Reversed-phase C18 Column Research Articles

Stability indicating eco-friendly HPLC method development and validation for the estimation of bisoprolol fumarate and telmisartan

BackgroundTelmisartan and bisoprolol fumarate together are two medications that diminish arterial pressure. The current study comprises an evaluation of the proposed methodology's greenness regarding the HPLC method used to govern the medication mixture regardless of dose form A novel stability suggesting HPLC method's environmental effect was evaluated using the greenness metrics. Stress conditions comprising acidic, alkaline, oxidative, thermal, and photolytic degradation were applied for both of the medications.ResultsThe RP—HPLC method employing a reversed-phase C18 column with a gradient approach, the HPLC chromatography was carried out. The mobile phase consisted of acetonitrile, methanol, and phosphate buffer (60:35:5, %v/v/v), with the stationary phase being the Unisphere C18 column Agela Tech. The RP-HPLC method uses UV detection at 224 nm with chromatographic purification spanning linearities of 2.5–12.5 μg/mL for bisoprolol fumarate and 40.0–200.0 μg/mL for telmisartan, correspondingly. The procedure is accurate and precise, as demonstrated by an outcome that % RSD inside the permissible range. Additionally, various stressors were introduced to the medications. The approach's green credentials with respect to solvent utilization, chemical substances, expenditure of energy, and waste formation have been verified by the greenness data collected during the evaluation. No chromatographic or spectrum impediments caused by formulation additives have been observed.ConclusionBisoprolol fumarate and telmisartan could be measured simultaneously using the devised RP-HPLC method, which was simple, quick, sensitive, accurate, precise, linear, and stability indicating. The proposed approach showed ecological friendliness, robustness, sensitivity, and ease of use. As a result, the devised method could be applied to the regular quality checking of tablets and bulk medications.

A Novel High-Conjugated Probe of 4-(Acridone-10-yl)-Phenylethyl Chloroformate (APE-Cl) for Rapid Detection of Amino Compounds Using HPLC with Fluorescence Detection.

The fluorescence detection of amino compounds and the evaluation of their content in environmental samples are vital, not only for assessing food quality but also for studying soil organic matter. Here, we present the synthesis and application of a novel fluorescent probe, 4-(9-acridone)benzylmethyl carbonochloride (APE-Cl), for detecting amino compounds via a chloroformate reaction with fluorescence detection. The complete derivatization reaction of APE-Cl with amino compounds can be accomplished in aqueous acetonitrile within 5min at room temperature, using 0.2M borate buffer (pH = 9.0). APE-amine derivatives exhibited intense fluorescence with an excitation maximum at λex 254nm and an emission maximum at λem 418nm. All derivatives demonstrated high stability, strong fluorescence, and elevated ionization potential under atmospheric pressure chemical ionization (APCI-MS) in positive ion detection mode. The method, combined with gradient elution, provides baseline resolution of common amine derivatives on a reversed-phase C18 column. The LC separation for the derivatized amines shows good reproducibility with aqueous acetonitrile as the mobile phase. The relative standard deviations (n = 6) for each amine derivative are < 3.99%. The detection limits (at a signal-to-noise ratio of 3) per injection ranged from 1.68 to 11.2 femtomole. The established pre-column derivatization method for determining amino compounds in practical samples proved to be satisfactory.

A simple HPLC method for the determination of plasma progesterone levels in the third trimester of human pregnancy.

Progesterone is a steroid hormone primarily associated with pregnancy. A simple, rapid, and reliable high-performance liquid chromatography (HPLC) method has been developed and validated for the quantification of progesterone in human plasma. The method consists of a simple liquid-liquid extraction of progesterone and internal standard (estriol) from human plasma using a mixture of hexane and diethyl ether. The chromatographic determination of progesterone was performed using an acetonitrile-water (70:30, v/v) mobile phase with a C18 reversed-phase column. The method achieved an extraction recovery of greater than 96.4% from spiked plasma samples. Intra- and inter-day precision were generally acceptable, with relative SD% less than ≤6.60% and accuracy (relative error %) better than 3.64%. The developed and validated method was used to successfully quantify progesterone levels in plasma samples collected from women during the third trimester of pregnancy. Furthermore, a statistical comparison was conducted between progesterone concentrations in plasma samples obtained from 2 groups of pregnant women: group 1 (n = 9) at 30-35 weeks and group 2 (n = 9) at 36-41 weeks. The developed and validated HPLC method described in this study enables the successful determination of progesterone in human plasma, offering advantages such as shorter analysis time, simplicity, cost-effectiveness, and potential routine use during pregnancy.

Quantification of ∆9-tetrahydrocannabinol (THC), 11-OH-THC, THC-COOH, hexahydrocannabinol and cannabidiol in human plasma and blood by liquid chromatography-tandem mass spectrometry.

Ongoing legalization of cannabis for recreational use contributes to increasing numbers not only of incidents of driving under the influence, but within all forensic fields. In addition, newly emerging cannabinoids such as hexahydrocannabinol (HHC) and the increasing use of cannabidiol (CBD) products have to be addressed. The aims of this study were first to extend laboratory analysis capacity for the "established" cannabinoid ∆9-tetrahydrocannabinol (THC) and its metabolites 11-OH-THC and THC-COOH in human plasma/blood, and second to develop analytical procedures concerning HHC and CBD. An LC-MS/MS method based on the available (low-end) instrumentation was used. Samples (250 µL) were prepared by protein precipitation and solid phase extraction. Chromatographic separation was achieved on a reversed-phase C18 column within 15 min. Detection was performed on a 3200 QTRAP instrument (Sciex) in positive multiple reaction monitoring (MRM) mode. Matrix matched six-point calibrations were generated applying deuterated internal standards for all analytes except HHC. The method was fully validated according to GTFCh guidelines. Linear ranges were 0.5-25 µg/L for THC, 11-OH-THC, HHC and CBD, and 2.0-100 µg/L for THC-COOH, respectively. Limits of detection and limits of quantification were 0.5 and 1.0 µg/L (THC, 11-OH-THC, HHC, CBD), and 2.0 and 4.0 µg/L (THC-COOH). Applicability of plasma calibrations to blood samples was demonstrated. Acceptance criteria for intra- and inter-day accuracy, precision, extraction efficiency and matrix effects were met. No interfering signals were detected for more than 60 pharmaceutical compounds. The presented method is sensitive, specific, easy to handle and does not require high-end equipment. Since its implementation and accreditation according to ISO 17025, the method has proven to be fit for purpose not only in DUID cases but also within post-mortem samples. Furthermore, the design of the method allows for an uncomplicated extension to further cannabinoids if required.

Robust In Vitro HPLC-DAD Method for Accurate Quantification of N-Carbamylglutamate in Drug Formulations

A comprehensive in vitro testing method has been developed and validated for accurately quantifying N-Carbamylglutamate (Carglumic acid) using a high-performance liquid chromatography instrument with a diode array detector. A method specifically developed for pharmaceutical quality control utilizes a reverse-phase C18 column for precise and reliable measurement of Carglumic acid. The chromatographic parameters were refined for peak resolution, employing a phosphate buffer and acetonitrile as the mobile phase, with detection setup at 205 nm. The method was validated under ICH Q2(R2) requirements. The approach exhibited a significant level of specificity, with no interference from excipients or other possible impurities. Linearity was achieved across the entire concentration range, yielding an R² value of 0.999. Precision (RSD ≤ 2) was demonstrated by intra- and inter-day variability being within acceptable limits. Recovery tests validated accuracy (98 – 102%). Determining the limit of detection (LOD, 0.1 ppm) and the limit of quantitation (LOQ, 0.5 ppm) was conducted. This in vitro test method was valid and effective for the routine quantification of Carglumic acidin pharmaceutical formulations, serving as a crucial tool for ensuring product quality and regulatory compliance. The methods' elevated sensitivity and endurance make it appropriate for bioanalytical applications, such as dissolution testing and stability analysis.

LITERATURE REVIEW ON HPLC METHODS FOR ESTIMATION OF EMTRICITABINE AND TENOFOVIR DISOPROXIL FUMARATE TABLETS

Emtricitabine and Tenofovir Disoproxil Fumarate (TDF) are essential components of antiretroviral therapy (ART) for HIV infection. Accurate quantification of these drugs in pharmaceutical formulations is crucial for ensuring therapeutic efficacy and patient safety. High-Performance Liquid Chromatography (HPLC) is a widely used analytical technique for this purpose. Several validated HPLC methods have been developed, employing diverse chromatographic conditions and detection techniques. These methods typically utilize reversed-phase C18 columns and UV detection at 260 nm or 270 nm. Key considerations for method development include selectivity, sensitivity, accuracy, precision, linearity, and robustness. Additionally, other analytical techniques such as UltraHigh Performance Liquid Chromatography (UHPLC), Thin-Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FTIR), and Ultraviolet-Visible (UV-Vis) Spectroscopy have been explored for the analysis of Emtricitabine and TDF. However, HPLC remains the preferred method due to its versatility, sensitivity, and reliability.

Investigation on the Retention and Separation of Glucosinolates With a Mixed-Mode Reversed-Phase/Weak Anion-Exchange Column.

Glucosinolates, a crucial group of secondary metabolites in Brassica vegetables, present significant chromatographic separation challenges due to their anionic form, structure diversities, and co-existence of other phenolic compounds. This study comparatively investigated the retention and separation of seven glucosinolates using a mixed-mode reversed-phase/weak anion-exchange column and a conventional reversed-phase C18 column. Separation factors for each glucosinolate with its adjacent peaks were over 1.0 on the mixed-mode column, while co-eluting was observed on the C18 column. The effects of mobile phase additives and pH on the separation and retention of glucosinolates were also investigated. Results showed that glucosinolate retention was inversely related to both buffer concentration and pH. The optimized method for the mix-mode column was applied to the complex Brassica vegetable samples. In addition to the 17 well-resolved glucosinolate peaks, 34 peaks for phenolic compounds were identified in broccoli microgreen, suggesting the successful application scenarios for qualitative analysis in comparison with the single mode reverse phase C18 column. This study demonstrates that the mixed-mode reversed-phase/weak anion-exchange column can be used as a promising separation tool for organic anions in a complex sample matrix.

LITERATURE REVIEW ON HPLC METHODS FOR ESTIMATION OF EMTRICITABINE AND TENOFOVIR DISOPROXIL FUMARATE TABLETS

Emtricitabine and Tenofovir Disoproxil Fumarate (TDF) are essential components of antiretroviral therapy (ART) for HIV infection. Accurate quantification of these drugs in pharmaceutical formulations is crucial for ensuring therapeutic efficacy and patient safety. High-Performance Liquid Chromatography (HPLC) is a widely used analytical technique for this purpose. Several validated HPLC methods have been developed, employing diverse chromatographic conditions and detection techniques. These methods typically utilize reversed-phase C18 columns and UV detection at 260 nm or 270 nm. Key considerations for method development include selectivity, sensitivity, accuracy, precision, linearity, and robustness. Additionally, other analytical techniques such as Ultra-High Performance Liquid Chromatography (UHPLC), Thin-Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FTIR), and Ultraviolet-Visible (UV-Vis) Spectroscopy have been explored for the analysis of Emtricitabine and TDF. However, HPLC remains the preferred method due to its versatility, sensitivity, and reliability.

Specific determination of (-)- and (+)-gossypol in vegetable oil by ultra-performance liquid chromatography-tandem mass spectrometry with chiral pre-column derivatization.

As the unique ingredient of cottonseed oil, gossypol is toxic and there are differences between the enantiomers. Although the determination of (-)- and (+)-gossypol in cotton plants or cottonseed was performed, the detection about the gossypol enantiomers in vegetable oil was rarely reported. To develop a specific inspection method for (-)- and (+)-gossypol in vegetable oil by ultra-performance liquid chromatography (UPLC) -tandem mass spectrometry with chiral pre-column derivatization. (-)- and (+)-gossypol were separated on an ACQUITY C18 reverse phase column (100 mm × 2.1 mm, 1.7 μm) maintained at 40 °C on an UPLC system with gradient elution. The mobile phases was composed of methanol (solvent A) and water containing 0.1% formic acid (solvent B). Analytes were detected with Electron Spray Ionization source in the positive mode. The contents of (-)- and (+)-gossypol standard derivatives were quantified by Multiple Reaction Monitoring with the transitions of m/z 633/483 for quantitative ion pairs and m/z 633/558 for qualitative ion pairs. The results indicated that the quantitative analysis could be accomplished within 12.5 minutes and the limits of detection of (-)- and (+)-gossypol were 25.55 μg/kg and 14.67 μg/kg, respectively. When this method was applied to olive oil spiked with gossypol standards, good recoveries (95.37%-105.84% for (-)-gossypol, 98.96%-105.14% for (+)-gossypol) and RSDs (3.41%-6.02% for (-)-gossypol, 4.50%-6.94% for (+)-gossypol) were achieved. The present study confirms the feasibility of determining the gossypol enantiomers and achieves trace determination of (-)- and (+)-gossypol in vegetable oil. These results make this method more suitable for the qualitative identification of whether the vegetable oil has been mixed with cottonseed oil. The trace determination of (-)- and (+)-gossypol in vegetable oils has been achieved. This method provides technical support for further identification of whether commercially available vegetable oil has been mixed with cottonseed oil.

B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase

Abstract Background Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol that are excreted in urine. These two metabolites are often tested for to determine if an individual has recently consumed alcohol. EtG and EtS are very polar analytes, which make them difficult to chromatograph using reversed-phase chromatography. These analytes often elute early, which can lead to irreproducible results. When EtG and EtS are analyzed by LC/MS with a mobile phase that contains formic acid, the signal of these two analytes can be suppressed. This leads to increased LOQ and cutoffs. We examined the perfluorophenyl (PFP) stationary phase for its utility in analyzing EtG and EtS. This stationary phase has been shown to have greater retention of polar compounds than C18 reversed-phase columns. In addition, we compared the effect of formic acid to acetic acid on the separation. Methods Urine samples containing EtG and EtS were diluted 10:1, using 50mM acetic acid or 0.1% formic acid, into a 96-well microtiter filter plate containing a 0.2micron filter. Internal standards were added to the microtiter plate containing the urine samples. The samples were filtered through the filter plate and analyzed by LC/MS using the PFP column (50 x 2.7micron) with a 50mM acetic acid or 0.1% formic acid/acetonitrile mobile phase. The analytes were detected using negative mode ionization. The total analysis time was 4.5minutes. Results EtG and EtS showed improved retention using the PFP column. Retention times for EtG and EtS were 0.75 and 1.20 minutes respectively using the PFP column, compared to 0.4 and 0.3 minutes using the C18 column. The greater retention time improved the LOQ for both analytes.The LOQ for EtS and EtG were determined to be 125 and 250 ng/mL, respectively. Greater retention of EtG was observed by switching the modifier from formic acid to acetic acid. The retention time increased from 0.45 to 0.75 minutes. There was less signal suppression using acetic acid in place of formic acid. Improvement in peak shape was observed when the injection volume was reduced to 5microliters. The method was validated over a range of 250 to 10000 ng/mL for EtG and 125 to 5000 ng/mL for EtS. Acceptable linearity was obtained for both analytes. Accuracy was performed at three concentration levels. All three levels demonstrated acceptable accuracy with the difference between the measured and expected concentration being with in 20% of each other. Conclusions The PFP stationary can be used to test EtG and EtS. This stationary phase gave greater retention of EtS and EtG. The use of acetic acid improved the limit of detection by decreasing the ion suppression, which decreased the limit of detection. The assay was accurate and precise for both analytes, demonstrated in the validation.

Method Development and Validation for the Simultaneous Estimation of Lobeglitazone Sulphate and Glimepiride by HPLC Method.

A simple, precise, and accurate reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed for the simultaneous estimation of lobeglitazone sulfate and glimepiride in bulk and tablet dosage forms. The method involved selecting various chromatographic parameters. Specifically, a new method was developed using a 250 mm × 4.6 mm (HSS) reverse-phase C18 column with 5 µm particle size (Shim-pack C18, 250 × 4.6 mm; 5 µm). The mobile phase consisted of 40 volumes of phosphate buffer (pH 3) and 60 volumes of acetonitrile, with methanol as the diluent, running as an isocratic elution. The flow rate was set at 1.0 mL/min, and UV detection was performed at 254 nm. The injection volume was 2 µL, and the total runtime was 10 minutes. The recoveries for lobeglitazone sulfate were found to be in the range of 101% to 100.6%, while those for glimepiride were in the range of 101.5% to 100%. The method’s accuracy is evident from these results. The inter-day and intra-day precision of the new method were both below the maximum allowable limit (RSD% ≤ 2.0), as per International Council on Harmonisation (Q2 R1) guidelines. The method exhibited a linear response, with correlation coefficient (r2) values of 0.9972% for pioglitazone and 0.998 for glimepiride. The validation parameters, such as accuracy, precision, linearity, specificity, stability in the analytical solution and robustness, met the acceptance criteria. Hence, this method can be effectively used for the simultaneous estimation of lobeglitazone sulfate and glimepiride in both bulk and pharmaceutical dosage forms.

Development and Validation of New Stability Indicating Analytical RPHPLC Method for Simultaneous Estimation of Metformin and Alogliptin.

The current study focuses on developing and validating a reverse phase-high performance liquid chromatography (RP-HPLC) method for quantifying metformin HCL (MET) and alogliptin (ALO) in both dosage and bulk drug forms. ALO and MET in tablet form were determined simultaneously using an RP-HPLC technique. The Reverse Phase (Waters) C18 (4.6 mm x 150 mm; 5 µ) column, a 20-injection loop are the components of the gradient system for the RP manufactured by Agilent Technologies. In the procedure, a 50:50 volumetric combination of methanol and water (0.1% acetic acid) was used as the mobile phase at pH 4.5. The developed approach yielded retention times of 3.856 minutes for MET and 6.302 minutes for ALO. The reliability of the established technique was demonstrated in compliance with the International Council of Harmonization (ICH) standards. In general, all of the metrics including linearity, accuracy, range, and robustness, were found to be within the acceptable ranges. Consequently, the methodology was assessed to be simple, precise, cost-effective, and replicable. Hence, the analysis of MET and ALO in both bulk medication and formulated states can be regularly conducted utilising the procedures specified for the purpose of quality control.

A Novel Method for Development and Validation of the Degradation Products Analysis of N-Carbamylglutamate with UHPLC by Using Design of Experiment Approach.

Carglumic acid, also known as N-carbamyl-l-glutamic acid, is a medication used in the treatment of a rare genetic disorder called N-acetylglutamate synthase (NAGS) deficiency. To the authors' knowledge, there was no method reported in the literature for the determination of degradation products suitable for quality control analyses of carglumic acid. Thus, the aim of the presented study is to develop an impurity method with a UHPLC/DAD detector configuration compatible with industrial standards from the European Pharmacopeia and the United States Pharmacopeia, making the drug more accessible for developing and underdeveloped countries through its precise evaluation. The method involved the separation of carglumic acid and its degradation products using a reverse-phase C18 column (Waters, BEH 150 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.39 mL/min with a stop time of 10 min. To separate all unknown and known impurities, a gradient elution (phosphate buffer, pH 2.4, and acetonitrile) system was used. The detection was performed at 214 nm. Forced degradation studies were conducted under different stress conditions, including acidic, basic, oxidative, thermal, and photolytic stress. Placket-Burman statistical experimental design was used to demonstrate the robustness of this method, and the suitability of the method was confirmed under the applied conditions. Box-Behnken design was used to provide the optimum resolution between the peaks determined to be critical during the optimization. The developed method was validated according to ICH guidelines for specificity, linearity, accuracy, precision, and robustness. The limit of detection and limit of quantification were 0.7 and 0.15 μg/mL for carglumic acid, respectively.

Stability Evaluation and Pharmacokinetic Profiling of Vepdegestrant in Rodents Using Liquid Chromatography-Tandem Mass Spectrometry.

Vepdegestrant (formerly ARV-471), a novel proteolysis-targeting chimera (PROTAC), targets estrogen receptor alpha (ERα) for degradation, offering a promising option to treat advanced ER-positive breast cancer. We developed and validated a sensitive and rapid liquid chromatography-tandem mass spectrometry method to quantify vepdegestrant in rodent plasma using bavdegalutamide (formerly ARV-110) as an internal standard. Plasma samples were prepared with protein precipitation using acetonitrile and analyzed using reverse-phase C18 columns and a mobile phase of 10 mM ammonium formate in distilled water and acetonitrile. The method demonstrated linearity from 1 to 1000 ng/mL in mouse and rat plasma, meeting all validation criteria, and successfully applied to in vivo and in vitro studies. Pharmacokinetic analysis revealed low-to-moderate clearance (313.3, 1053 mL/h/kg) and oral bioavailability (17.91, 24.12%) of vepdegestrant in mice and rats, respectively. It was unstable in buffer solutions across pH 2-10 and in phosphate-buffered saline (pH 7.4), likely due to adsorption, but remained stable in mouse and rat plasma at varying temperatures. In liver microsomes, vepdegestrant exhibited moderate stability in rats but was stable in mice, dogs, and humans. These findings enhance the understanding of pharmacokinetic properties of vepdegestrant supporting further development of PROTAC drugs.

Moojecin: The first disintegrin from Bothrops moojeni venom and its antitumor activity in acute myeloid leukemia

Disintegrins are a class of peptides found in snake venom that inhibit the activity of integrins, which are essential cell adhesion receptors in tumor progression and development. In this work, moojecin, a RGD disintegrin, was isolated from Bothrops moojeni snake venom, and its antitumor potential in acute myeloid leukemia (AML) HL-60 and THP-1 cells was characterized. The isolation was performed using a C18 reverse-phase column in two chromatographic steps, and its molecular mass is 7417.84 Da. N-terminal and de novo sequencing was performed to identify moojecin. Moojecin did not show cytotoxic or antiproliferative activity in THP-1 and HL-60 at tested concentrations, but it exhibited significant antimigratory activity in both cell lines, as well as inhibition of angiogenesis in the tube formation assay on Matrigel in a dose-dependent manner. A stronger interaction with integrin αVβ3 was shown in integrin interaction assays compared to α5β1, and the platelet aggregation assay indicated an IC50 of 5.039 μg/mL. Preliminary evaluation of disintegrin toxicity revealed no incidence of hemolysis or cytotoxic effects on peripheral blood mononuclear cells (PBMCs) across the tested concentrations. Thus, this is the first study to report the isolation, functional and structural characterization of a disintegrin from B. moojeni venom and bring a new perspective to assist in AML treatment.

Liquid Chromatography with Orbitrap High-Resolution Mass Spectrometry for the Determination of Glucose in Saliva Following Derivatization with Dibenzylamine

As an important circulating metabolite in the body, the accurate determination of glucose in body fluids is very important for the evaluation of physiological status. However, liquid chromatography–mass spectrometry (LC–MS) methods present challenges for glucose due to poor ionization combined with weak chromatographic retention. Here, a method for determining salivary glucose using LC–MS after derivation by dibenzylamine is described in which 3-O-methyl-D-glucopyranose was used as internal standard. Saliva samples were prepared by protein precipitation, and the analytes were separated on a polar-copolymerized C18 reversed phase column. Detection took place by high-resolution orbitrap mass spectrometry after ionization in the positive mode. The calibration function was linear from 0.35 to 50 ng/mL. The recoveries were from 97.82 to 104.51% for intraday assays and 98.96 to 106.61% for interday assays. The intraday precision of salivary glucose was from 0.37% to 1.43% and the interday precision from 1.33% to 1.43%. The limits of detection and quantification in saliva were 0.2 and 0.6 ng/mL. The samples after derivatization were stored at 4 °C and 25 °C and analyzed every two days with five repeats. The relative standard deviations were 0.09% and 0.27% at 4 °C and 25 °C. This method was employed for the determination of the glucose in saliva for healthy male college students after exercise.

Proximate Composition, Comparative Phytochemical Analysis, and HPLC Profiling of Various Solvent Extracts of Anthocleista djalonensis Leaves

Choosing the right solvent in herbal preparation is very important for extracting bioactive compounds from medicinal plant, as it greatly affects the yield and quality of phytochemicals obtained. This study aimed to investigate the proximate composition, phytochemical analysis and HPLC profiling of various solvents extracts of Anthocleista djalonensis leaves, a medicinal plant used in herbal remedies. The dried leaves of Anthocleista djalonensis were analyzed for their nutritional profile using standard methods. The leaves were then extracted by maceration using three different solvents: 95% analytical ethanol, hot distilled water, and local gin (78% alcohol). Various phytochemicals in the extracts were identified and quantified using standard analytical methods. Additionally, High-Performance Liquid Chromatography (HPLC) was used to compare the chemical compositions of the extracts, using a reverse-phase C18 column and UV detection at 254nm. The results of the proximate analysis revealed good nutritional value, with protein (15.90%), moisture content (9.08%), total ash (10.37%), crude fibre (4.00%), crude fat (2.40%), carbohydrate (61.53%), and an energy value of 331.33 KJ. All tested phytochemicals were found in the ethanol and local gin extracts, while the distilled water extract showed the absence of terpenoids and steroids. In the quantitative analysis, the local gin extract had the highest concentrations of alkaloids, terpenoids, flavonoids, and tannins. On the other hand, the HPLC fingerprint analysis identified seventeen (17), twelve (12), and nine (9) compounds in ethanol, local gin and water extracts respectively. Four (4) shared peaks with similar retention time were observed across all extracts, indicating the presence of common phytocompounds. In conclusion, this study shows that Anthocleista djalonensis leaves have good nutritional values, suggesting their potential as dietary phytonutrient. Local gin stands out as the best solvent among those tested for extracting beneficial compounds from Anthocleista djalonensis leaves supporting its traditional use as solvent in herbal preparation.

Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

Simultaneous quantification of terpenes and cannabinoids by reversed-phase LC-APCI-MS/MS in Cannabis sativa L. samples combined with a subsequent chemometric analysis

Cannabis sativa L. has been the most discussed medicinal plant in recent years. In particular, the dynamic shift from a formerly illicit and tightly controlled substance to a plant recognized for both medicinal and recreational purposes has brought C. sativa into the global spotlight. Due to the ongoing international legalization processes, fast and convenient analytical methods for the quality control of C. sativa flowers for medicinal and recreational purposes are of tremendous interest. In this study, we report the development and validation of a liquid chromatography-tandem mass spectrometry (LC–MS/MS)-based method applying atmospheric pressure chemical ionization (APCI) to fully quantify 16 terpenes and 7 cannabinoids including their acidic forms by a single chromatographic method. The method presented here is unique and simple, as it eliminates the need for derivatization reactions and includes the unconventional analysis of volatile compounds by liquid chromatography. Samples were prepared by a simple and fast ethanolic extraction. Separation was accomplished within 25 min on a reversed-phase C18 column. Method validation was conducted according to international guidelines regarding selectivity, accuracy, precision, robustness, and linearity. Detection was done in multiple reaction monitoring, which allowed the simultaneous quantification of co-eluting analytes applying two selective mass transitions. In addition, due to reproducible in-source decarboxylation, the acidic forms of cannabinoids were reliably quantified using mass transitions of the neutral forms. The accuracy given as the bias was below 15% for all analytes. Matrix effects for cannabinoids were studied by spiking Humulus lupulus extracts with the analytes at varying concentrations. APCI did not show susceptibility toward ion suppression or enhancement. In addition, the recovery effect after spiking was between 80 and 120% for terpenes. Further, 55 authentic C. sativa extracts were fully quantified, and the obtained results for the terpene profiles were compared to state-of-the-art gas chromatography coupled to flame ionization detection. Comparable results were achieved, emphasizing the method’s applicability for cannabinoids and terpenes. Further, acquired metabolite patterns for C. sativa samples were studied, identifying a relationship between cannabinoid and terpene patterns, as well as the abundance of myrcene in CBD-dominant C. sativa strains.Graphical

DEVELOPMENT AND VALIDATION OF A HPLC/PDA METHOD FOR SIMULTANEOUS QUANTIFICATION OF AMLODIPINE BESYLATE AND VALSARTAN IN DISSOLUTION MEDIA

Background: Valsartan, an angiotensin blocker, is often combined with amlodipine (a calcium channel antagonist) in the current treatment of hypertension. There is a generic brand-name drug Exforge® which has been proven to be clinically effective. However, its price is high, making it quite difficult to access, especially in low- and middle-income countries. Therefore, research and development of generic drugs is essential. In addition, research on simultaneous quantification of amlodipine and valsartan in the dissolution medium must be performed to serve as a basis for comparing in vitro equivalence between the two finished products. Objectives: Investigation of the mobile phase for separating amlodipine and valsartan using the isocratic elution method and validation of the simultaneous quantification of amlodipine and valsartan in three different dissolution media with pH of 1.2, 4.5, and 6.8. Materials and methods: Experimental methods on finished tablets containing amlodipine and valsartan. Results: A procedure for simultaneous quantification of amlodipine and valsartan has been developed and validated in three dissolution media using the HPLC method with DAD detector, ZORBAX Eclipse Plus C18 reversed-phase column (4.6 x 250 mm; 5m), isocratic elution method, detection wavelength of 237 nm, flow rate of 1 mL/min, an injection volume of 20 µL, and mobile phase composed of acetonitrile-triethylamine 0.7% (adjusted to pH 3.0 with 0.05% of phosphoric acid) with a ratio of 40:60. The quantitative method achieves linearity with a correlation coefficient R2&gt;0.999 and the linearity for amlodipine and valsartan was determined in the range at pH of 1.2 (0.25-10 µg/mL and 2-160 µg/mL), at pH of 4.5 and 6.8 (0.5-10 µg/mL and 8-160 µg/mL). The values of amlodipine and valsartan content in the test sample on the same day and between two different days were less than 2.0%. The recovery rate ranged from 98% to 102% with the relative standard deviation (RSD) not exceeding 2.0%. Conclusions: The HPLC method with the above chromatographic conditions can be applied for simultaneous quantification of amlodipine and valsartan in three dissolution media with various pH.