HPLC Method For Analysis Research Articles

CIM monolithic chromatography as a useful tool for endotoxin reduction and purification of bacteriophage particles supported with PAT analytics

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.

The Comparative Study of Different Pharmacopoeias: Indian Pharmacopoeia, British Pharmacopoeia, United States Pharmacopoeia

The objective of the study is to ensure Indian pharmacopoeia monographs are on par with United States, British pharmacopoeia standards. A few monographs were selected on random and compared. It has been observed that Indian pharmacopoeia has replaced titrimetrry assay method of analysis to sophisticated chromatographic techniques. As a whole the comparative study reveals that Indian pharmacopoeia is on par with United States, British pharmacopoeia and it is necessary that global countries also recognize Indian pharmacopoeia. General monographs available in Indian pharmacopeia 2014 were compiled and analyzed to identify the number of analytical method employed especially in the assays. HPLC Method of analysis is highest used where ever necessary UV method is made available instead especially for formulation, Indian pharmacopeia Commission is periodically updating indian Pharmacopoeia on par with ICH Q4 pharmaceutical harmonization and has its unique identity over years and being recognized globally.

Liquid Chromatographic Determination of <i>o</i>-Phosphoethanolamine in Human Plasma Using Fluorescent Derivatization

In this study, an HPLC analysis method using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was developed for the determination of o-phosphoethanolamine (PEA), which is a potential biomarker for the diagnosis of major depressive disorder, in human plasma sample. After PEA was derivatized with AQC under mild conditions, the obtained derivative was subjected to purification with a titanium dioxide-modified monolithic silica spin column (MonoSpin® TiO). The eluate from the MonoSpin® TiO was directly injected into an amide-type hydrophilic interaction liquid chromatography (HILIC) column-equipped HPLC system, and the resulting derivative could be separated on the column under alkaline mobile phase conditions and subsequently detected fluorometrically at excitation and emission wavelengths of 250 and 395 nm, respectively. The limit of detection and limit of quantification for a 10 µL injection volume of PEA were 0.052 and 0.17 µM, respectively. The method was validated at 0.2, 1.0, and 5.0 nmol/mL levels in plasma sample, and the precision values were 2.0-6.6% as relative standard deviation and the correlation coefficient (r) of the calibration curve was 0.9995. Furthermore, applicability of this method was demonstrated by analyzing PEA levels in plasma samples from mental illness patients.

An Efficient 4-Step Synthesis of Favipiravir with Industrial Potential

An efficient, economical synthetic route is developed for the antiviral drug favipiravir using a four-steps instead of a six-step protocol. Starting with 6-bromo-3-hydroxypyrazine-2-carboxamide the method offers an overall 65% molar yield. The prime intermediate 3,6-difluoropyrazine-2-carbonitrile was synthesized by reacting 3,6-dichloropyrazine-2-carbonitrile with potassium fluoride using tetrabutyl-ammonium bromide (TBAB) as a phase transfer catalyst in toluene and DMSO medium at reflux temperature (120 ºC). A simple purification method from fluoro intermediate was developed by making dicyclohexylamine salt and a neutral compound using hydrogen peroxide. The prepared pure favipiravir is fully characterized using NMR Mass and IR techniques. Validated analytical HPLC methods assessed the purity of the drug. The quality of the drug synthesized in terms of assay and impurities is well within the ICH and pharmacopeia standards.

Laccase의 (+)-Catechin 기질을 이용한 Catechin-중합체 생성

When the reaction condition of polymer production from (−)-catechin was examined, the catechin-polymer was produced, but the anti-oxidative activity was lower than that of (−)-catechin. Therefore, in this study, (+)-catechin, which has the highest content in green tea catechin, was used to establish the reaction condition for production of (+)-catechin-polymer having anti-oxidative activity by laccase. In addition, the absorption regions of supernatant and precipitates were compared using a scanning method in the UV-visible region (250~450 nm) to identify the presence of (+)-catechin-polymer along with HPLC analysis method. The optimal temperature and pH for laccase-induced (+)-catechin-polymer production were 30°C and pH 5.0. Also, the optimal (+)-catechin-polymer production was achieved at the 1.5% laccase and 0.5% substrate content. In substrate specificity of laccase, (+)-catechin and epicatechin were efficient, whereas EGCG and removed-GA were not. However, despite (+)-catechin-polymer production, it did not increase anti-oxidative activity as in the case of (−)-catechin-polymer, so we will try to find a suitable substrate among green tea catechins in the future.

RP HPLC Method Development and Validation for Estimation of Orlistat in Pharmaceutical Dosage Forms

Orlistat is an anti-obesity drug that inhibits the secretion of gastric and pancreatic lipases. Normally lipase breakdown triglyceride in the intestine as a result of metabolism. Despite the numerous advantages of this drug, researchers are facing problems with estimation by HPLC because limited methods are available; only United State Pharmacopoeia (USP) method is most reliable and effective. The present work aimed to develop an RP- HPLC method for determining orlistat in floating microcapsules. Considering the virtual reality in drug testing, quality control of dosage forms is the main criterion to be followed in the Pharmaceutical industry, which is to establish the procedures undertaken to ensure the identity and purity of a particular dosage form. Syncronis C18, 150mm x 4.6mm, 3μm column were used. Isocratic elution was performed with ACN:Water mixture. The flow rate was 2.0 mL min-1 and UV detection was at 205 nm. Caffeine was used as an internal standard. The developed method was validated according to the ICH guidelines and found to be linear within the range 150-600 ppm of Orlistat. In addition, this method was validated for its suitability, accuracy, precision, selectivity, robustness, and ruggedness per the ICH guidelines. The results HPLC analytical method validation was suitable for the quantitative and qualitative analysis of orlistat and it showed that this method could be successfully utilized for the identification and quantification of orlistat in any dosage form, with high resolution, accuracy, and precision.

Effect of Ripening Stage on Biochemical Composition of Seeds from three Species of Canavalia spp Consumed as Protein Substitutes in Côte d’Ivoire

Aims: The present study was aimed at investigating the nutritional properties of legume-seeds from Canavalia genus as a function of their ripening stage.
 Methodology: Seeds of three species of canavalia seeds notably Canavalia gladiata, Canavalia ensiformis and Canavalia rosea were obtained from an experimental plot located in Bouaké area’s (7°41′00″ N, 5°01′59″ W) in Côte d’Ivoire. The seeds of these three species were collected at different ripening stages. The seeds were sun dried and ground to obtain the crude flour. Chemical composition and functional properties were investigated using standard methods while amino acid profiles were performed by using HPLC analytical methods.
 Results: Results reveal that the profiles of nutrients in the three Canavalia species varied as a function of ripening stages. The highest fat (3.27 ± 0.01 %), protein (33.20 ± 0.17), fibre (7.51 ± 0.06 %), ash (6.67 ± 0.01 %) and total sugar (25.40 ± 0.05 %) contents were obtained at the last ripening stage. All the legume-seeds investigated are excellent sources of proteins mainly consisting of high amounts of essential amino acids such as lysine (100.23 ± 0.04 mg/g protein), leucine (103.51 ± 0.04 mg/g protein), valine (83.68 ± 0.04 mg/g protein), methionine (27.36 ± 0.04 mg/g protein), tryptophan (74.32 ± 0.04 mg/g protein), cysteine (44.24 ± 0.04 mg/g protein), isoleucine (63.61 ± 0.04 mg/g protein), threonine (40.22 ± 0.04 mg/g protein) and histidine (17.76 ± 0.04 mg/g protein). As regards mineral contents of these seeds, they were higher at penultimate ripening stage and, they partially meet dietary allowance of infants.
 Conclusion: These seeds could be ranked as protein rich food for human and then, used as valuable substitutes for meat or fish.

Anti-adipogenic Effects of Sulforaphane-rich Ingredient with Broccoli Sprout and Mustard Seed in 3T3-L1 Preadipocytes.

Glucoraphanin (GRA) is a precursor of sulforaphane (SFN), which can be synthesized by the enzyme myrosinase. In this study, we developed and validated HPLC analytical methods for the determination of GRA and SFN in mustard seed powder (MSP), broccoli sprout powder (BSP), and the MSP-BSP mixture powder (MBP), and evaluated their anti-adipogenic effects in 3T3-L1 adipocytes. We found that the analysis methods were suitable for the determination of GRA and SFN in MSP, BSP, and MBP. The content of GRA in BSP was 131.11 ± 1.84 µmol/g, and the content of SFN in MBP was 162.29 ± 1.24 µmol/g. In addition, BSP and MBP effectively decreased lipid accumulation content without any cytotoxicity. Both BSP and MBP significantly inhibited the expression of adipogenic proteins and increased the expression of proteins related to lipolysis and lipid metabolism. BSP and MBP inhibited the expression of adipocyte protein 2 (aP2), CCAAT/enhancer-binding protein-α (C/EBP-α), and peroxisome proliferator-activated receptor-γ (PPAR-γ) in 3T3-L1 adipocytes, and inhibited the expression of fatty acid synthase (FAS) through AMP-activated protein kinase (AMPK). Meanwhile, BSP and MBP also increased the expression of the lipolysis-related proteins, uncoupling protein-1 (UCP-1) and carnitine palmitoyltransferase-1 (CPT-1). Moreover, MBP exerted anti-adipogenic to a greater extent than BSP in 3T3-L1 preadipocytes.

HPLC Analytical Method Development and Validation of Gabapentin through Chemical Derivatization with Catechol as a Chromophore.

Background Gabapentin is a drug with anticonvulsant activity and has been widely used in the treatment of epilepsy. Gabapentin chemical structure lacks a chromophore which makes its absorption very low and hence complicates its analysis and reduces the sensitivity of the method. Adding a chromophore by chemical derivatization makes the drug easily identified and quantified at a much lower concentration using chromatographic analysis such as HPLC. Methodology. The derivatization of gabapentin was done by adding a chromophore to the structure by introducing an auxochrome group. Suitable coupling reagents were used to introduce catechol group to gabapentin. The analytical method has been developed using HPLC with UV/Vis detector. Moreover, the method was validated for parameters such as linearity, range, precision, accuracy, LOD, and LOQ. Result The developed method adapted derivatization of gabapentin using catechol reagent measured at λmax 300 nm. The method used HPLC using mobile phase methanol water 50 : 50. The eluted peak of the derivatized gabapentin was separated from other used derivatization reagents. The analytical method showed to be a validated method, and all the tested validation parameters were within the accepted limits. The developed method was found to be linear (R2 = 0.9917), precise (RSD = 0.91) and accurate (% recovery = 105). Moreover, the developed method was sensitive with LOD (0.5∗10−6 mg/mL) and LOQ (1.5∗10−6 mg/mL). Conclusion The developed method is simple and feasible with high sensitivity and selectivity. It can be applied in the analysis of gabapentin in different dosage forms and raw materials including active pharmaceutical ingredients (API). This research work can be continued in the future, and the developed method can be used for testing gabapentin in biological systems.

Development and Validation of RP-HPLC Method for Quantification of Total, Free and Entrapped Ritonavir in Lipid Nanocarriers and Drug content of Film Coated Fixed Dose Formulation

Abstract: Aim: The current study was aimed to develop and validate a simple isocratic, precise, sensitive, accurate and robust reverse phase HPLC analytical method for the quantification of total, free and entrapped ritonavir in lipid nanocarriers and drug content of the film-coated fixed-dose formulation. Materials and Methods: The method was developed by using Inertsil ODS-3V C18 column as stationary phase, orthophosphoric acid (OPA) in water (pH 3.0) and acetonitrile as mobile phase. Further, the method was validated following ICH Q2(R1) guidelines. Ritonavir loaded lipid nanocarriers were developed using hot-emulsion and probe-sonication method. A solid phase extraction (SPE) method was developed for the separation of free and entrapped ritonavir. Total ritonavir was extracted from lipid nanocarriers (LNCs) and film-coated tablets using methanol and analyzed with validated method. Results: The responses were found to be linear and precise over a range of 0.25 μg/mL to 16 μg/mL. The limit of detection (LOD) and limit of quantification (LOQ) of the RP-HPLC method were found to be 14.06 ng/mL and 42.60 ng/mL, respectively which emphasizes the sensitivity of the method. The recovery of ritonavir from LNCs was found to be within 3% of the total drug present in LNCs. Similar, amount of drug recovery was found from assay of marketed formulation i.e., > 97%. Conclusion: The SPE method is capable of separating free and entrapped ritonavir from LNCs. The developed HPLC method is sensitive, robust, economical and gives high recovery. The developed HPLC method can also be employed for routine quantitative analysis of ritonavir in bulk formulation. Key words: HPLC method, Ritonavir, Validation, SPE, NLC, Bulk formulation.

Development and Validation of a Sensitive HPLC Assay for Determination of Sparfloxacin According to the European Medicines Agency Guideline

Sparfloxacin, a synthetic antibiotic belonging to the third-generation fluoroquinolones, has numerous pharmacokinetic and microbiological advantages which can make it an excellent candidate for the treatment of infections in sheep. The objective of this study was to develop and validate an analytical HPLC method to quantify sparfloxacin in sheep plasma, following the recommendations set out in the Guideline on Bioanalytical Method Validation of the European Union (EMEA/CHMP/EWP/192217/2009). The HPLC mobile phase consisted of acetonitrile and monopotassium phosphate buffer (1.36 g/L) 49:51 (v/v). Genabilic acid was used as internal standard. Mean retention times for sparfloxacin and genabilic acid were 2.6 and 5.8 min, respectively. The method met all specifications of the EMA guideline, being selective and linear in the range of 0.2–10 µg/mL (R2 ≥ 0.99). Within-run precision ranged between 0.00 and 0.88%, with an accuracy of 90.3–118.0% for the lower limit of quantitation (LLOQ). The LLOQ was 0.2 µg/mL, and no interference from the biological matrix was found. The stability of sparfloxacin in the biological matrix was demonstrated under different storage conditions. Therefore, the method can be used to determine sparfloxacin concentrations in sheep plasma in different types of studies.

Potential Application of Carbon‐based Electrical Sensor for the Highly Sensitive Diltiazem HCl Quantification in its Pharmaceutical Products and Biological Samples

AbstractDiltiazem (DTZ) hydrochloride, a calcium channel blocker compound, is a very well‐known drug used by many clinicians to treat important diseases playing a role in increased morbidity and mortality among adults worldwide, namely hypertension, cardiac arrhythmia, and ischemic heart disease (angina pectoris). These diseases are a common public health concern, implying that the construction of a specific, accurate, and simple sensor for DTZ determination is needed. Herein, an innovative, portable, and sensitive modified carbon paste electrode (MCPE) was seamlessly developed based on the affordable green compound N‐bromosuccinimide (NBS) as a sensing material and tricresyl phosphate as a solvent mediator for the voltammetric determination of DTZ. The proposed NBS‐MCPE exhibited an excellent electrocatalytic activity towards the oxidation of DTZ molecules in a phosphate buffer solution of pH 5.0 in presence of 20 μM sodium dodecyl sulfate to improve the developed sensor performance. The sensor was found to respond linearly to the DTZ drug over a wide concentration ranging from 1 μM to 300 μM with a low limit of detection. LSV and EIS measurements were also used to scrutinize our sensor, in addition to using scanning electron microscopy and energy dispersive X‐ray analysis techniques for its surface morphology inspection before and after soaking in the drug‐containing solution. The applicability of the proposed sensor for the rapid, sensitive, and selective determination of DTZ in pharmaceutical preparation and human urine samples was assessed and the obtained results were compared with that of the reported HPLC analytical method.

Phytochemical and pharmacological study of Rosa x gallica L. georgian cultivar essential oil production waste

The study aimed to assess chemical composition and pharmacological properties of Rosa x gallica cultivated in Georgia for application in medicine and cosmetology. The quantitative and qualitative composition of secondary metabolites of the aqueous extract obtained during the production of essential oil of R. gallica (AERGP) was analysed using HPLC analytical methods, and its pharmacological activity was evaluated in in vitro and in vivo experiments. Five prevalent compounds have been established in AERGP: Glycyrrhetinic acid (0.83%), Hyperoside (1, 08%), Caffeic acid (0, 85%), Quercetin (6, 17%), and Ferulic acid (0, 28%). Studies of antioxidant activity of AERGP revealed its dose-dependent radical scavenging effect in both ABTS (IC50 20.0 µg/mL), and DPPH (IC50 39.0 µg/mL) assays. AERGP appeared active against Plasmodium falciparum 3D7 strain (IC50 47.2 µg/mL). In in vivo experiments AERGP showed pronounced analgesic (124%) and anti-inflammatory effects (67.58%), as well as a moderate gastroprotective efficacy (28, 9%).

Box–Behnken Design-Based Development and Validation of a Reverse-Phase HPLC Analytical Method for the Estimation of Paclitaxel in Cationic Liposomes

Stability-indicating reverse-phase HPLC analytical method for the quantification of Paclitaxel (PTX) in the bulk and cationic liposomes was developed. The optimized method was validated according to the ICH Q2 (R1) guidelines by following a 2-level–4-factor interaction Box–Behnken design using Design-Expert® software. The responses measured at 228 nm were retention time (Rt), peak area, tailing factor (Tf10%), and the number of theoretical plates (NTP). PTX was eluted best using the Luna® C18 LC Column along with a mobile phase of methanol and 25 mM ammonium acetate buffer (pH 6) 75:25 v/v mixture at 25 ± 2 °C temperature. The currently developed method was linear in the 2.5–100 µg/mL range with a detection limit of 0.062 µg/mL and a quantification limit of 0.188 µg/mL. The optimized method was utilized to evaluate the stability of PTX in different stress conditions by performing forced degradation studies. The results from the degradation study stipulated that on exposure to various stressors, namely acid, alkali, oxidative, thermal, and UV light, the PTX did not show considerable degradation except alkali exposure. Further, the method was successfully used for the quantification of PTX in cationic liposomes. The particle size, zeta potential, and polydispersity index of the PTX-loaded liposomes were 219.25 ± 7.566 nm, 57.15 ± 12.374 mV, and 0.807 ± 0.1958 respectively. The percent of drug entrapped was quantified and was found to be 59 ± 1.414%.

DoE Engineered Development and Validation of an RP-HPLC Method for Simultaneous Estimation of Temozolomide and Resveratrol in Nanostructured Lipid Carrier

Abstract Background Temozolomide is drug of choice for the treatment of glioblastoma, but dose-related side effects limit its use. Resveratrol suppresses tumor growth and promotes apoptosis. Many studies showed synergistic activity of resveratrol and temozolomide against glioblastoma. There are methods reported for the assessment of temozolomide and resveratrol individually, but no analytical method has been reported for assessment of temozolomide and resveratrol simultaneously. Objective Therefore, the present study aimed to develop and optimize an HPLC analytical method for the simultaneous assessment of temozolomide and resveratrol in a developed nanostructured lipid carrier. Method A Central composite rotable design was used to optimize the method. The method was developed using a C18 column. The composition of the mobile phase was 30% methanol and 70% glacial acetic acid (0.1% v/v in HPLC grade water); detecting wavelength was 310 nm. Forced degradation test was also performed to demonstrate the proposed HPLC method’s ability to indicate stability. Results The LOD for temozolomide and resveratrol was found to be 1.10 and 0.83 µg/mL, respectively, while LOQ was 3.33 and 2.52 µg/mL, respectively. The drug loading and entrapment efficacy of the formulation, as determined using the aforementioned method, was found to be 6.73 and 96.28% for temozolomide and 3.45 and 89.39% for resveratrol, respectively. Conclusions The developed HPLC method was simple, rapid, economical, precise, accurate, and reproducible, and it had high selectivity with good detection limits. Standard guidelines of ICH Q2 (R1) including linearity, specificity, system suitability, robustness, precision, accuracy, the LOQ, and LOD gave satisfactory results. Forced degradation studies showed a good stability-indicating capacity of the developed HPLC method. Highlights Analytical Quality by Design is a powerful tool that could be used for the development of the analytical method. Central composite rotable design was used for optimizing the method. The percent of methanol and concentration of glacial acetic acid were selected as two independent variables for optimization.

Ferrocene‐modified imidazoles: One‐pot oxalyl chloride‐assisted synthesis, HPLC enantiomeric resolution, and in vivo antitumor effects

AbstractHighlights on the development of ferrocene biochemistry in Russia since the 1950s are given. Here, avoiding high‐toxic precursors, ferrocene‐based imidazoles and benzimidazoles (16 compounds) were synthesized in good to high yields via the reaction of the α‐(hydroxy)alkyl ferrocenes with modified heterocycles using oxalyl chloride to activate imidazole system. Mild conditions (room temperature, chloroform, stirring for up to 1.5 h) allowed preparing a number of various ferrocene‐containing imidazoles, which are promising as antitumor agents. The analytical HPLC method was successfully applied to enantioseparation of the resulting ferrocene enantiomers (Chiralcel OD column). Antitumor activities of the preparing compounds against murine solid tumours such as Acatol adenocarcinoma and Ca755 carcinoma have been studied in vivo. Some of the tested compounds have been strong effective against Acatol adenocarcinoma. The value of the tumour growth inhibition coefficient was about 90% versus control in this strain.

Antiinflammatory and Anticancer Properties of Grewia asiatica Crude Extracts and Fractions: A Bioassay-Guided Approach.

The study was an extension of our earlier work on antiinflammatory and anticancer properties of G. asiatica fruit. We aimed to develop a bioassay guided multistep purification technique for producing bioactive fractions of G. asiatica crude extracts. Dried fruit powder was sequentially fractionated with 100% dichloromethane, 100% methanol (MeOH), and 50% MeOH. Active extracts were subjected to liquid-liquid partitioning followed by subfractionation using RP-HPLC. Antioxidant, antiinflammatory, and anticancer activities of the fruit extracts, and their potent fractions were evaluated in vitro, while identification of compounds from the bioactive fractions was performed by ESI-MS/MS analysis. The amount of the identified compounds present was confirmed using external standards adopting a simple, accurate, and rapid analytical HPLC method. The results showed that 100% and 50% MeOH extracts possessed bioactivity; one of which (the 50% MeOH extract) displayed potent activity in all in vitro bioassays. MeOH extract (50%) derived fraction C and hydroalcoholic fraction 5 (GAHAF5) were observed to possess higher antioxidant, antiinflammatory, and in vitro anticancer activity. IC50 of GAHAF5 against MCF-7, HEp-2, and NCI-H522 cancer cells was recorded as 26.2, 51.4, and 63 μg/mL, respectively. ESI-MS/MS and HPLC analysis identified catechin, chlorogenic acid, caffeic acid, and morin as potential bioactive compounds in the GAHAF5 fraction with concentrations of 1230, 491, 957, and 130 μg/g, respectively. The findings indicated that G. asiatica bioactive fractions possessed antiinflammatory activity in vitro and were cytotoxic against breast cancer, lung cancer, and laryngeal cancer cell lines.

Determination of methylxanthine contents and risk characterisation for various types of tea in Korea

Tea leaves contain methylxanthines, such as caffeine, which have various physiological and pharmacological effects in vivo. Caffeine, theobromine and theophylline were analysed in various teas to determine the exposure amount and health risk of methylxanthine intake from tea. All the determined validation parameters of the HPLC analysis method, which included specificity, linearity of the calibration curve, detection limit, reproducibility and accuracy, showed satisfactory results. When 83 leached extracts of 11 different teas were analysed by the HPLC method, the average contents of caffeine, theobromine and theophylline were 5561.5, 407.3 and 24.8 mg/kg, respectively. Pu-erh tea contained the highest average levels of caffeine, while hibiscus tea presented the highest average levels of theobromine and theophylline. Based on the seventh Korea National Health and Nutrition Examination Survey data on food intake, the daily intake of caffeine, theobromine and theophylline was calculated as 30.819, 1.408 and 0.011 μg/kg body weight/day, respectively, showing low levels of methylxanthine intake compared to the recommended daily intakes.

Effects of dihydroartemisinin, a metabolite of artemisinin, on colon cancer chemoprevention and adaptive immune regulation.

Artemisinin (ART) is an anti-malaria natural compound with a moderate anticancer action. As a metabolite of ART, dihydroartemisinin (DHA) may have stronger anti-colorectal cancer (CRC) bioactivities. However, the effects of DHA and ART on CRC chemoprevention, including adaptive immune regulation, have not been systematically evaluated and compared. Coupled with a newly-established HPLC analytical method, enteric microbiome biotransformation was conducted to identify if the DHA is a gut microbial metabolite of ART. The anti-CRC potential of these compounds was compared using two different human CRC cell lines for cell cycle arrest, apoptotic induction, and anti-inflammation activities. Naive CD4+T cells were also obtained for testing the compounds on the differentiation of Treg, Th1 and Th17. Using compound extraction and analytical methods, we observed for the first time that ART completely converted into its metabolites by gut microbiome within 24h, but no DHA was detected. Although ART did not obviously influence cancer cell growth in the concentration tested, DHA very significantly inhibited the cancer cell growth at relatively low concentrations. DHA included G2/M cell cycle arrest via upregulation of cyclin A and apoptosis. Both ART and DHA downregulated the pro-inflammatory cytokine expression. The DHA significantly promoted Treg cell proliferation, while both ART and DHA inhibited Th1 and Th17 cell differentiation. As a metabolite of ART, DHA possessed stronger anti-CRC activities. The DHA significantly inhibited cell growth via cell cycle arrest, apoptosis induction and anti-inflammation actions. The adaptive immune regulation is a related mechanism of actions for the observed effects.

Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [68Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method.

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in β-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R–avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic β-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [68Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a 68Ge/68Ga Generator (GalliaPharma®) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP®). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 μg/mL) and [68Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [68Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5–0.75 μg/mL for both the analytes (NODAGA-exendin-4 and 68Ga-NODAGA-exendin-4), with a correlation coefficient (R2) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [68Ga]Ga-NODAGA-exendin-4 was linear with a R2 of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 μg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [68Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [68Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [68Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.