (Shimadzu, LC-2030) consisted of Hypersil ODS - C18 (250 mm, 4.6 mm, 5 μm- Thermo Scientific) was used for the isocratic elution using mobile phase composed of sodium phosphate buffer (pH 3.5) and acetonitrile in the ratio of 40:60% (v/v). Both sofosbuvir and ledipasvir were detected at 260 nm with a flow rate of 1 mL/min. The mean retention time of sofosbuvir was 3.094, whereas ledipasvir was 6.22 minutes. A linear relationship was found in the concentration ranged from 0.54 to 162 μg/mL for sofosbuvir (r2 = 0.9993) and 0.14 to 42 μg/mL for ledipasvir (r2 = 0.9994). The RSD values for recovery test were found 0.99% for sofosbuvir and 0.22% for ledipasvir. The lower limit of detection (LOD) were 0.054 mcg/mL and 0.012 mcg/mL, and the limit of quantification (LOQ) were 0.135 mcg/mL and 0.030 mcg/mL for sofosbuvir and ledipasvir, respectively. The method was successfully applied to the assay and in-vitro dissolution studies of sofosbuvir and ledipasvir in tablet dosage form and all the results were found within the acceptance limits.

Assessment of metabolic interaction between curcumin and tramadol using the isolated perfused rat liver

IntroductionThe presence of phytochemicals in herbal medicines can lead to herb-drug interactions, altering the levels of these compounds and conventional drugs in the bloodstream by influencing CYP450 activity. Considering curcumin's effect on the CYP enzymes responsible for tramadol metabolism, it is essential to assess the potential interaction between curcumin and tramadol when administered together. Materials and methodsThe pharmacokinetics of tramadol were examined in rats receiving either single or multiple doses of curcumin (80 mg/kg) compared to rats without curcumin treatment. Tramadol liver perfusion was conducted on all rat groups and perfusate samples were collected at specified intervals. Tramadol and its main metabolite were detected using an HPLC system coupled with a fluorescence detector. ResultsTramadol concentrations were notably higher in the co-administered group compared to both the control and treatment groups. Conversely, lower concentrations of M1 were observed in the co-administered and treatment groups compared to the control group. The AUC0-60 parameters for tramadol were as follows: 32944.8 ± 1355.5, 22925.7 ± 1650.1, and 36548.0 ± 2808.4 ng⋅min/ml for the control, treatment, and co-administered groups, respectively. Both the co-administered and treatment groups exhibited a lower AUC0-60 of M1 compared to the control group. The lack of significant difference in Cmax and AUC0-60 of M1 between the treatment and co-administered groups suggests that single and multiple doses of curcumin have comparable effects on CYP2D6. ConclusionsThese results indicate a potential for drug interactions when curcumin and tramadol are taken together. Furthermore, the influence of curcumin on tramadol metabolism varied between single and multiple oral administrations of curcumin. Hence, it is vital to highlight this interaction in clinical settings and conduct additional research to fully understand the clinical implications of combining curcumin and tramadol.

A new sensitive and robust method development for estimation of tolcapone and quinapril in the bulk and formulations

Tolcopone and Quinapril are anti-hypersensitive drugs and, in combined dosages, are effective in managing hypertension. The RP-HPLC method was developed using OPA and Acetonitrile in 50:50 v/v as Mobile phase and 250*4.6mm, 5µ RP-HPLC column measured with a wavelength of 240nm. The HPLC system was fixed with a flow rate of 1.0ml/min; Both drugs, TOLCO and QUINA, were successfully detected at 2.153 and 3.203 min, respectively. The validation of the method as per ICH Linearity r2 lies at 0.999, and Precision, system suitability, and selectivity RSD values are within permissible limits. The accuracy of the process was found to be 99.73 and 99.52 for TOLCO and QUINA, respectively. The LOD 0.72 and 0.3 µl/ml and LOQ 0.84 and 0.13 µl/ml. The robustness and degradation studies show that the selected method is acceptable per the Guidelines.Bottom of Form

To Study Force Degradation Of Ozenoxacin Using RP-HPLC

In this study a simple, robust and accurate RP-HPLC was developed and validated for the force degradation study of ozenoxacin as per ICH guidelines. Chromatographic separation and elution of peak was achieved by isocratic elution on agilant – 1260 infinite II HPLC system. An analysis C18X150X4.6mm 5mm, eclipse XDB column was used at flow rate 1ml/min in the mobile phase ratio methanol: 10 mM (adjusted pH -3 in the ratio of 42:52 %v/v at wavelength 250 nm and the temperature 25ºc. The force degradation study was performed on ozenoxacin under acidic, basic, oxidation, thermal, photodegradation,. The result obtained from the experiments was proved that the developed method suitable for routine analysis.