Reliable Reversed-phase High-performance Liquid Chromatography Research Articles

Analytical Method Development and Validation of Faricimab Injection by RP-HPLC

A robust and reliable Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative analysis of Faricimab in Vabysmo injections. Faricimab, an IgG1-based bispecific antibody targeting VEGF-A and Ang-2, is used for treating retinal vascular disorders like age-related macular degeneration and diabetic macular edema. The method development involved the use of an Inertsil C18 column (250 x 4.6 mm, 5 µm) with a mobile phase composed of KH2PO4 (pH 4.6) and methanol in a 1:1 ratio, sonicated for 20 minutes. The developed method was evaluated for various validation parameters as per ICH guidelines, including linearity, precision, accuracy, and sensitivity. Linearity was demonstrated within the concentration range of 50-150 µg/ml with a correlation coefficient (R²) of 0.999. The limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.050 µg/ml and 0.166 µg/ml, respectively, indicating high sensitivity. Precision, expressed as relative standard deviation (RSD), was below 0.1%, confirming the method's reliability. Accuracy was assessed, with mean assay values ranging between 97-101%, and RSD below 0.2%, indicating the method's precision. Robustness tests confirmed the method's resilience against slight variations in analytical conditions such as acetonitrile ratio, flow rate, detection wavelength, and pH. Stability studies showed that Faricimab remains stable under various stress conditions, including exposure to 0.1N HCl, 0.1N NaOH, 30% peroxide, heat, and sunlight, with assay values ranging from 88.93% to 94.07%. The optimized RP-HPLC method ensures good peak shape and resolution, making it suitable for routine quality control of Faricimab in pharmaceutical formulations. The validated method adheres to ICH guidelines, ensuring its reliability for consistent and accurate quantification of Faricimab

Simultaneous quantification of 4-hydroxytamoxifen and hesperidin in liposomal formulations: Development and validation of a RP-HPLC method

Breast cancer treatment options are diverse, with tamoxifen commonly used as a selective estrogen receptor modulator (SERM) for hormone receptor-positive breast cancer. However, tamoxifen can have adverse systemic effects. Local transdermal therapy offers a potential solution by delivering the drug directly to the breast and minimizing systemic exposure. Hesperidin, a flavonoid, exerts synergistic effects when combined with anticancer agents. This combination therapy may be a more effective approach to breast cancer management. Analytical methods have been developed to quantify 4-Hydroxytamoxifen (4-HT) and hesperidin separately; however, no method currently exists for their simultaneous quantification in pharmaceutical formulations. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous quantification of 4-HT and hesperidin in liposomal formulations. A Design of Experiments (DoE) approach was employed using a Box-Behnken design (BBD) to optimize the RP-HPLC method. BBD allowed for a reduction in the number of required tests by creating a statistical model to estimate the significance of various factors and interactions. The methanol concentration, flow rate, and injection volume were considered as independent variables for optimization. A mobile phase (90:10 ratio of methanol: 0.1% v/v orthophosphoric acid) with a flow rate of 0.4 mL/min, and an injection volume of 10 μL was selected as optimized chromatographic condition. 4-HT showed a retention time (Rt) of 5.05 min and hesperidin showed an Rt of 7.11 min using an optimized analytical method and was detected at 275 nm. The developed RP-HPLC method was validated according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines, confirming its accuracy, precision, linearity, selectivity, and robustness. The validated method was then successfully applied to determine the entrapment efficiency and permeation of 4-HT and hesperidin into loaded liposomes. This study fills a gap in the literature by providing a simple and reliable RP-HPLC method for the simultaneous quantification of 4-HT and hesperidin in liposomal formulations.

Donepezil and Quercetin Simultaneous Estimation in Rat Plasma Using Developed Bioanalytical HPLC Method: Relevance in Pharmacokinetic Studies

The objective of this investigation was to create and apply a reliable reverse phase high performance liquid chromatography (RP-HPLC) technique for the concurrent measurement of donepezil (DPZ) and quercetin (QT) in rat blood samples for pharmacokinetic research. This is the first publication that introduces a method for DPZ and QT simultaneous determination in rat plasma by high-performance liquid chromatography (HPLC). Using a mobile phase of methanol and HPLC grade water (pH 2.8; adjusted with 0.05% v/v orthophosphoric acid) 45:55 v/v with 2-3 drops of triethylamine (TEA) in an isocratic elution mode at flow rate of 1.0ml/min, DPZ and QT were successfully separated chromatographically on a Hypersil gold C-18 column (250 mm × 4.6 mm, 5μm). The retention time for DPZ and QT was observed to be 6.3 and 12.3 minutes and was detected at an isobestic wavelength of 273 nm using a UV detector. The method was shown to be precise (%RSD < 2%), accurate (96–100%), and specific for the simultaneous detection of DPZ and QT. Several freeze-thaw cycles of the treated plasma samples did not significantly affect the analyte’s stability. The method’s applicability was subsequently confirmed through an oral pharmacokinetic investigation in rats. Since the results were deemed trustworthy, the validated RP-HPLC method may be used to simultaneously detect and quantify both drugs. The method worked well for evaluating the pharmacokinetic characteristics in wistar rats following a single oral dose of 5 mg/kg of QT and 10 mg/kg of DPZ. It is well acknowledged that the chromatographic process is straightforward, robust, accurate, exact, and repeatable.

Development of a robust and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method using analytical quality by design principles for the accurate determination of esculin in its bulk form

BackgroundAnalytical quality by design is a proactive, holistic, and data-driven approach to quality that emphasizes risk assessment and management. This can lead to more robust and reliable methods than traditional approaches. Using principles of analytical quality by design for method development can help to assure the quality and consistency of analytical methods. This is important for the pharmaceutical industry, where accurate and reproducible analytical methods are essential for ensuring drug safety, shelf life, and efficacy. Esculin is a naturally occurring derivative of coumarin that is found in the stems of the plant Aesculus indica. The present study describes the use of an analytical quality by design approach to develop and validate a reliable RP-HPLC method for the analysis of esculin bulk form.ResultA central composite design was employed to optimize the percent of methanol in the mobile phase and flow rate for the analysis of a compound esculin using the RP-HPLC method. The optimized conditions were 43% methanol and 0.9 ml/min flow rate, with a retention time of 3.78 min, and Phenomenex Luna (5 µm × 250 mm, 4.6 mm) column was used. The method was found to be linear with a correlation coefficient of 0.9998 for a concentration range of 4–20 μg/ml. The parameters of the system suitability test were within the acceptable range (0.0612–0.1398%), and the precision for both intra-day and inter-day measurements was below 2%. The robustness and ruggedness of the method were also good, with changes in the flow rate and mobile phase composition having a minimal impact on the method's performance. The limit of detection (LOD) and limit of quantification (LOQ) values were reported to be 0.82891 μg/ml and 2.511 μg/ml, respectively. The validation parameters of the method adhered to the specified limit following the ICH guidelines.ConclusionIn summary, an AQbD-based efficient and robust RP-HPLC chromatographic method has been developed for the quantification of the esculin compound. The method is linear, precise, and reproducible, and it has good LOD and LOQ values. The method could be used for repetitive analysis of the compound in pharmaceutical formulations.

Stability Indicating Assay Method for the Quantitative Determination of Olaparib in Bulk and Pharmaceutical Dosage Form.

Olaparib is an orally active poly (ADP-ribose) PARP (polymerases) inhibitor known to destroy cancer cells with BRCA1 or BRCA2 deficiency. An authentic, fast, distinct, and reliable reverse phase-high performance liquid chromatography (RP-HPLC) method was developed and promptly validated in tablet formulations for olaparib estimation. The proposed method focuses on the separation of olaparib in reverse phase mode using a Waters symmetry C18 (150 x 4.6 mm, 5 μm) analytical column with a flow rate of 1.0 mL/min and the injection volume was kept at 20 μL. The optimized mobile phase consists of ammonium acetate buffer (pH adjusted to 3.5 by glacial acetic acid): methanol in the ratio of 50:50 v/v. The eluents were measured at 254 nm and the retention time for the drug encircled was about 4.32 min. The stress degradation studies of olaparib were conducted under acidic, alkaline, oxidative, photolytic and thermal conditions to demonstrate the stability of the drug. The regression value of 0.998 showed that the developed method was linear over the range of 80 μg/mL to 120 μg/mL. The developed RP-HPLC method is accurate and precise. The method was statistically validated as per International Conference on Harmonization guidelines. The proposed method is suitable and can be applied for the quantitative estimation of olaparib without any interference of the excipients used in the drug formulations.

Development and validation of a reversed-phase high-performance liquid chromatography-ultraviolet method for abemaciclib-related substance detection in bulk drugs.

Abemaciclib is an effective selective cyclin-dependent kinases 4 and 6 inhibitors for cancer therapy. The abemaciclib-related substances influence its efficacy and safety, and are important in process preparation studies and quality control. Thus, a reversed-phase high-performance liquid chromatography method was developed and validated for the detection of related substances in its bulk drug. The separation of abemaciclib and related substances was performed on a Phenomenon Gemini C18 column (4.6 × 250 mm, 5 μm) with a flow rate of 1.0ml/min. The ultraviolet detection wavelength was 280 nm. Mobile phase A was composed of a mixed solution of aqueous solution and acetonitrile (9:1, v/v). The aqueous solution (pH 2.5) contained 0.025-mM potassium dihydrogen phosphate solution and 0.4% triethylamine. Mobile phase B was composed of acetonitrile. This novel method exhibits good system suitability, specificity, precision, stability, linearity (0.1-20 μg/ml), repeatability, and durability. Among abemaciclib and related substances, the lowest limit of detection and quantitation were 0.02 and 0.06 μg/ml, respectively, for abemaciclib. The recovery rates for related substances were above 95%. In addition, a novel degradation product was found during the process. In summary, a reliable reversed-phase high-performance liquid chromatography method was developed for abemaciclib-related substance detection in bulk drugs.

Quantification of Anti-Osteoporotic Anabolic Peptide in Stealth Lipid Nanovesicles Through Validated RP-HPLC Method.

Teriparatide is a recombinant fragment of human parathyroid hormone, a potent osteoanabolic agent used for osteoporosis. The present study endeavored to develop a simple, rapid, and reliable reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of teriparatide in pegylated lipid nanovesicles for rapid formulation development/optimization. A rapid RP-HPLC-based analytical method was developed for the quantification of teriparatide in pegylated lipid nanovesicles. The method was optimized on a Waters XBridge C18 (4.6 × 150 mm, 10 μm) column with a mobile phase consisting of 0.1% formic acid in water and acetonitrile both in a linear gradient program. In the method, a short run time of 9 min was achieved at a flow rate of 1.0 mL/min with an injection volume of 50 µL at a detection wavelength of 210 nm. The developed method was validated according to the ICH Q2 (R2) guideline. The method was applied for the quantification of teriparatide in prepared pegylated lipid nanovesicles. Teriparatide encapsulated pegylated lipid nanovesicles were prepared by the ethanol injection method. Further, these vesicles were characterized for % entrapment efficiency (%EE), particle size, zeta potential, and morphology by Cryo-SEM. The teriparatide was eluting at 4.8 min in the run. Further, for the method validation, the linear relationship between concentration and response was established over the concentration range of 50-250 µg/mL with the R2 > 0.999. The method sensitivity was shown with LOD and LOQ with the value of 100 ng/mL and 500 ng/mL, respectively. The method was found to be accurate and precise with the recovery ranging in 100 ± 2% and RSD <2%, respectively. Minor deliberate changes proved the robustness of the developed method. These results indicated that the developed and validated method is accurate, precise, rapid, reliable, and fit for the quantification of teriparatide in different formulations. The RP-HPLC method was developed and validated for the quantification of teriparatide from novel pegylated lipid nanovesicles.

Development and Validation of a Rapid and Reliable HPLC-FLD Method for the Quantification of Ciprofloxacin and Enrofloxacin Residues in Zea mays

The accumulation of antibiotics by plants is a currently concern associated to yield reduction and transference of antibiotic residues along the food web. Maize is a staple food in many parts of the world; it is also considered an important ingredient in animal nutrition. Considering the lack of validated analytical methods for the analysis of ciprofloxacin (CIPRO) and enrofloxacin (ENRO) in the shoot of Zea mays maize, we developed a reliable reversed-phase high-performance liquid chromatography method with fluorescence detection (HPLC-FLD). This method was validated for linearity, matrix effects, precision, accuracy, limits of quantification (LOQ) and detection (LOD), and robustness. The analytical curves were linear with coefficient of determination (R2 ) of 0.9907 for CIPRO and 0.9962 for ENRO. The LOD values were 16.65 and 6.57 μg kg−1 for CIPRO and ENRO, respectively, whereas LOQ values were 50.44 μg kg−1 (CIPRO)and 19.92 μg kg−1 (ENRO). HPLC-FLD also displayed good precision and accuracy. Therefore, the proposed method can be considered a reliable and useful tool for the analysis of ciprofloxacin and enrofloxacin in the shoot of maize.

A Validated RP-HPLC Method for the Simultaneous Detection and Quantification of Pyridoxine and Terizidone in Pharmaceutical Formulations

Tuberculosis (TB) remains a life-threatening infection, and it is well-known that effective TB treatment is associated with multiple drugs administered to infected patients on a daily basis. Terizidone (TZD) is an anti-TB drug used in the treatment of multi-drug resistant and extensively drug-resistant TB but presents with polyneuropathic adverse effects in some patients. To counteract these adverse effects, TZD is typically prescribed with pyridoxine (PDX), well known as Vitamin B6. As part of a pre-formulation study investigating the potential to co-formulate these two compounds, it became necessary to have a simple and reliable reversed-phase high-performance liquid chromatography (RP-HPLC) method. Optimal, simultaneous separation and detection of TZD and PDX were obtained using an isocratic mobile phase setup, consisting of ultrapure water and acetonitrile (30:70% v/v), with 1 mL glacial acetic acid added to the mobile phase mixture. A Discovery® C18, 150 × 4.6 mm, 5 μm column maintained at ambient temperature was utilized, with a detection wavelength of 260 nm. The method was validated in terms of linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ), specificity, robustness, and solution stability. Validation proved this method to be acceptable and reliable for the simultaneous accurate detection and quantification of TZD and PDX.

Development of a Validated Stability Indicating Method for Quantification of Amoxicillin, Clarithromycin and Lansoprazole in Bulk and Pharmaceutical Dosage form by RP-HPLC

Objective: The main objective of the present work is to develop an efficient, unique, reliable Reverse Phase High Performance Liquid Chromatography (RP-HPLC) method for the simultaneous quantification of Amoxicillin (AMX), Clarithromycin (CTM) and Lansoprazole (LPZ) in bulk and pharmaceutical formulations. &#x0D; Methods: The chromatographic separation was achieved by using Kinetex column C18 (100 x 4.6 mm, 2.6 µm) with Buffer (2.5 g of hexane sulphonic acid and 1ml of Triethylamine which are added to 1000 ml of HPLC water and adjusted its pH at 5.0 with Ortho phosphoric acid) and acetonitrile in the ratio of 70: 30 (%v/v) as a mobile phase at flow rate of 1.0 ml/min. The column effluents were monitored by a photodiode array detector at wavelength predetermined at 240 nm.&#x0D; Results: The method produced reliable results at optimized chromatographic conditions. The method was linear at concentration range of 15-225 µg/ml of AMX, 15-225 µg/ml of CTM and 0.9-13.5 µg/ml of LPZ with regression coefficients of 0.9999, 0.9999, and 0.9999 respectively. The retention times of AMX, CTM, LPZ were obtained as 1.513, 3.124, 3.770 min respectively. Results obtained for system suitability, precision, LOD and LOQ were in acceptable range and were validated according to the guidelines of the International Council for Harmonization (ICH).&#x0D; Conclusion: The proposed method was validated in accordance with ICH and all the obtained results were found satisfactory and were successfully applicable to the analysis of the bulk and the pharmaceutical formulations.

Separation and Identification of Oligomeric-Hindered Amine Light Stabilizer Uvasorb HA 88 by High-performance Liquid Chromatography (HPLC) Coupled With Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALSI-TOF-MS)

Uvasorb HA 88 is widely used as a light stabilizer to prevent plastic polymer degradation. Its high molecular weight and oligomeric characters provide challenge for quality control. In this study, high-performance liquid chromatography (HPLC) coupled with electrospray ionization time of flight mass spectrometry (ESI-TOF MS) was applied for separation and detection of Uvasorb HA 88. The synthesis scheme was deduced and confirmed by the characterization of Uvasorb HA 88 products from different batches. Direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was employed for the detailed characterization of chemical composition of Uvasorb HA 88. Furthermore, a reliable reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with MALDI-TOF MS method was established and the weight-average and number-average molecular masses were calculated. The results revealed that six oligomers with repeat unit numbers from 1 to 4 were present in Uvasorb HA 88. A molecular weight of up to 6808 g mol−1 was detected and two new series of oligomers were reported for the first time. Different series of oligomers and positional isomers were observed in Uvasorb HA 88. This work provides a suitable method to evaluate the technical grade of Uvasorb HA 88 as well as comprehensive characterization of complex oligomeric hindered amine light stabilizers.

DETERMINATION OF SOME PESTICIDE RESIDUES IN APPLE JUICE BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

A new, simple and reliable reversed-phase high-performance liquid chromatography (RP-HPLC) method for determination of 2,4-D ((2,4-dichlorophenoxy)acetic acid), atrazine, malathion, fenitrothion and parathion residues in apple juices has been developed and validated. Successful separation and quantitative determination of analytes were performed on Purospher STAR RP-8e (30 x 4 mm, 3 μm) analytical column, with mobile phase consisted of acetonitrile/water (45/55, V/V), flow rate of 1 mL/min, constant column temperature at 25 oC and UV detection at 220 nm and 270 nm. A solid-phase extraction (SPE) was used for concentration and clean-up of analytes. Specificity, selectivity, linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) were tested for the method validation according to European Commission guidelines for pesticide residue analytical methods, and all performance characteristics were found within acceptance criteria. Calibration curves were linear within the range less than 50 % of the maximum residue limit (MRL) values and 20 % above for all investigated pesticides (R2 &gt; 0.95). Under the stipulated conditions, values for relative standard deviations (RSD) of retention times and peak areas were 0.12 % - 0.28 % and 0.08 % - 8.50 %, respectively. The obtained values for recoveries ranged from 93.65 % - 118.62 %, with RSD ≤ 5.64 %. This method was successfully applied for determination of investigated pesticide residues in apple juice samples, which were taken from Macedonian markets. The run time of assay obtained from this chromatography condition was about 4 min.

Optimized determination of phenolic compounds in Dendrobium officinale stems by reverse-phase high performance liquid chromatography

Introduction: Phenolic compounds have been widely credited as an important part in various human ailments and plants’ disease resistances. Few studies, however, focused on the method to extract and detect the contents of phenolic compounds in the stems of Dendrobium officinale, a prized medicinal plant and functional food in China.Materials and methods: In this work, a very rapid, sensitive and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established for the separation and quantification of two phenolic compounds, i.e., quercetin and erianin in D. officinale stems, but also an optimized extraction process.Result and discussion: Results showed that quercetin and erianin were eluted before around 35 min, providing a time-saving separation. Furthermore, the steeper gradient rate in the range of 2.66–4.85% acetonitrile/min was the best for the elution. Meanwhile, the maceration of 30 min by the extraction solvent should be performed for removing polysaccharides that interfered the detection of phenolic components and resulted in the blockage of the chromatographic column, followed by a shorten ultrasonic-assisted extraction time of 30 min. The solid-liquid ratio was 1:100.Conclusions: This method is broadly applicable for chemical analyses of phenolic components in other Dendrobium species, for which similar problem may emerge.

Development and Validation of a Rapid and Reliable RP-HPLC-PDA Method for the Quantification of Six Diterpenes in Copaifera duckei, Copaifera reticulata and Copaifera multijuga Oleoresins

Oleoresins from Copaifera species are extensively used in folk medicine in Brazil, which are employed mainly in the production of cosmetic formulations in Brazil, North America and Europe. Considering the lack of validated analytical methods for the analyses of diterpenes in Copaifera oleoresins, it was developed a validated and reliable reversed-phase high-performance liquid chromatography with photodiode array detection (RP-HPLC-PDA) method for the analysis of six diterpenes, including: (-)-polyalthic acid; (13E)-ent-labda-7,13-dien-15-oic acid; ent-8(17)-labden-15,18-dioic acid; (-)-copalic acid; (-)-3β-acetoxycopalic acid and (-)-3β-hydroxycopalic acid. These compounds were isolated from C. duckei, C. reticulata and C. multijuga oleoresins by chromatographic means. The analytical curves were linear with regression coefficients (r2) between 0.9903 and 0.9999. The limits of detection (LOD) and quantification (LOQ) values were 0.35 to 3.09 µg mL-1 and 1.05 to 9.36 µg mL-1, respectively. The method also displayed good precision and accuracy. The developed analytical method is reliable and a useful tool for the analysis of Copaifera oleoresin and its products.

RP-HPLC METHOD DEVELOPMENT, VALIDATION, AND QUANTIFICATION OF LORNOXICAM IN LIPID NANOPARTICLE FORMULATIONS

Objective: A simple, reliable, sensitive and validated reversed phase-high performance liquid chromatography (RP-HPLC) method was developed for quantification of lornoxicam (LX) in rat plasma.Methods: Solid lipid nanoparticle (SLN) and nanostructured lipid carriers (NLC) gel formulations containing lornoxicam were prepared using high-speed homogenization followed by ultra-sonication. Pharmacokinetic study of formulated LX loaded SLN and NLC were performed on Wister albino rats.Results: The chromatographic separation was performed on hypersil octadecylsilane (ODS)-18 column using a mobile phase of 10 mmol. Phosphate buffer (pH, 4.5) and acetonitrile (65:35 v/v). Elute was monitored at 377 nm with a flow rate of 1 ml/min. Calibration curve was linear over the concentration range of 25.38–2046.45 ng/ml. Retention times of LX and internal standard (piroxicam) were 9.3 and 10.2 min, respectively. Maximum plasma drug concentration, the area under the plasma drug concentration versus time curve and elimination half-life for LX loaded SLN gel were found 6381.51±971.27ng/ml, 19917.21±7111.24 ng h/ml and 7.27±1.21h and 8558.13±1564.08 ng/ml, 21317.99±4568.71 ng/ml and 6.22±2.16 h. respectively. In vivo in vitro correlation study, the fraction of drug dissolved from nanoparticle in pH 7.4 was plotted against the fraction of drug absorbed and a linear correlation (R2= 0.9987) was obtained.Conclusion: A novel simple, simple, sensitive, precise, rapid, accurate, and economical and reliable RP-HPLC method was developed and validated for the estimation of LX in rat plasma.

Boswellia serrataProtects Against Glutamate-Induced Oxidative Stress and Apoptosis in PC12 and N2a Cells

This study was designed to investigate whether the extract from Boswellia serrata oleo-gum resin (BSE) can protect against glutamate-induced oxidative damage and cytotoxicity in PC12 and N2a cell lines. Using a simple and reliable reverse-phase high-performance liquid chromatography (HPLC), the amount of 3-acetyl-11-keto-β-boswellic acid (AKBA) in the BSE was found to be 18.5% w/w. The results confirmed that BSE and AKBA, at concentrations as high as 100 μg/mL or 10 μM, respectively, caused no significant cytotoxicity or apoptotic cell death. Co- and pretreatment with BSE (25-100 μg/mL) or AKBA (5 μM) restored the viability of PC12 and N2a cells under glutamate toxicity (8 mM). Treatment with BSE and AKBA also attenuated the toxic effects of glutamate on intracellular reactive oxygen species, lipid peroxidation, superoxide dismutase activity, and oxidative DNA damage compared with the untreated glutamate-injured cells. Furthermore, BSE and AKBA decreased the apoptotic cell population in the sub-G1 region and the rate of both early and late-stage apoptosis induced by glutamate in the cells. Our data suggest that the protective effects of Boswellia extract and AKBA against glutamate toxicity in PC12 and N2a cells may be mediated through the amelioration of the oxidative stress and the resultant apoptosis.

Simultaneous Determination of Two Isomers of Asarone in &lt;i&gt;Piper sarmentosum&lt;/i&gt; Roxburgh (Piperaceae) Extracts using Different Chromatographic Columns

Purpose: To develop a rapid and reliable reverse phase high performance liquid chromatography (RPHPLC) method to quantify the two isomers of asarones in P. sarmentosum extracts using two different columns under similar analytical conditions. Methods: Two isomers, α- and β-asarone, were analyzed using two types of C 18 columns with 0.1 % orthophosphoric acid: acetonitrile: methanol (50: 40: 10) as mobile phase. The developed method was applied to determine the contents of α- and β-asarone in extracts of different parts of P. sarmentosum. Results: Column A retention times for the elution of α- and β-asarone were 11.890 ± 0.008 and 10.80 ± 0.004 min, respectively, and were significantly shorter than those of column B (15.110 ± 0.024 and 13.290 ± 0.018, respectively, p < 0.001). Column B showed better resolution (1.82 ± 0.025 of the isomers than column A (1.10 ± 0.01, p < 0.001). Both columns showed comparable sensitivity, precision and selectivity of the compounds investigated. α-Asarone level was in the range 0.36 - 5.14 % in ethanol and 50 % ethanol extracts, but absent in all water extracts. β-Asarone occurred in the range of 0.01 - 0.15 % in ethanol and 50 % ethanol extracts but was absent in all water extracts of P. sarmentosum . Conclusion: The results indicate that the developed method is a suitable quality assurance method for determining α- and β-asarone isomers in herbal extracts and food preparations. Keywords: α- Asarone, Isomers, Piper sarmentosum , Herbal extracts, Retention time

DEVELOPMENT AND VALIDATION OF RAPID REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR SIMULTANEOUS ESTIMATION OF STIGMASTEROL AND β-SITOSTEROL IN EXTRACTS OF VARIOUS PARTS (LEAVES, STEMS, AND ROOTS) OF XANTHIUM STRUMARIUM LINN.

ABSTRACTObjective: Xanthium strumarium is a cocklebur or burweed belonging to family Asteraceae and commonly found as a weed, is widely distributed inNorth America, Brazil, China, Malasia, and hotter part of India. The herb is traditionally used mostly in treating several ailments. The present studydeals with development and validation of a reliable reverse phase high-performance liquid chromatography (RP-HPLC) method for the simultaneousestimation of Stigmasterol and β-sitosterol in the various extracts of the plant.Methods: The proposed method utilizes a Qualisil Gold C18 column (250×4.6 mm), 5 µm particle size, under isocratic elution conditions with themixture of acetonitrile:ethanol:water (85:14:1 v/v/v) at 25° as a mobile phase. An effluent flow rate of 1 ml/minute and ultraviolet detection at202 nm was used for the analysis of Stigmasterol and β-sitosterol.Results: The described method was linear in the range of range of 100-500 µg/ml and 10-500 µg/ml for stigmasterol and β-sitosterol respectively,with excellent correlation coefficients. The precision, robustness, and ruggedness values were also within the prescribed limits (&lt;2%). The recoveryvalues were within the range, which indicates that the accuracy of the analysis was good and that the interference of the matrix with the recovery ofphytosterols was low. The phytosterols were found to be stable in a stock solution for 24 hrs (percentage relative standard deviation was below 2%)and no interfering extra peaks were observed under controlled stress conditions.Conclusion: The proposed method is simple, specific, precise, accurate, and reproducible and thus can be used as appropriate method for routineanalysis of X. strumarium phytosterols in quality control laboratories.Keywords: Xanthium strumarium, Reverse phase high-performance liquid chromatography method, Precision, Phytosterols, Quality control.

Determinação de ácido rosmarínico em Cordia verbenacea por cromatografia líquida: aplicabilidade em estudo sazonal

RESUMO Neste estudo, uma técnica de cromatografia líquida de alta resolução em fase reversa (CLAE-FR) para a determinação de ácido rosmarínico em Cordia verbenacea foi desenvolvida e validada. A análise de regressão foi avaliada, com observação de uma boa linearidade (r = 0,9997). Os valores obtidos para a precisão e exatidão estão de acordo com as diretrizes do ICH e com a legislação brasileira. Os valores de repetibilidade e precisão intermediária foram 2,79% e 4,76%, respectivamente. Os limites de detecção e de quantificação de ácido rosmarínico foram de 1,92 µg/mL e 5,81 µg/mL, respectivamente. Os resultados mostraram que o método desenvolvido é uma técnica por CLAE-FR de confiança para a determinação de ácido rosmarínico em tintura de C. verbenacea. Além disso, essa metodologia foi aplicada em estudo sazonal, que revela uma correlação positiva relativamente forte entre o período de chuvas e o teor de ácido rosmarínico.

Heterologous expression of human cytochrome P450 (CYP) 2C19 in Escherichia coli and establishment of RP‐HPLC method to serve as activity marker

In this study, a simple and reliable reverse-phase high-performance liquid chromatography (RP-HPLC) method was established and validated to analyze S-mephenytoin 4-hydroxylase activity of a recombinant CYP2C19 system. This system was obtained by co-expressing CYP2C19 and NADPH-CYP oxidoreductase (OxR) proteins in Escherichia coli (E. coli) cells. In addition to RP-HPLC, the expressed proteins were evaluated by immunoblotting and reduced CO difference spectral scanning. The RP-HPLC assay showed good linearity (r(2) = 1.00) with 4-hydroxymephenytoin concentration from 0.100 to 50.0 μm and the limit of detection was 5.00 × 10(-2) μm. Intraday and interday precisions determined were from 1.90 to 8.19% and from 2.20 to 14.9%, respectively. Recovery and accuracy of the assay were from 83.5 to 85.8% and from 95.0 to 105%. Enzyme kinetic parameters (Km , Vmax and Ki ) were comparable to reported values. The presence of CYP2C19 in bacterial membranes was confirmed by immunoblotting and the characteristic absorbance peak at 450 nm was determined in the reduced CO difference spectral assay. Moreover, the activity level of co-expressed OxR was found to be comparable to that of the literature. As a conclusion, the procedures described here have generated catalytically active CYP2C19 and the RP-HPLC assay developed is able to serve as CYP2C19 activity marker for pharmacokinetic drug interaction study in vitro.