Isolation of N-Ethyl-2-pyrrolidinone-Substituted Flavanols from White Tea Using Centrifugal Countercurrent Chromatography Off-Line ESI-MS Profiling and Semi-Preparative Liquid Chromatography

N-Ethyl-2-pyrrolidinone-substituted flavanols (EPSF) are marker compounds for long-term stored white teas. However, due to their low contents and diasteromeric configuration, EPSF compounds are challenging to isolate. In this study, two representative epimeric EPSF compounds, 5′′′R- and 5′′′S-epigallocatechin gallate-8-C N-ethyl-2-pyrrolidinone (R-EGCG-cThea and S-EGCG-cThea), were isolated from white tea using centrifugal partition chromatography (CPC). Two different biphasic solvent systems composed of 1. N-hexane-ethyl acetate-methanol-water (1:5:1:5, v/v/v/v) and 2. N-hexane-ethyl acetate-acetonitrile-water (0.7:3.0:1.3:5.0, v/v/v/v) were used for independent pre-fractionation experiments; 500 mg in each separation of white tea ethyl acetate partition were fractionated. The suitability of the two solvent systems was pre-evaluated by electrospray mass-spectrometry (ESI-MS/MS) analysis for metabolite distribution and compared to the results of the CPC experimental data using specific metabolite partition ratio K_D values, selectivity factors α, and resolution factors R_S. After size-exclusion and semi-preparative reversed-phase liquid chromatography, 6.4 mg of R-EGCG-cThea and 2.9 mg of S-EGCG-cThea were recovered with purities over 95%. Further bioactivity evaluation showed that R- and S-EGCG-cThea possessed in vitro inhibition effects on α-glucosidase with IC₅₀ of 70.3 and 161.7 μM, respectively.     

Phytochemical Analysis and Anti-Cancer Properties of Extracts of Centaurea castriferrei Borbás & Waisb Genus of Centaurea L

The Centaurea L. (Asteraceae) genus includes many plant species with therapeutic properties. Centaurea castriferrei Borbás & Waisb is one of the least known and least described plants of this genus. The aim of the study was the phytochemical analysis of water and methanol–water extracts (7:3 v/v) obtained from the aerial parts of the plant as well as evaluation of their anticancer activity. Quantitative determinations of phenolic compounds and flavonoids were performed, and the antioxidant potential was measured using the CUPRAC method. The RP-HPLC/DAD analysis and HPLC-ESI-QTOF-MS mass spectroscopy were performed, to determine the extracts’ composition. The antiproliferative activity of the obtained extracts was tested in thirteen cancer cell lines and normal skin fibroblasts using MTT test. Regardless of the extraction method and the extractant used, similar cytotoxicity of the extracts on most cancer cell lines was observed. However, the methanol–water extracts (7:3 v/v) contained significantly more phenolic compounds and flavonoids as well as showing stronger antioxidant properties in comparison to water extracts. Centaurea castriferrei Borbás & Waisb is a rich source of apigenin and its derivatives. In all tested extracts, chlorogenic acid and centaurein were also identified. In vitro research revealed that this plant may be a potential source of compounds with anticancer activity.     

Novel β-Cyclodextrin and Catnip Essential Oil Inclusion Complex and Its Tick Repellent Properties

Cyclodextrin inclusion complexes have been successfully used to encapsulate essential oils, improving their physicochemical properties and pharmacological effects. Besides being well-known for its effects on cats and other felines, catnip (Nepeta cataria) essential oil demonstrates repellency against blood-feeding pests such as mosquitoes. This study evaluates the tick repellency of catnip oil alone and encapsulated in β-cyclodextrin, prepared using the co-precipitation method at a 1:1 molar ratio. The physicochemical properties of this inclusion complex were characterized using GC-FID for encapsulation efficiency and yield and SPME/GC-MS for volatile emission. Qualitative assessment of complex formation was done by UV-Vis, FT-IR, ¹H NMR, and SEM analyses. Catnip oil at 5% (v/v) demonstrated significant tick repellency over time, being comparable to DEET as used in commercial products. The prepared [catnip: β-CD] inclusion complex exerted significant tick repellency at lower concentration of the essential oil (equivalent of 1% v/v). The inclusion complex showed that the release of the active ingredient was consistent after 6 h, which could improve the effective repellent duration. These results demonstrated the effective tick repellent activity of catnip essential oil and the successful synthesis of the inclusion complex, suggesting that β-CDs are promising carriers to improve catnip oil properties and to expand its use in repellent formulations for tick management.     

Extraction of Phenolic Compounds from Fresh Apple Pomace by Different Non-Conventional Techniques

Red Delicious apple pomace was produced at laboratory scale with a domestic blender and different non-conventional extraction techniques were performed to isolate phenolic compounds, such as ultrasound-assisted extraction (UAE), ultraturrax extraction (UTE), accelerated solvent extraction (ASE) and pulsed electric field (PEF) extraction pre-treatment. Total phenolic content (TPC) was determined by Folin–Ciocalteu assay. Phloridzin, the main phenolic compound in apples, was determined by chromatographic analysis Q-TOF-LC/MS. The results obtained with these techniques were compared in order to identify the most efficient method to recover polyphenols. The highest value of TPC (1062.92 ± 59.80 µg GAE/g fresh apple pomace) was obtained when UAE was performed with EtOH:H₂O (50:50, v/v), while ASE with EtOH:H₂O (30:70, v/v) at 40 °C and 50% of flush was the most efficient technique in the recovery of phloridzin. The concentration of the main phenolic compounds ranged from 385.84 to 650.56 µg/g fresh apple pomace. The obtained results confirm that apple pomace represents an interesti-ng by-product, due to the presence of phenolic compounds. In particular, phloridzin could be considered a biomarker to determine the quality of numerous apple products. Therefore, this research could be a good starting point to develop a value-added product such as a functional food or nutraceutical.     

Ultrasound-Assisted Water Extraction of Gentiopicroside,Isogentisin, and Polyphenols from Willow Gentian “Dust” Supported by Hydroxypropyl-β-Cyclodextrin as Cage Molecules

The residue after sieving (“dust”) from the willow gentian underground parts is an unexploited herbal tea by-product, although it contains valuable bioactive compounds. Cyclodextrins as efficient green co-solvents, cage molecules, and multifunctional excipients could improve the extraction and contribute to the added value of the resulting extracts. The objective of this study was to determine the optimal conditions for the extraction of gentiopicroside, isogentisin, and total phenolics (TPC) from willow gentian “dust” using ultrasound-assisted water extraction coupled with hydroxypropyl-β-cyclodextrin (HPβCD). The influence of extraction temperature (X₁: 20–80 °C), time (X₂: 20–50 min), and HPβCD concentration (X₃: 2–4% w/v) was analyzed employing the response surface methodology (RSM). The optimal extraction conditions for simultaneously maximizing the extraction yield of all monitored responses were X₁: 74.89 °C, X₂: 32.57 min, and X₃: 3.01% w/v. The experimentally obtained response values under these conditions (46.96 mg/g DW for gentiopicroside, 0.51 mg/g DW for isogentisin, and 12.99 mg GAE/g DW for TPC) were in close agreement with those predicted, thus confirming the suitability and good predictive accuracy of the developed RSM models. Overall, the developed extraction system could be an applicable alternative strategy to improve the extraction of bioactive compounds from the underutilized “dust” of willow gentian underground parts.     

A New and Sensitive HPLC-UV Method for Rapid and Simultaneous Quantification of Curcumin and D-Panthenol: Application to In Vitro Release Studies of Wound Dressings

Curcumin (CUR) and D-panthenol (DPA) have been widely investigated for wound-healing treatment. In order to analyse these two compounds from a dosage form, such as polymer-based wound dressings or creams, an analytical method that allows the quantification of both drugs simultaneously should be developed. Here, we report for the first time a validated high-performance liquid chromatographic (HPLC) method coupled with UV detection to quantify CUR and DPA based on the standards set by the International Council on Harmonization (ICH) guidelines. The separation of the analytes was performed using a C₁₈ column that utilised a mobile phase consisting of 0.001% v/v phosphoric acid and methanol using a gradient method with a run time of 15 min. The method is linear for drug concentrations within the range of 0.39–12.5 μg mL⁻¹ (R² = 0.9999) for CUR and 0.39–25 μg mL⁻¹ for DPA (R² = 1). The validated method was found to be precise and accurate. Moreover, the CUR and DPA solution was found to be stable under specific storage conditions. We, therefore, suggest that the HPLC-UV method developed in this study may be very useful in screening formulations for CUR and DPA within a preclinical setting through in vitro release studies.     

A Box–Behnken Extraction Design and Hepatoprotective Effect of Isolated Eupalitin-3-O-β-D-Galactopyranoside from Boerhavia diffusa Linn.

The objectives of this study were to optimize and quantify the maximum percentage yield of eupalitin-3-O-β-D-galactopyranosidefrom Boerhavia diffusa leaves using response surface methodology (RSM), as well as to demonstrate the hepatoprotective benefits of the bioactive compound. The Box–Behnken experimental design was utilized to optimize the eupalitin-3-O-β-D-galactopyranoside extraction procedure, which also looked at the extraction duration, temperature, and solvent concentration as independent variables. Boerhaviadiffusa leaves were extracted, and n-hexane, chloroform, ethyl acetate, and water were used to fractionate the dried extracts. The dried ethyl acetate fraction was thoroughly mixed in hot methanol and stored overnight in the refrigerator. The cold methanol was filtered, the solid was separated, and hot methanol was used many times to re-crystallize the solid to obtain pure eupalitin-3-O-β-D-galactopyranoside (0.1578% w/w). The proposed HPTLC method for the validation and quantification of eupalitin-3-O-β-D-galactopyranosidewassuccessfully validated and developed. The linearity (R₂ = 0.994), detection limit (30 ng), and quantification limit (100 ng) of the method, as well as its range (100–5000 ng), inter and intraday precision (0.67% and 0.991% RSD), specificity, and accuracy (99.78% RSD), were all validated as satisfactory. The separation of the eupalitin-3-O-β-D-galactopyranoside band was achieved on an HPTLC plate using toluene:acetone:water (5:15:1 v/v) as a developing system. The Box–Behnken statistical design was used to determine the best optimization method, which was found to be extraction time (90 min), temperature (45 °C), and solvent ratio (80% methanol in water v/v) for eupalitin-3-O-β-D-galactopyranoside. Standard silymarin ranged from 80.2% at 100 µg/mL to 86.94% at 500 µg/mL in terms of significant high hepatoprotection (cell induced with carbon tetrachloride 0.1%), whereas isolated eupalitin-3-O-β-D-galactopyranoside ranged from 62.62% at 500 µg/mL to 70.23% at 1000 µg/mL. More recently, it is a source of structurally unique flavonoid compounds that may offer opportunities for developing novel semi-synthetic molecules.     

Bauhinia forficata Link Infusions: Chemical and Bioactivity of Volatile and Non-Volatile Fractions

This study aimed to evaluate Bauhinia forficata infusions prepared using samples available in Rio de Janeiro, Brazil. As such, infusions at 5% (w/v) of different brands and batches commercialized in the city (CS1, CS2, CS3, and CS4) and samples of plant material botanically identified (BS) were evaluated to determine their total phenolic and flavonoid contents (TPC and TFC), antioxidant capacity (ABTS^•+, DPPH^•, and FRAP assays), phytochemical profile, volatile compounds, and inhibitory effects against the α-amylase enzyme. The results showed that infusions prepared using BS samples had lower TPC, TFC and antioxidant potential than the commercial samples (p < 0.05). The batch averages presented high standard deviations mainly for the commercial samples, corroborating sample heterogeneity. Sample volatile fractions were mainly composed of terpenes (40 compounds identified). In the non-volatile fraction, 20 compounds were identified, with emphasis on the CS3 sample, which comprised most of the compounds, mainly flavonoid derivatives. PCA analysis demonstrated more chemical diversity in non-volatile than volatile compounds. The samples also inhibited the α-amylase enzyme (IC₅₀ value: 0.235–0.801 mg RE/mL). Despite the differences observed in this work, B. forficata is recognized as a source of bioactive compounds that can increase the intake of antioxidant compounds by the population.     

Simultaneous Determination of Escitalopram Impurities including the R-enantiomer on a Cellulose tris(3,5-Dimethylphenylcarbamate)-Based Chiral Column in Reversed-Phase Mode

A high-performance liquid chromatographic method was developed for the simultaneous determination of the related substances—three potential synthesis-related chemical impurities and the distomer—of escitalopram. The separation capacity of seven different polysaccharide-type chiral columns, including three amylose-based (Lux Amylose-1, Lux i-Amylose-1, Lux Amylose-2) and four cellulose-based columns (Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, and Lux Cellulose-4) were screened in the polar organic and reversed-phase modes. Lux Cellulose-1, based on cellulose tris(3,5-dimethylphenylcarbamate) as the chiral selector with an acetonitrile-water mixture containing 0.1% diethylamine was identified as the most promising separation system. Using the “one factor at a time” optimization approach, the effect of column temperature, flow rate, and mobile phase constituents on separation performance was evaluated, and the critical resolution values were determined. A U-shaped retention pattern was obtained when plotting the retention factors of the citalopram enantiomers versus the water content of the binary mobile phases on the Lux Cellulose-1 column. A thermodynamic analysis revealed enthalpy-driven enantioseparation in both the polar organic and reversed-phase modes. For further method optimizations, an L9 orthogonal array table was employed. Using the optimized parameters (Lux Cellulose-1 column with 0.1% (v/v) diethylamine in water/acetonitrile 55/45 (v/v); 0.8 mL/min flow rate at 25 °C), baseline separations were achieved between all compounds. Our newly developed HPLC method was validated according to the ICH guidelines and its application was tested with a commercially available pharmaceutical formulation. The method proved to be suitable for routine quality control of related substances and the enantiomeric purity of escitalopram.     

Automated Optimized Synthesis of [18F]FLT Using Non-Basic Phase-Transfer Catalyst with Reduced Precursor Amount

3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) is a positron emission tomography (PET) tracer useful for tumor proliferation assessment for a number of cancers, particularly in the cases of brain, lung, and breast tumors. At present [¹⁸F], FLT is commonly prepared by means of the nucleophilic radiofluorination of 3-N-Boc-5′-O-DMT-3′-O-nosyl thymidine precursor in the presence of a phase-transfer catalyst, followed by an acidic hydrolysis. To achieve high radiochemical yield, relatively large amounts of precursor (20–40 mg) are commonly used, leading to difficulties during purification steps, especially if a solid-phase extraction (SPE) approach is attempted. The present study describes an efficient method for [¹⁸F]FLT synthesis, employing tetrabutyl ammonium tosylate as a non-basic phase-transfer catalyst, with a greatly reduced amount of precursor employed. With a reduction of the precursor amount contributing to lower amounts of synthesis by-products in the reaction mixture, an SPE purification procedure using only two commercially available cartridges—OASIS HLB 6cc and Sep-Pak Alumina N Plus Light—has been developed for use on the GE TRACERlab FX N Pro synthesis module. [¹⁸F]FLT was obtained in radiochemical yield of 16 ± 2% (decay-corrected) and radiochemical purity >99% with synthesis time not exceeding 55 min. The product was formulated in 16 mL of normal saline with 5% ethanol (v/v). The amounts of chemical impurities and residual solvents were within the limits established by European Pharmacopoeia. The procedure described compares favorably with previously reported methods due to simplified automation, cheaper and more accessible consumables, and a significant reduction in the consumption of an expensive precursor.     

A Rapid Nano-Liquid Chromatographic Method for the Analysis of Cannabinoids in Cannabis sativa L. Extracts

Cannabis sativa L. is an herbaceous plant belonging to the family of Cannabaceae. It is classified into three different chemotypes based on the different cannabinoids profile. In particular, fiber-type cannabis (hemp) is rich in cannabidiol (CBD) content. In the present work, a rapid nano liquid chromatographic method (nano-LC) was proposed for the determination of the main cannabinoids in Cannabis sativa L. (hemp) inflorescences belonging to different varieties. The nano-LC experiments were carried out in a 100 µm internal diameter capillary column packed with a C18 stationary phase for 15 cm with a mobile phase composed of ACN/H₂O/formic acid, 80/19/1% (v/v/v). The reverse-phase nano-LC method allowed the complete separation of four standard cannabinoids in less than 12 min under isocratic elution mode. The nano-LC method coupled to ultraviolet (UV) detection was validated and applied to the quantification of the target analytes in cannabis extracts. The nano-LC system was also coupled to an electrospray ionization–mass spectrometry (ESI-MS) detector to confirm the identity of the cannabinoids present in hemp samples. For the extraction of the cannabinoids, three different approaches, including dynamic maceration (DM), ultrasound-assisted extraction (UAE), and an extraction procedure adapted from the French Pharmacopeia’s protocol on medicinal plants, were carried out, and the results achieved were compared.     

Chemical Fingerprint and Quantitative Analysis of Cirsium setosum by LC

A reverse phase liquid chromatography method with diode array detection was developed to evaluate the quality of Cirsium setosum through establishing chromatographic fingerprint and simultaneous determination of six phenolic compounds, namely chlorogenic acid, caffeic acid, rutin, linarin, luteolin and apigenin. The chromatographic separation was performed on an Agilent SB-C₁₈ column (250 × 4.6 mm, 5.0 μm) with a gradient elution program using a mixture of acetonitrile and 0.5% aqueous acetic acid (v/v) as mobile phase within 25 min at 326 nm wavelength. The correlation coefficients of similarity were determined from the LC fingerprints, and they shared a close similarity. The LC with electrospray ionization mass spectrometry experiment was performed to further confirm the identity of phenolic compounds. The six phenolic compounds showed good regression (R ² > 0.9995) within test ranges and the recovery of the method was in the range of 95.8–102.8%. In addition, the content of those six phenolic compounds in C. setosum growing in different locations of China was determined to establish the effectiveness of the method. The results indicated that the developed method by having a combination of chromatographic fingerprint and quantification analysis could be readily utilized as a quality control method for C. setosum and its related traditional Chinese medicinal preparations.     

A Study on Synthesis and Upscaling of 2′-O-AECM-5-methyl Pyrimidine Phosphoramidites for Oligonucleotide Synthesis

2′-O-(N-(Aminoethyl)carbamoyl)methyl-modified 5-methyluridine (AECM-MeU) and 5-methylcytidine (AECM-MeC) phosphoramidites are reported for the first time and prepared in multigram quantities. The syntheses of AECM-MeU and AECM-MeC nucleosides are designed for larger scales (approx. 20 g up until phosphoramidite preparation steps) using low-cost reagents and minimizing chromatographic purifications. Several steps were screened for best conditions, focusing on the most crucial steps such as N³ and/or 2′-OH alkylations, which were improved for larger scale synthesis using phase transfer catalysis (PTC). Moreover, the need of chromatographic purifications was substantially reduced by employing one-pot synthesis and improved work-up strategies.     

ZnO Nanoparticles of Rubia cordifolia Extract Formulation Developed and Optimized with QbD Application,Considering Ex Vivo Skin Permeation,Antimicrobial and Antioxidant Properties

The objective of the current research is to develop ZnO-Manjistha extract (ZnO-MJE) nanoparticles (NPs) and to investigate their transdermal delivery as well as antimicrobial and antioxidant activity. The optimized formulation was further evaluated based on different parameters. The ZnO-MJE-NPs were prepared by mixing 10 mM ZnSO₄·7H₂O and 0.8% w/v NaOH in distilled water. To the above, a solution of 10 mL MJE (10 mg) in 50 mL of zinc sulfate was added. Box–Behnken design (Design-Expert software 12.0.1.0) was used for the optimization of ZnO-MJE-NP formulations. The ZnO-MJE-NPs were evaluated for their physicochemical characterization, in vitro release activity, ex vivo permeation across rat skin, antimicrobial activity using sterilized agar media, and antioxidant activity by the DPPH free radical method. The optimized ZnO-MJE-NP formulation (F13) showed a particle size of 257.1 ± 0.76 nm, PDI value of 0.289 ± 0.003, and entrapment efficiency of 79 ± 0.33%. Drug release kinetic models showed that the formulation followed the Korsmeyer–Peppas model with a drug release of 34.50 ± 2.56 at pH 7.4 in 24 h. In ex vivo studies ZnO-MJE-NPs-opt permeation was 63.26%. The antibacterial activity was found to be enhanced in ZnO-MJE-NPs-opt and antioxidant activity was found to be highest (93.14 ± 4.05%) at 100 µg/mL concentrations. The ZnO-MJE-NPs-opt formulation showed prolonged release of the MJE and intensified permeation. Moreover, the formulation was found to show significantly (p < 0.05) better antimicrobial and antioxidant activity as compared to conventional suspension formulations.     

Setup of an Ultrasonic-Assisted Extraction to Obtain High Phenolic Recovery in Crataegus monogyna Leaves

Hawthorn leaves are a rich source of phenolic compounds that possess beneficial activities for human health. Ultrasonic-assisted extraction (UAE) is an extraction technique frequently used for the isolation of phenolic compounds in plants. Thus, in this study, a Box–Behnken design was used to optimize UAE conditions such as the percentage of acetone, the extraction time and solvent-to-solid ratio (v/w) in order to obtain the maximum content of total compounds by Folin–Ciocalteu and the maximum in vitro antioxidant activity by DPPH, ABTS and FRAP assays in Crataegus monogyna leaves. The optimum conditions to obtain the highest total phenolic content and antioxidant activities were 50% acetone, 55 min and 1/1000 (w/v). A total of 30 phenolic compounds were identified and quantified in C. monogyna leaf extract obtained at these optimum UAE conditions. HPLC-MS allows the identification and quantification of 19 phenolic compounds and NP-HPLC-FLD analyses showed the presence of 11 proanthocyanidins. According to the results, the most concentrated phenolic compounds in C. monogyna leaf extract obtained at optimum UAE conditions were phenolic acid derivatives such as protocatechuic acid-glucoside, dihydroxy benzoic acid pentoside and chlorogenic acid, flavones such as 2″-O-rhamnosyl-C-hexosyl-apigenin, flavonols such as hyperoside and isoquercetin and proanthocyanidins such as monomer and dimer. As a result, the optimized UAE conditions could be used to obtain an extract of C. monogyna leaves enriched with phenolic compounds.     

RP-HPLC Determination of Famotidine and its Potential Impurities in Pharmaceuticals

A simple, sensitive, and rapid reversed-phase high-performance liquid chromatographic method has been developed for determination of famotidine (FMT) and its impurities in pharmaceutical formulations. Separations were performed on a Supelcosil LC18 column with an isocratic mobile phase—13:87 (v/v) acetonitrile–0.1 M dihydrogen phosphate buffer containing 0.2% triethylamine (pH 3.0). The mobile phase flow rate was 1 mL min^–1 and the detection wavelength was 265 nm. Response was linearly dependent on concentration between 1 and 80 g mL^–1 (regression coefficient, R², from 0.9981 to 0.9999). RSD from determination of method repeatability (intraday) and reproducibility (interday) were <2% (n=6). Lowest detectable concentrations ranged from 0.08 to 0.14 g mL^–1. The proposed liquid chromatographic method can be satisfactorily used for routine quality control of famotidine in pharmaceutical formulations.     

Development of Improved High-Performance Liquid Chromatography Method for the Determination of Residual Caprylic Acid in Formulations of Human Immunoglobulins

Quality control of human immunoglobulin formulations produced by caprylic acid precipitation necessitates a simple, rapid, and accurate method for determination of residual caprylic acid. A high-performance liquid chromatography method for that purpose was developed and validated. The method involves depletion of immunoglobulins, the major interfering components that produce high background noise, by precipitation with acetonitrile (1:1, v/v). Chromatographic analysis of caprylic acid, preserved in supernatant with no loss, was performed using a reverse-phase C18 column (2.1 × 150 mm, 3 μm) as a stationary phase and water with 0.05% TFA–acetonitrile (50:50, v/v) as a mobile phase at a flow rate of 0.2 mL/min and run time of 10 min. The developed method was successfully validated according to the ICH guidelines. The validation parameters confirmed that method was linear, accurate, precise, specific, and able to provide excellent separation of peaks corresponding to caprylic acid and the fraction of remaining immunoglobulins. Furthermore, a 2⁴⁻¹ fractional factorial design was applied in order to test the robustness of developed method. As such, the method is highly suitable for the quantification of residual caprylic acid in formulations of human immunoglobulins for therapeutic use, as demonstrated on samples produced by fractionation of convalescent anti-SARS-CoV-2 human plasma at a laboratory scale. The obtained results confirmed that the method is convenient for routine quality control.     

Simultaneous chemical fingerprint and quantitative analysis of Ginkgo biloba extract by HPLC–DAD

A reverse-phase liquid chromatography method with diode array detection was developed to evaluate the quality of Ginkgo biloba extract through establishing chromatographic fingerprint and simultaneous determination of eight flavonoid compounds, namely rutin, myricetin, quercitrin, quercetin, luteolin, kaempferol, apigenin, and isorhamnetin. The chromatographic separation was performed on an Agilent SB-C18 column (250 × 4.6 mm, 5.0 μm) with a gradient elution program using a mixture of methanol and 0.1% formic acid (v/v) as mobile phase within 55 min at 360-nm wavelength. The correlation coefficients of similarity for different batches of G. biloba extract from the same manufacturer and G. biloba extract from different manufacturers were determined from the LC fingerprints, and they shared a close similarity. The eight flavonoid compounds showed good regression (R ² > 0.9995) within test ranges, and the recovery of the method was in the range of 94.1–101.4%. In addition, the content of those eight flavonoid compounds in G. biloba extract prepared by different manufacturers of China was determined to establish the effectiveness of the method. The results indicated that the developed method by having a combination of chromatographic fingerprint and quantification analysis could be readily utilized as a quality control method for G. biloba extract and its related traditional Chinese medicinal preparations.     

Development and Validation of an LC-MS/MS Method for Simultaneous Determination of Short Peptide-Based Drugs in Human Blood Plasma

A novel HPLC-ESI-MS/MS method for simultaneous gonadotropin-releasing hormone (GnRH) analogs and somatostatin analog quantitation was developed and validated. The developed method was successfully applied to pharmacokinetic studies. The sample preparation process included solid-phase extraction (SPE). Effective chromatographic separation of the analytes and internal standard (dalargin) was achieved with a C18 column, using a gradient elution with two mobile phases: 0.1% v/v formic acid (aqueous solution) and 0.1% v/v formic acid (acetonitrile solution). The linearity of the method was demonstrated within a concentration range of 0.5–20 ng/mL, with correlation coefficients between 0.998–0.999 for goserelin, buserelin, triptorelin, and octreotide, respectively. The relative standard deviation (RSD, %) values for method accuracy and precision did not exceed 20% at the lower level of quantitation (LLOQ) or 15% at other concentration levels.