A Comparative Study of Two Oroxylum indicum (L.) Kurz. Phenotypes Based on Phytochemicals and Antioxidant Effects, and the Anti-Inflammatory Activity of Leaf and Pod Extracts.

Indian trumpet tree Oroxylum indicum (L.) Kurz exhibits a wide range of biological activities in all plant parts, including anti-inflammation, antioxidant, and wound-healing activities. In Thailand, there are tall- and short-stem phenotypes. The latter are preferred for commercial cultivation due to their fast growth and lower harvesting cost. This study aimed to compare the chemical profiles and antioxidant effects of leaves and young pods between two phenotypes using principal component analysis (PCA) and then to evaluate the anti-inflammatory potential of the selected phenotype's plant parts. The biomarker contents were quantified by HPLC. The antioxidants were determined using the DPPH, ABTS, and FRAP models. Nitric oxide (NO) production assays in LPS-induced RAW264.7 macrophages were performed to determine the anti-inflammatory property of the extracts. The PCA revealed that there were no differences in total phenolic content, total flavonoid content, or antioxidant activities between short- and tall-stem phenotypes. Higher potency of the NO-inhibitory effect was achieved from the leaf extract than the pod extract. These results support using the short-stem phenotypes for utilizing the leaf and pod of O. indicum, and suggest choosing the leaf part for further anti-inflammatory product development.

A new method for quantification of tomatine-enriched extracts.

BACKGROUND: Green tomato extracts, an agro-food industry waste, are rich in the glycoalkaloid tomatine, which presents activity against several diseases. High-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is one of the most used techniques for quantification of bioactive compounds. The aim of this study was to optimize and validate a selective HPLC method with diode array detector (DAD) for the quantitative analysis of tomatine extracted from green tomatoes by subcritical water. RESULTS: Chromatographic runs were performed on a InertSustain Phenyl (250 mm × 4.6 mm, 5 µm) analytical column, at a wavelength of 205 nm. A concentration range of 50-580 µg mL-1 was used. The validation process was performed considering the linearity, precision, trueness, limit of detection (LOD) and limit of quantitation (LOQ) of the method. The selected mobile phase composed of acetonitrile and a solution of 20 mmol L-1 potassium dihydrogen phosphate (KH2PO4) pH 3, resulted in suitable retention times and a standard calibration curve with adequate linearity (R2 = 0.9999). The method trueness was evaluated by the recovery assay, obtaining a mean recovery of 105% and the precisions were 1.4% and 0.9% (percentage relative standard deviation, RSD%) for the tomatine standard and extract samples, respectively. The inter-day variability was 2.7-9.0% (RSD%) for the standards and 6.9% (RSD%) for extract. The LOD and the LOQ of the method were determined at 8.0 and 24.1 µg mL-1, respectively. CONCLUSION: The herein described method was successfully used for the quantification of tomatine in a tomato-derived extract. Furthermore, the method constitutes a simple and rapid analytical approach able to be used as a routine protocol. © 2024 Society of Chemical Industry.

Development and validation of a HPLC method for the determination of chemical and radiochemical purity of O-(2-[18F]fluoroethyl-l-tyrosine ([18F]FET)), a PET radiotracer for the imaging of brain tumor.

A novel HPLC method was developed and validated to determine radiochemical identity, radiochemical purity and chemical purity for the analysis of O-(2-[18F]fluoroethyl-l-tyrosine ([18F]FET). In this method, an analytical Phenomenex Gemini C18 column was used with an isocratic eluent of 7 % ethanol and 93 % 50 mM potassium phosphate buffer (pH = 6.9). The flow rate was 1.0 mL/min and the injection volume was 10 µL. A photo-diode array detector set at 220 nm was used for UV mass detection and a single channel, high sensitivity radiation detector was used. The method validation assays including specificity, linearity, precision, accuracy, and robustness were evaluated. Results show that the method was suitable for qualitative and quantitative determination of radiochemical and chemical purity of [18F]FET. This system has been routinely used for the analysis of more than 120 batches of [18F]FET with radiochemical yield 23.7 ± 6 % (no decay corrected) and molar activity 593 ± 284 GBq/µmole in our facility to support human use.

The organic acid environmental capacity of mangrove ecosystem in Dongzhai harbor, Hainan, China.

To establish a method for studying the organic acid environmental capacity of mangrove ecosystems, high-performance liquid chromatography was used to measure the organic acid detoxification agent; Using different cultivation methods to determine the toxicity threshold of organic acids on mangrove plants; Calculate the environmental capacity of organic acids by combining the toxicity threshold of organic acids with the volume of water in the study area. The results showed the range of toxicity thresholds of organic acids to 25.29-30 mg/L would have an inhibitory effect on the development of mangrove plant hypocotyls; The organic acid environmental capacity of Dongzhai harbor Mangrove Wetland Protection Area is 7.76 × 10^4 kg/d ~ 8.73 × 10^4 kg/d, while the estimated organic acid emissions from shrimp ponds around Dongzhai harbor are 7.06 × 10^3 kg/d ~ 7.83 × 10^3 kg/d. Therefore, the organic acid emissions from shrimp ponds around Dongzhai harbor are within the carrying range of the mangrove wetland ecosystem in Dongzhai harbor.

Validation of an Analytical Method of 3',4',5-Trihydroxy-3-Methoxy-6,7-Methylenedioxyflavone 4'-Glucuronide for Standardization of Spinacia oleracea.

Spinach (Spinacia oleracea) is one of the most famous vegetables worldwide, rich in essential metabolites for various health benefits. It is a valuable plant source that has the potential to be a nutraceutical. This study aimed to evaluate the single characteristic marker compound to establish the validation of HPLC-DAD methods applied to the development of a nutraceutical using spinach samples. Six metabolites (1-6) were identified from the spinach samples such as freeze-dried spinach (FDS) and spinach extract concentrate (SEC) by LC-Q-TOF/MS analysis. Among the six metabolites, 3',4',5-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4'-glucuronide (TMG) was selected as a marker compound due to its highest abundance and high selectivity. The specificity, accuracy, linearity, precision, repeatability, limit of detection (LOD), and limit of quantification (LOQ) of TMG in the spinach samples (FDS and SEC) were validated according to AOAC international guideline. The specificity was confirmed by monitoring the well separation of the marker compound from other compounds of spinach samples in the base peak intensity (BPI) and ultraviolet (UV) chromatogram. The calibration curve of TMG (15.625~500 µg/mL) had reasonable linearity (R2 = 0.999) considered with LOD and LOQ values, respectively. Recovery rate of TMG was 93-101% for FDS and 90-95% for SEC. The precision was less than 3 and 6% in the intraday and interday. As a result, the HPLC-DAD validation method of TMG in the spinach samples (FDS and SEC) was first established with AOAC and KFDA regulations for approving functional ingredients in functional foods.

Developed and Validated for the Estimation of Bupropion and Dextromethorphan in a Fixed Dose Combination of the Tablet.

Objectives: The aim of this study was to develop a simple, accurate, and precise method for the estimation of bupropion and dextromethorphan in a fixed-dose combination of tablets and robust high-performance liquid chromatography for assay analysis of such a fixed combination. Materials and Methods: Chromatographic analysis was performed and separations were achieved on a Denali C18 150 × 4.6 mm, 5 micron using a mobile phase composition of ortho-phosphoric acid and acetonitrile in the ratio of 600:400 (v/v), flow rate of 1.0 mL/min, injection volume is 10 µL and run time of 6 min in isocratic elution. Ultraviolet (UV) detection was performed at a wavelength of 221 nm. The temperature was maintained at 30 °C. Well-resolved peaks were observed with a high number of theoretical plates, lower tailing factor, and reproducible relative retention time. The method was validated, and all validation parameters were found to be within the acceptance limits. Results: A simple, accurate, and precise method has been developed for estimating bupropion and dextromethorphan in a fixed dose combination of tablets. The optimized method included the following parameters: column temperature of 30 °C, 40% acetonitrile as the mobile phase, and flow rate of 1.0 mL/min. Retention times were 2.25 min and 3.12 min for bupropion and dextromethorphan, respectively. The method was found to be linear in the range of 17.5-105 µg/mL [for R2 < 0.999) and 7.5-45 µg/mL (for R2 > 0.999] for bupropion and dextromethorphan, respectively. Both active pharmaceutical ingredients dissolved more than 90% within 5 min. Conclusion: The current study describes a new, simple, reliable, and economical elution reversed-phase high performance liquid chromatography method for estimating bupropion and dextromethorphan in a fixed combination tablet dosage form. The forced degradation studies were conducted using several degradation conditions such as acidic, alkali, oxidation, thermal, UV, and neutral conditions; the proposed method was effectively employed from the resolution of sample peaks. To the best of our knowledge, no such detailed and stability-indicating method has been reported for a fixed tablet dosage form.

Novel RP-HPLC method for estimation of a newly developed combination of tizanidine and etoricoxib in rat plasma: Eight criteria for greens evaluation.

A novel environmentally friendly reversed-phase high-performance liquid chromatography (RP-HPLC) method has been effectively validated for simultaneously measuring a prospective conjunction of tizanidine (TIZ) and etoricoxib (ETC), the combined medicine, in rat plasma. The technique employs diclofenac potassium as the internal standard, guaranteeing dependable and precise outcomes. This study aimed to assess the impact of the suggested combination therapy on treating inflammation resulting from rheumatoid arthritis (RA) in a rat model. The procedure was performed using an Agilent series 1200 model HPLC apparatus. The chromatographic conditions consist of isocratic elution mode, C18 column with dimensions of 150 mm × 4.6 mm × 5 µm, flow rate of 1.5 mL/min, wavelength of 230 nm, temperature of 50°C, and injection volume of 10 µL. The elution was performed using a mobile phase consisting of a phosphate buffer with a pH of 3.5 and acetonitrile in a ratio of 80:20 v/v. Calibration curves were conducted for TIZ and ETC within the 1-50 µg/mL range, demonstrating linear trends with R2 values over 0.999. The effectiveness and eco-friendliness of the proposed method were evaluated using eight separate environmentally conscious metrics. The addition of TIZ and ETC to arthritic rodents amplified these effects significantly. Furthermore, TIZ and ETC significantly reduced serum levels in arthritic rodents, and safety investigations revealed normal complete blood count, liver, and renal functions. TIZ and ETC appear to have antiarthritic, anti-inflammatory, and safe combinations, making them viable future treatment options for RA that are also safe and efficacious. Following validation by United States Food and Drug Administration (US-FDA) rules, all goods met the criteria.

Enantioselective polar-organic mode high-performance liquid chromatographic separation of lifitegrast on immobilized polysaccharide stationary phase and its application to pH-dependent chiral interconversion studies.

(S)-Lifitegrast (LFT) is the novel integrin antagonist, approved by the Food and drug administration, to treat signs and symptoms of dry eye disease. Synthesis of racemic LFT, preparative and analytical enantiomer separation, and chiral interconversion studies are lacking in the literature. Hence, in our study, synthesis of LFT racemate, chiral preparative purification procedure of enantiomer, and comprehensive analytical advancements are focused on rapid enantioselective separation and pH-dependent chiral interconversion studies. The synthesis of LFT racemate employed 2-amino-3-(3-(methylsulfonyl)phenyl)propanoic acid hydrochloride and 2-(benzofuran-6-carbonyl)-5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carbonyl chloride as starting materials. (R)-LFT was isolated from the racemate by preparative chiral HPLC and characterized using Q-TOF, FT-IR, NMR spectroscopy, and chiral HPLC. The purity of (R)-LFT was determined to have an enantiomeric excess of 99.12%. A precise, accurate, rapid HPLC-DAD enantioselective analytical method has been developed on Chiralpak IC [tris(3,5-dichloro phenyl carbamate) immobilized on cellulose] using water and methanol as mobile phase. The chiral interconversion study reveals 0.22% and 0.21% of interconversion of (S)-LFT into (R)-LFT at 80°C in pH 7.4 and 9.5 buffers, respectively, on the 24th day. An alternative route to enantioselective synthesis of LFT enantiomers by chromatographic separation is proposed. The validated enantioselective HPLC method will help to test the regular quality control samples.

Development of chromatographic methods to determine multivitamins formulation depending on their solubility and polarity: comparative study using three greenness assessment tools.

High performance liquid chromatography is one of the techniques of choice for the separation and quantitative determination of drugs in mixture form. Ipriflavone, ascorbic acid, pyridoxine, vitamin D3, and lysine are formulated together as an adjuvant combination in osteoporosis. In this work, we developed and validated two complementary high performance liquid chromatographic methods to determine the five compounds in their pharmaceutical dosage form. The first method (method A) was capable of determining ipriflavone, ascorbic acid, pyridoxine, and vitamin D3 in their bulk and combined pharmaceutical formulation. The method is based on Liquid Chromatographic separation with UV detection at 254 nm using Agilent Eclipse XDB-C18 column with a mobile phase consisting of 25 mM ammonium acetate buffer (pH 4.2): methanol in gradient mode. Due to the high polarity of lysine, it was difficult to achieve satisfactory retention on reversed phase columns. So, we separated it on a strong cation exchange column (Exsil 100 SCX) without derivatization with a mobile phase consisting of 10 mM sodium dihydrogen phosphate and 200 mM sodium chloride (pH 6) with UV detection at 210 nm (method B). Validation of the proposed methods was performed according to ICH guidelines Q2(R1). The proposed methods proved to be valid for selective analysis of the stated drugs in their bulk and combined pharmaceutical formulation. Greenness assessment of the developed methods was evaluated using three assessment tools: ESA, GAPI and the most recently developed tool AGREE, showing a satisfactory comprehensive guide of the greenness of the developed methods.

Computational quality-by-design strategy to validate high-performance liquid chromatography method for the estimation of meloxicam in bulk dosage forms and milk samples.

Bovine clinical mastitis has significant repercussions for farmers across the globe. Meloxicam, a COX-2 inhibitor, attenuates mastitis symptoms and is also approved for veterinary use. An RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) method development and validation is essential in the pharmaceutical industry to assess API (Active Pharmaceutical Ingredient) quantity present in the pharmaceutical dosage forms. RP-HPLC method of meloxicam was developed and optimized with the aid of QbD (Quality by Design) using Box-Behnken design (BBD). The pH of the aqueous mobile phase, acetonitrile (ACN) percentage, and column temperature were chosen as influence variables, and retention time (RT) and tailing factor (Tf) were selected as response variables. The optimum experimental conditions were selected as pH ~ 3 of the aqueous mobile phase, 65% v/v ACN, and 30˚C as column temperature. The drug was eluted at 6.02 min RT with 1.18 as Tf. The method was subjected to validation for accuracy, linearity, precision, range, sensitivity, and robustness and was found to comply with ICH Q2 (R1) guidelines. The in vitro bioequivalent studies were performed in hydrochloric acid, pH ~ 1.2; acetate buffer, pH ~ 4.5; and phosphate buffer, pH ~ 6.8 for two veterinary brands of meloxicam tablets, and their release profiles were compared by mathematical models. Both the brands, brand 1 and 2 exhibited significant (Unpaired t-test, P < 0.05) differences in dissolution efficiency (DE) and mean dissolution time (MDT) except DE at pH 1.2. However, brands 1 and 2 showed similarity (f2 > 50) in terms of release of meloxicam except at pH 6.8 (f2 = 47.01). The in vitro release of meloxicam followed Peppas kinetics except for brand 2 at pH 6.8, where it followed the Higuchi model. Moreover, the recovery of meloxicam extracted with ACN in the milk sample was estimated to be 99.67 ± 0.58% significantly (Unpaired t-test, P < 0.05) higher than 90.34 ± 6.77% extracted with methanol. In conclusion, the optimized and validated RP-HPLC method of meloxicam may further be used for the analysis of drug content in pharmaceutical dosage forms in addition to biological fluids.

Implementation of Quality by Design Approaches for Development and Validation of Reverse-Phase High-Performance Liquid Chromatography Assay Method for Determination of Glycyrrhizin in Nanoformulation.

Glycyrrhizin (GL) is the principal constituent of Glycyrrhiza glabra, having antiallergic, anticancer, anti-inflammatory, and antimicrobial action. The reverse-phase high-performance liquid chromatography (RP-HPLC) analytical method was used to quantitatively estimate GL in a nanoformulation and validated as per International Conference on Harmonization Q2 (R1) standards. A stationary phase of the C18-HL reversed-phase column and a mobile phase of acetonitrile and water were used for effective elution. The chromatographic conditions of RP-HPLC were optimized utilizing a quality-by-design approach to accomplish the required chromatographic separation of GL from its nanoformulation with minimal experimental runs. Optimized RP-HPLC conditions for the assay method consist of acetonitrile (41%) and water, pH 1.8, balanced with phosphoric acid (0.1%) as a mobile phase with a flow rate of 1 mL/min. The retention time was found at 7.25 min, and method validation confirmed its sensitivity, preciseness, accuracy, and robustness.

HPLC method for quantifying verbascoside in Stizophyllum perforatum and assessment of verbascoside acute toxicity and antileishmanial activity.

We report the chemical composition of the crude leaf extracts obtained from Stizophyllum perforatum (Cham.) Miers (Bignoniaceae), a simple high-performance liquid chromatography-diode array detection (HPLC-DAD) method based on mangiferin as an internal standard to quantify verbascoside, and the verbascoside acute oral toxicity and antileishmanial activity. HPLC-high-resolution mass spectrometry-DAD (HPLC-HRMS-DAD) analyses of the crude ethanol S. perforatum leaf extracts (CE-1 and CE-2) revealed that verbascoside was the major constituent in both extracts. CE-1 was purified, and verbascoside and casticin, among other compounds, were isolated. The developed HPLC-DAD method was validated and met the required standards. Investigation of the CE-2 acute toxicity indicated a lethal dose (LD50) greater than 2,000 mg/kg of body weight. Both CE-1 and CE-2 exhibited antileishmanial activity. The isolated compounds, verbascoside and casticin, also displayed antileishmanial activity with effective concentrations (IC50) of 6.23 and 24.20 µM against promastigote forms and 3.71 and 18.97 µM against amastigote forms of Leishmania amazonensis, respectively, but they were not cytotoxic to J774A.1 macrophages. Scanning electron microscopy of the L. amazonensis promastigotes showed that the parasites became more rounded and that their plasma membrane was altered in the presence of verbascoside. Additionally, transmission electron microscopy demonstrated that vacuoles emerged, lipids accumulated, kinetoplast size increased, and interstitial extravasation occurred in L. amazonensis promastigotes exposed to verbascoside. These findings suggest that S. perforatum is a promising candidate for further in vivo investigations against L. amazonensis.

PLGA Nanoparticles Containing Natural Flavanones for Ocular Inflammation.

Flavanones are natural compounds that display anti-inflammatory activity. The aim of this work was to prepare PLGA nanoparticles (NPs) containing natural flavanones I ((2S)-5,7-dihydroxy-6-methyl-8-(3-methyl-2-buten-1-il)-2-phenyl-2,3-dihydro-4H-1-Benzopyran-4-one) and II (2S)-5,7-dihydroxy-2-(4'-methoxyphenyl)-6-methyl-8-(3-methyl-2-buten-1-yl)-2,3-dihydro-4H-1-Benzopyran-4-one) (NP I and NP II, respectively) so as to evaluate their potential for topical anti-inflammatory ocular therapy. An in silico study was carried out using the Molinspiration® and PASS Online web platforms before evaluating the in vitro release study and the ex vivo porcine cornea and sclera permeation. The HPLC analytical method was also established and validated. Finally, the in vitro anti-inflammatory efficacy of NPs was studied in the HCE-2 model. The flavanones I and II could be released following a kinetic hyperbolic model. Neither of the two NPs was able to permeate through the tissues. NP I and NP II were found to be respectful of any changes in the tissues' morphology, as evidenced by histological studies. In HCE-2 cells, NP I and NP II were not cytotoxic at concentrations up to 25 µM. NP I showed higher anti-inflammatory activity than NP II, being able to significantly reduce IL-8 production in LPS-treated HCE-2 cells. In summary, ocular treatment with NP I and NP II could be used as a promising therapy for the inhibition of ocular inflammation.

A Simple and Rapid High-Performance Liquid Chromatography Method for Preparation and Content Detection of the Mainly Numbing Taste Substances of Zanthoxylum bungeanum Maxim.

As the characteristic numbing taste substances, hydroxyl-α-sanshool (HAS) and hydroxyl-ß-sanshool (HBS) were considered vital indicators to evaluate the quality of Zanthoxylum bungeanum Maxim. However, it is very difficult to obtain their high-purity monomers individually, as the only difference between HAS and HBS is that C-6 cis-trans isomerism. In our study, a simple and rapid Ag +-HPLC method was developed to pure the standard chemicals of Z. bungeanum with numbing taste, and 1H NMR and 13C NMR were employed to determine the purity and structure. Moreover, an HPLC method was established to determine the content of numbing taste components of 16 varieties of Z. bungeanum from different regions. The analytical methods were validated for accuracy, precision, and linearity, respectively. The validated method was accurate (spiked recoveries 0.94-1.10), precise in terms of peak area (intra-day RSDs <1.25% and inter-day RSDs <1.61%), and linear (r2 ≥ 0.999). It was found that there were significant differences in the content of HAS and HBS among different types of Z. bungeanum, with HAS content ranging from 60.06 ± 1.14 to 164.13 ± 3.28 mg/g and HBS ranging from 7.81 ± 0.36 to 21.11 ± 0.75 mg/g. The RSDs of HAS range were 1.73-3.80% and that of HBS range 2.03-4.73% (RSDs ≤5%), which indicated that the measurements of HAS and HBS were reliable.

Development and Validation of a Stability-indicating UPLC-DAD Method for the Simultaneous Determination of Ivermectin and Praziquantel in Pharmaceutical Tablets and Dissolution Media.

Currently, there is no single rapid and accurate stability-indicating quantitative method that can simultaneously determine both ivermectin and praziquantel and their related compounds. Thus, the goal of this research is to develop and validate a new rapid, accurate, and stability-indicating ultra-performance liquid chromatography (UPLC) method. The method uses a water, acetonitrile, and methanol gradient. The chromatographic separation was achieved on a C18 (1.7 µm, 2.1 × 50 mm) column with a flow rate of 0.7 mL/min, and the column temperature was maintained at 40°C. Analytes are detected at 245 nm. The method was validated in accordance with ICH Q2R1 guidelines. The linearity (R2) was >0.9987 and 0.9997 for praziquantel and ivermectin, respectively. The corresponding accuracy ranged between 98.0 and 102.0%. Intermediate precision (assessed as inter-day precision) was determined by calculating the cumulative %CV of eighteen assay preparations and was less than 2.0% for both praziquantel and ivermectin. The specificity of the method was shown by the resolution of the two active pharmaceutical ingredients (APIs) from any interfering excipients, impurities, or degradation products. The limit of detection and quantitation for ivermectin was 26.80 ng/mL and 81.22 ng/mL, respectively. The limit of detection and quantitation for praziquantel was 1.39 µg/mL and 4.22 µg/mL, respectively. The robustness study proved that method performance is stable against small variations in sample processing parameters (shaking, sonication time, and acetonitrile % in solvent solution) and also against small variations in the initial % of mobile phase components and gradient slope. Using ICH Q2R2 criteria, the method was demonstrated to be specific, accurate, stability indicating, and robust to small variations of chromatographic variables.

Stability-indicating method development and validation for quantitative estimation of organic impurities of the antidiabetic drug glipizide in drug substance and pharmaceutical dosage form using HPLC.

Glipizide is an antidiabetic drug used for the treatment of type 2 diabetes. A simple, reliable and robust reverse-phase liquid chromatographic method (RP-HPLC) was developed and validated as per International Conference on Harmonization Q2(R1) for estimating the impurities of glipizide in pharmaceutical formulations. The chromatographic separation was carried out on a Phenomenex Luna C18 (2), 250 × 4.6 mm, 5 µm with a binary solvent delivery system [MP-A, a homogenous mixture of water and acetonitrile in a ratio of 90:10 (v/v) and 1 ml of orthophosphoric acid; and MP-B, a homogenous mixture of water and acetonitrile in a ratio of 10:90 (v/v) and 1 ml of orthophosphoric acid] with a detection wavelength of 225 nm, a column temperature of 30°C, a flow rate of 1.5 ml/min, and an injection volume of 20 µl. All process, degradant and unknown impurities were separated well with a resolution >2.2 and were estimated accurately without any interference. The recovered values and regression values were 98.7-100.5% and R2 > 0.9999, respectively. The recovery and linearity studies covered the quantitation limit to 150% of the specification limit. The stability-indicating properties of the developed RP-HPLC method was assessed from the forced degradation studies. The developed method was successfully applied for real-time sample analysis of the glipizide dosage form.

ß-Carotene and ß-apo-8'-carotenal contents in processed foods in Korea.

A robust and rapid HPLC method for ß-carotene and ß-apo-8'-carotenal analyses in various processed foods was developed. The analysis method was validated for low-fat, moderate-fat, and high-fat food matrices. The two carotenoids were identified by LC-MS/MS. The detection limits for ß-carotene and ß-apo-8'-carotenal in the three food matrices were 0.08-0.27 µg/g and 0.09-0.18 µg/g, respectively. The inter- and intra-day accuracy and precision were in accordance with the Codex guidelines. The validated method was applied to 57 processed food samples, possibly containing ß-carotene and ß-apo-8'-carotenal, obtained in Korea. The detected ß-carotene and ß-apo-8'-carotenal levels in the samples ranged from not detected (ND) to 6.92 µg/g and ND to 1.63 µg/g, respectively. Chocolate and cheese samples had the highest ß-carotene and ß-apo-8'-carotenal levels, respectively. Notably, several samples with no labeled carotenoid additives contained ß-carotene. Moreover, the developed analytical method was compatible with various processed food matrices. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01285-2.

Development and Validation of a Simple, Fast, and Accessible HPLC-UV Method for Cannabinoids Determination in Cannabis sativa L. Extracts and Medicinal Oils.

INTRODUCTION: Cannabis sativa L. is a well-recognized medicinal plant. Cannabis regulations in Argentina are insufficient to solve the problem of patient access to full-spectrum cannabis-based products. So, the market of artisanal products with unknown quality and dosage of cannabinoids is increasing, and so is the local demand and need for analyzing these products. However, much of the latest validated methodologies for cannabinoid quantification include expensive instrumentation that is not always available in laboratories of health institutions in Argentina. METHODS: The aim of this work was to develop and validate a simple and rapid HPLC-UV method for the identification and quantification of principal cannabinoids in cannabis resins, inflorescences, and medicinal oils using standard HPLC equipment. The cannabinoids selected for validation were cannabidiol acid (CBDA), cannabigerol (CBG), cannabidiol (CBD), cannabinol (CBN), delta-9-tetrahydrocannabinol (Δ9-THC), cannabichromene (CBC), and tetrahydrocannabinol acid (THCA). A method for the simultaneous identification and quantification of these 7 main cannabinoids was developed and then validated. Some data parameters were comparable to other reports with more sophisticated analytical instruments for the analysis of cannabis. The assessed limits of detection and the limits of quantitation ranged from 0.9 to 3.66 µg/mL and 2.78 to 11.09 µg/mL, respectively. The concentration-response relationship of the method indicated a linear relationship between the concentration and peak area with R2 values of > 0.99 for all 7 cannabinoids. RESULTS: The relative standard deviation (RSD%) varied from 2.34 to 4.82 for intraday repeatability and from 1.16 to 3.15 for interday repeatability. The percentage of recovery values was between 94 to 115% (resins) and 80 to 103% (inflorescence extract). The cannabis industry is growing rapidly, and there is a need for reliable testing methods to ensure the safety and efficacy of cannabis products. In addition, current methods for cannabinoid analysis are often time-consuming and expensive, while the HPLC-UV method herein reported is a simple, rapid, accurate, and cost-effective alternative for the analysis of cannabinoids in cannabis resins, inflorescences, and medicinal oils. CONCLUSION: This method will be proposed to be included in the Cannabis sativa L. monograph of the Argentine Pharmacopoeia.

Identification and structural characterization of major stress degradation products of halcinonide by liquid chromatography-high-resolution mass spectrometry.

Halcinonide is a topical corticosteroid approved by the US Food and Drug Administration (FDA), known for its anti-inflammatory and antipruritic properties. The therapeutic benefits of halcinonide have rendered it an effective treatment regimen for various dermatological conditions such as psoriasis, dermatitis, and eczema. However, stability of the drug substance is a prerequisite in determining the therapeutic efficacy and plays a crucial role during formulation development and long-term storage. As corticosteroids are highly susceptible to degradation, the current study aims to expose halcinonide to different stress conditions and understand its stability behavior. An HPLC method was developed for the separation of halcinonide and its degradation products. Separation was accomplished in gradient mode using an Eclipse Plus C18 column (250 × 4.5 mm, 5 µm) with ammonium formate (10 mM, pH 6.5) and acetonitrile as the mobile phases. LC-Q-TOF/MS/MS studies were conducted on halcinonide, which led to the identification of degraded products using optimized mass parameters. A potential mechanistic degradation pathway for halcinonide, along with the major identified degradation products has been established. The chromatographic method that was developed has been validated in compliance with the Q2(R1) guideline of the International Council for Harmonization. ProTox-II was used to perform in silico toxicity studies in order to evaluate the toxicity potential of both halcinonide and the identified degradation products.

Theoretical charge plots as a tool for targeted and accelerated ion exchange chromatography method development of NANOBODYⓇ molecules.

NANOBODYⓇ molecules are an innovative class of biotherapeutics based on heavy chain only VHH immunoglobulins. Much like canonical antibodies, they are prone to the formation of charge variants and other post-translational modifications, which can potentially impact their critical quality attributes. Therefore, establishing high-resolution product-specific methods, such as IEX chromatography, is essential for evaluating the purity of these molecules. However, due to the lower surface charge of NANOBODYⓇ molecules, their charge-based elution behavior can differ considerably from that of classical antibodies, resulting in a more extensive method development set-up for these smaller molecules. Using an initial pH screening gradient based on theoretical protein charge plots, we investigated the IEX retention behavior of eight NANOBODYⓇ molecules with a wide range of pI values (pI 5.0 to 10.0). Our findings reveal that the charge-based chromatographic behavior of NANOBODYⓇ molecules cannot be solely attributed to the isoelectric point (pI) of the protein. Rather, a molecule-specific charge threshold was identified as a critical parameter for NANOBODYⓇ molecule retention. Furthermore, the protein charge plot also showed that NANOBODYⓇ molecule elution can be characterized by a charge plateau where the net charge of the protein remains constant over a certain pH range (∼ pH 5.5 to pH 8.0), further challenging the paradigm that elution pH and pI are fixed values. The application of this theoretical approach using protein charge plots to define NANOBODYⓇ molecule charge threshold and charge plateau parameters, can reduce overall IEX method development turnaround time by at least 2-fold.