A comprehensive review of capillary electrophoresis-based techniques for erythropoietin isoforms analysis.

Different CE techniques have been used to analyze erythropoietin. These techniques have been shown to be effective in differentiating and quantifying erythropoietin isoforms, including natural and recombinant origins. This review provides a comprehensive overview of various capillary electrophoresis-based techniques used for the analysis of erythropoietin isoforms. The importance of erythropoietin in clinical practice and the necessity for the accurate analysis of its isoforms are first discussed. Various techniques that have been used for erythropoietin isoform analysis are then described. The main body of the review focuses on the different capillary electrophoresis-based methods that have been developed for erythropoietin isoform analysis, including capillary zone electrophoresis and capillary isoelectric focusing. The advantages and drawbacks of each method as well as their applications are discussed. Suggestions into the future directions of the area are also described.

Evaluation of aloins, pH and moisture in aloe leaf gel-based personal care products.

OBJECTIVE: Some easily applicable analytical methods were explored to evaluate the quality of personal care products containing aloe leaf gel. Aloins should be absent in these products in view of their side effects. To check this, liquid chromatography (LC) was applied. METHODS: The LC method used a C18 monolithic column combined with gradient elution and ultraviolet (UV) detection. The mobile phase consisted of a mixture of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The method was validated with respect to specificity, linearity, precision and accuracy. Next, it was practically applied for the analysis of commercial samples. In addition, the pH and moisture content were determined. RESULTS: The LC results indicated that aloins were detected in 25% of the analysed commercial samples. Further, it turned out that 42% of the test samples were found to be in the basic pH range and 33% of them contained excessive moisture. CONCLUSION: Proper quality control and adequate labelling of aloe leaf gel-based cosmetics are mandatory to avoid side effects.

OBJECTIF: Certaines méthodes analytiques facilement applicables ont été explorée pour évaluer la qualité des produits de soins personnels contenants du gel de feuille d'aloe. Les aloïnes doivent être absentes de ces produits en raison de leurs effets secondaires. Pour vérifier cela, la chromatographie liquide (CL) a été appliquée. MÉTHODES: La méthode CL a utilisé une colonne monolithique C18 combinée à une élution par gradient et une détection ultraviolette (UV). La phase mobile était constituée d'un mélange de 0,1 % d'acide formique dans l'eau (A) et de 0,1 % d'acide formique dans l'acétonitrile (B). La méthode a été validée en ce qui concerne la spécificité, la linéarité, la précision et l'exactitude. Ensuite, elle a été appliquée pour l'analyse d'échantillons commerciaux. De plus, le pH et la teneur en humidité ont été déterminés. RÉSULTATS: Les résultats CL ont indiqué que des aloïnes ont été détectées dans 25 % des échantillons commerciaux analysés. Il s'est avéré que 42 % des échantillons se trouvaient dans la plage de pH basique et que 33 % d'entre eux contenaient une humidité excessive. CONCLUSION: Un contrôle de qualité approprié et un étiquetage adéquat des cosmétiques à base de gel de feuille d'aloe sont obligatoires pour éviter des effets secondaires.

Antihyperglycemic Activity of TLC Isolates from the Leaves of Aloe megalacantha Baker in Streptozotocin-Induced Diabetic Mice.

BACKGROUND: Diabetes mellitus (DM) is a group of metabolic disorders that are characterized by hyperglycemia which results from defects in insulin release or its efficient use by the human body. Although significant progress has been made to manage DM and related complications, it remains a major global health problem. To this end, the search for new antidiabetic drugs from traditionally claimed medicinal plants is important. Aloe megalacantha Baker is an endemic plant used traditionally to treat diabetes in Ethiopia. This study aimed to investigate antidiabetic activity of isolates from the leaf of A. megalacantha Baker in streptozotocin-induced diabetic mice. METHODS: The exudate of A. megalacantha was collected by cutting the leaves and scraping the yellow sap and then dried at room temperature. The dried exudate was subjected to repeated thin layer chromatographic (TLC) separations using a mixture of solvent system to isolate the major compounds identified on the TLC plate. A single dose of streptozotocin (50 mg/kg) was injected intraperitoneally to overnight fasted mice to induce diabetes. Antidiabetic activity of TLC isolates was assessed by in vivo methods including oral glucose tolerance test, antihyperglycemic and hypoglycemic activity tests. RESULTS: Three major isolates were obtained from the TLC analysis of the exudate of A. megalacantha. Exudate and TLC isolates were found to be non-toxic up to a dose of 2000 mg/kg. The TLC isolates (Ia and Ib) significantly (p<0.05) reduced blood glucose levels and also increased body weight change of the diabetic mice compared with control groups. CONCLUSION: The present study demonstrated the ability of the exudate of A. megalacantha and its TLC isolates to significantly decrease blood glucose levels and increase body weights in mice, thus strengthening the claim of its traditional use in DM-related disorders.

Fast and easily applicable LC-UV method for analysis of bioactive anthrones from Aloe leaf latex.

Aloe leaf latex is a commonly used plant preparation in traditional medicine. However, quality control on the content of medicinally important constituents is often limited. Hence, establishing a reliable quality control method to identify and quantify bioactive markers is important to ensure safety and efficacy. In the present study, a novel liquid chromatographic (LC) method was developed and validated for efficient analysis of bioactive markers to evaluate the quality of aloe leaf latex. Quantification of marker compounds was possible in only 7 min on a monolithic column using gradient elution with 0.1 % formic acid in acetonitrile and water as mobile phases. The major compounds (aloins A and B) could be baseline separated together with related compounds within 10 min. The method showed excellent linearity with determination coefficients (r2) of 0.9999. Detection limits were 0.017 and 0.013 µg/mL, while quantification limits were 0.057 and 0.043 µg/mL for aloin A and aloin B, respectively. Relative standard deviation (RSD) values for intra- and inter-day precision were less than 2% and recoveries for both aloins were close to 100 %. The robustness was evaluated using an experimental design. The method was applied to some aloe leaf latex samples from Ethiopia. Aloin contents varied from 14 to 35 % and two unknown peaks were tentatively identified as aloinoside and microdontin.

Formulation and Optimization of Monolithic Fixed-Dose Combination of Metformin HCl and Glibenclamide Orodispersible Tablets.

The treatment of type II DM involves the use of combination of drugs, especially at the chronic stage. However, the pill burden of this combination therapy combined with swallowing difficulties, occurring at a later stage of DM, has been the major challenge for successful treatment outcomes. This study was aimed at formulating and optimizing a monolithic fixed-dose combination (FDC) of metformin (MET) and glibenclamide (GLB) orodispersible tablets (ODTs) to overcome both the pill burden and swallowing problems. The FDC ODTs were prepared by the melt granulation technique using polyethylene glycol (PEG) 6000 as a binding agent and crospovidone as a superdisintegrant. In the preliminary study, the effects of sodium lauryl sulphate (SLS), PEG 6000, crospovidone, and compression force on friability, disintegration time, and drug release of tablets were investigated. The FT-IR studies showed that there were no incompatibilities between MET and GLB as well as within excipients. The preliminary studies revealed that PEG 6000 and compression force significantly affect both the friability and the disintegration time, while SLS and crospovidone only affect the disintegration time. Therefore, the effects of PEG 6000, crospovidone, and compression force were further studied and optimized using the central composite design. Accordingly, the most desirable optimal values were obtained at 3.82% of PEG 6000, 9.83% of crospovidone, and 10.6 kN compression force having a friability of 0.302% and a disintegration time of 18.7 seconds. From these results, it can be concluded that a monolithic FDC of MET and GLB ODTs having adequate mechanical strength and faster disintegration time was successfully formulated.

Antimicrobial Evaluation of Latex and TLC Fractions from the Leaves of Aloe adigratana Reynolds.

BACKGROUND: The highest prevalence and emergence of microbial infections coupled with the threat of antimicrobial resistance constitute a global concern, which entails searching for novel antimicrobial agents. Medicinal plants are among the major sources of medicines for novel drug discovery. Aloe adigratana is one of the endemic Aloe species in Ethiopia where the leaf latex of the plant is traditionally used for the treatment of various pathogenic conditions such as wound, dandruff, malaria, and diabetes. In spite of such claims, there was no scientific study done so far. The aim of the current study was, therefore, to evaluate the antimicrobial effect of leaf latex of A. adigratana and its thin layer chromatography (TLC) fractions. METHODS: Thin layer chromatography (TLC) separation was employed for isolation of bioactive compounds. Agar well diffusion and microdilution assay method were used to evaluate the antimicrobial actions of the leaf latex and TLC fractions against six bacterial strains and four Candida species of reference and clinical isolate microbial strains. RESULTS: Three major fractions, AA01, AA02, and AA03, were identified by TLC. Among the tested microbial strains, the reference strain of Staphylococcus aureus ATCC 29213 (MIC = 0.06 mg/mL) and clinical Candida krusei 242/18 (MIC = 0.14 mg/mL) exhibited higher susceptibility towards AA02, while reference strains of Klebsiella pneumoniae ATCC 700603 (MIC = 0.19 mg/mL) revealed the highest susceptibility towards AA01. The leaf latex displayed the highest activity against Staphylococcus aureus ATCC 29213 and clinical Candida krusei 242/18 with a MIC value of 0.19 mg/mL. CONCLUSION: The leaf latex and TLC fractions were found to be active against the tested bacterial and Candida species. Therefore, this finding supports the traditional claim of Aloe adigratana and the need for characterization of the TLC fractions to provide as lead compounds for further comprehensive antibacterial and antifungal activities.

Study of aldehyde oxidase with phthalazine as substrate using both off-line and on-line capillary electrophoresis.

An optimized and economical capillary electrophoretic method for both off-line and on-line study of the enzyme aldehyde oxidase and its substrate phthalazine was developed. The separation of the substrate phthalazine and its metabolite 1-phthalazinone was achieved using micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulphate in the background electrolyte (BGE). The BGE consists of 25 mM sodium phosphate buffer containing 50 mM sodium dodecyl sulphate at pH 7.4. A bare-fused-silica capillary with a capillary length of 40 cm, 50 µm ID and effective length of 30 cm was used to develop the capillary electrophoresis method. Improved separation conditions were elaborated and the separation method was validated based on the ICH and EMA guidelines. The limit of detection for phthalazine and 1-phthalazinone was 8 µM and 3 µM, respectively. The limit of quantification was 25 µM for phthalazine and 10 µM for 1-phthalazinone. The linearity of the detector response was checked for 1-phthalazinone at nine different concentrations in the range 10-500 µM and the determination coefficient was 0.9994. Accuracy was tested by comparing the corrected peak area of 1-phthalazinone reference solution at 20 µM and 50 µM with the corrected peak area of 20 µM and 50 µM 1-phthalazinone in the presence of human liver cytosol (HLC). Accuracy values of +5.3% and -2.5% were obtained at 20 µM and 50 µM, respectively. The on-line enzymatic reaction was successful with the application of the method of transverse diffusion of laminar flow profiles (TDLFP), which enables the mixing as well as separation of the enzyme and substrate inside the nanoliter-scale capillary. TDLFP is examined to be precise when performing 5 consecutive injections, with a relative standard deviation of 7.16% which is within the limitation of EMA standards. This miniaturized and low-cost incubation and separation method could be further introduced into industry and extended to other substrates.

New RP-HPLC Method Development and Validation for Dorzolamide in Ophthalmic Dosage Form.

A reversed phase liquid chromatographic method with UV detection at 254 nm for dorzolamide assay in ophthalmic solutions was developed and validated. Chromatographic separation was achieved on a Zorbax SB C18 (250 mm × 4.6 mm, 5 µm) column kept at 30°C with an isocratic mixture of mobile phase (phosphate buffer, pH 2.5, and acetonitrile, 90 : 10 v/v) at a flow rate of 0.8 mL/min. The method was validated for its specificity, linearity, accuracy, precision, limit of detection, limit of quantification, and robustness based on ICH guidelines. The validation studies revealed satisfactory results. The proposed method has been applied for the quantification of dorzolamide in commercial samples. The developed method is fast, simple, specific, accurate, and sensitive, hence can be applied for routine quality control analysis of dorzolamide in pharmaceutical dosage form.

Development and Validation of a High-Performance Liquid Chromatographic Method for the Determination of Cinitapride in Human Plasma.

A precise and reliable reversed-phase high-performance liquid chromatographic method with ultraviolet detection was developed and validated to determine cinitapride in human plasma. After liquid-liquid extraction, chromatographic separation was achieved on a Nucleosil C18 (25 cm × 4.6 mm, 5 µm) column with an isocratic elution consisting of 10 mM ammonium acetate (pH 5.2), methanol, and acetonitrile, 40 : 50 : 10, v/v/v. The developed method was validated as per US FDA guidelines for its linearity, selectivity, sensitivity, precision, accuracy, and stability. Satisfactory findings were obtained from the validation studies. The linearity range of the method was 1 to 35 ng/mL while the extraction recovery of cinitapride in human plasma was more than 86%. The percent coefficient of variation of both intraday and interday precision was ≤7.1%.

Infant Exposure to Metals through Consumption of Formula Feeding in Mekelle, Ethiopia.

This study aimed at determination of heavy metals (cadmium, lead, and zinc) in milk-based infant formulas collected from Mekelle, Ethiopia, and their associated health risks to the infants through consumption of these products. The infant feeding samples were dry-ashed in a muffle furnace followed by digestion in nitric acid and the resulting solutions were analyzed by flame atomic absorption spectrophotometer. Cadmium was not detected in the samples while the levels of lead and zinc ranged from not detected value to 0.103 mg/kg and from 27.888 to 71.553 mg/kg, respectively. The estimated daily intake values and the health risk indices of both metals were below their respective safety limits and the threshold of 1, respectively. These findings show low infant health risk of these metals through consumption of these products. Nevertheless, regular monitoring of infant formula for toxic metals is required since infants are potentially more susceptible to metals.

Characterization of mesoporous silica used for drug delivery by sorptive interaction - multiple headspace extraction-gas chromatography.

A multiple headspace extraction experiment coupled to gas chromatography (MHE-GC) is used for the classification and qualification of different mesoporous silica (MPSi) materials used for drug delivery. In this MHE experiment, a pure liquid solvent probe is fully evaporated in a sealed headspace vial in the presence of the MPSi sample, leading to a gas-solid partitioning that is theoretically described. The obtained results matched with the known characteristics of the studied samples, such as adsorption capacity due to differences in porosity and passivation treatments. Moreover, it proves the effectiveness of a poly dimethyl siloxane (PDMS) coating treatment over a thermal one in reducing the specific interactions of the MPSi. In addition, it evidences the important role of confinement effects when the pore diameter is close to the microporosity range. Finally, a simple experiment for fast screening is proposed by comparison of the results obtained for four different probes used as a mixture.

Development and Validation of an Extractive Spectrophotometric Method for Miconazole Nitrate Assay in Pharmaceutical Formulations.

A simple extractive spectrophotometric technique has been developed and validated for the determination of miconazole nitrate in pure and pharmaceutical formulations. The method is based on the formation of a chloroform-soluble ion-pair complex between the drug and bromocresol green (BCG) dye in an acidic medium. The complex showed absorption maxima at 422 nm, and the system obeys Beer's law in the concentration range of 1-30 µg/mL with molar absorptivity of 2.285 × 104 L/mol/cm. The composition of the complex was studied by Job's method of continuous variation, and the results revealed that the mole ratio of drug : BCG is 1 : 1. Full factorial design was used to optimize the effect of variable factors, and the method was validated based on the ICH guidelines. The method was applied for the determination of miconazole nitrate in real samples.

Chemometric-Assisted Spectrophotometric Method for the Simultaneous Determination of Ciprofloxacin and Doxycycline Hyclate in Pharmaceutical Formulations.

Two chemometrics methods-principal component regression and partial least squares-were developed for simultaneous spectrophotometric estimation of ciprofloxacin and doxycycline hyclate in pharmaceutical dosage forms without any pretreatment. The UV spectra of both drugs were recorded at concentrations within their linear ranges between 200 and 400 nm with the intervals λ = 2 nm at 100 wavelengths in distilled water. Beer's law was obeyed for both drugs in the concentration ranges of 1-10 µg/mL for ciprofloxacin and 5-25 µg/mL for doxycycline hyclate. Two sets of standard mixtures, 25 as a calibration set and 9 as a validation set, were prepared. The calibration models were evaluated by cross-validation and external validation over synthetic mixtures. The optimized models were successfully applied for chemometric analysis of ciprofloxacin and doxycycline hyclate in synthetic and pharmaceutical mixtures with satisfactory accuracy (recovery values from 97.50% to 101.87%) and precision (RSD < 2%).

Improved liquid chromatographic method for quality control of spiramycin using superficially porous particles.

This article describes the development and validation of a liquid chromatographic method for spiramycin using a column with superficially porous particles. Gradient elution was applied and the mobile phase consisted of phosphate buffer (0.2M; pH 8.3) - H2O - acetonitrile in a ratio 10:60:30 (v/v/v) for mobile phase A and 10:30:60 (v/v/v) for mobile phase B. UV detection was performed at 232nm. Compared to previous methods, the analysis time was about two times faster and impurities were better separated. Furthermore, impurities which were present above 0.25% were characterized using liquid chromatography coupled with mass spectrometry (LC/MS).

Development and validation of LC methods for the separation of misoprostol related substances and diastereoisomers.

Misoprostol is a synthetic prostaglandin E1 analogue which is mainly used for prevention and treatment of gastric ulcers, but also for abortion due to its labour inducing effect. Misoprostol exists as a mixture of diastereoisomers (1:1) and has several related impurities owing to its instability at higher temperatures and moisture. A simple and robust reversed phase liquid chromatographic (RPLC) method is described for the separation of the related substances and a normal phase (NP) LC method for the separation of misoprostol diastereoisomers. The RPLC method was performed using an Ascentis Express C18 (150 mm × 4.6 mm, 5 µm) column kept at 35 °C. The mobile phase was a gradient mixture of mobile phase A (ACN-H2O-MeOH, 28:69:3 v/v/v) and mobile phase B (ACN-H2O-MeOH, 47:50:3 v/v/v) eluted at a flow rate of 1.5 mL/min. UV detection was performed at 200 nm. The NPLC method was undertaken by using an XBridge bare silica (150 mm × 2.1 mm, 3.5 µm) column at 35 °C. The mobile phase contained 1-propanol-heptane-TFA (4:96:0.1%, v/v/v), pumped at a flow rate of 0.5 mL/min. UV detection was performed at 205 nm. This LC method can properly separate the two diastereoisomers (Rs > 2) within an analysis time of less than 20 min. Both methods were validated according to the ICH guidelines. Furthermore, these new LC methods have been successfully applied for purity control and diastereoisomers ratio determination of misoprostol bulk drug, tablets and dispersion.

Kinetic performance comparison of fully and superficially porous particles with a particle size of 5 µm: intrinsic evaluation and application to the impurity analysis of griseofulvin.

After the great commercial success of sub-3 µm superficially porous particles, vendors are now also starting to commercialize 5 µm superficially porous particles, as an alternative to their fully porous counterparts which are routinely used in pharmaceutical analysis. In this study, the performance of 5 µm superficially porous particles was compared to that of fully porous 5 µm particles in terms of efficiency, separation performance and loadability on a conventional HPLC instrument. Van Deemter and kinetic plots were first used to evaluate the efficiency and performance of both particle types using alkylphenones as a test mixture. The van Deemter and kinetic plots showed that the superficially porous particles provide a superior kinetic performance compared to the fully porous particles over the entire relevant range of separation conditions, when both support types were evaluated at the same operating pressure. The same observations were made both for isocratic and gradient analysis. The superior performance was further demonstrated for the separation of a pharmaceutical compound (griseofulvin) and its impurities, where a gain in analysis time of around 2 could be obtained using the superficially porous particles. Finally, both particle types were evaluated in terms of loadability by plotting the resolution of the active pharmaceutical ingredient and its closest impurity as a function of the signal-to-noise ratio obtained for the smallest impurity. It was demonstrated that the superficially porous particles show better separation performance for griseofulvin and its impurities without significantly compromising sensitivity due to loadability issues in comparison with their fully porous counterparts. Moreover these columns can be used on conventional equipment without modifications to obtain a significant improvement in analysis time.

Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics.

This paper presents a general overview of the application of hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics in different sample matrices including pharmaceutical, plasma, serum, fermentation broths, environmental water, animal origin, plant origin, etc. Specific applications of HILIC for analysis of aminoglycosides, ß-lactams, tetracyclines and other antibiotics are reviewed. HILIC can be used as a valuable alternative LC mode for separating small polar compounds. Polar samples usually show good solubility in the mobile phase containing some water used in HILIC, which overcomes the drawbacks of the poor solubility often encountered in normal phase LC. HILIC is suitable for analyzing compounds in complex systems that elute near the void in reversed-phase chromatography. Ion-pair reagents are not required in HILIC which makes it convenient to couple with MS hence its increased popularity in recent years. In this review, the retention mechanism in HILIC is briefly discussed and a list of important applications is provided including main experimental conditions and a brief summary of the results. The references provide a comprehensive overview and insight into the application of HILIC in antibiotics analysis.

Development and validation of a reversed phase liquid chromatographic method for analysis of griseofulvin and impurities.

A simple and robust reversed phase liquid chromatographic method was developed and validated for the quantitative determination of griseofulvin (GF) and its impurities in drug substances and drug products (tablets). Chromatographic separation was achieved on a Discovery C18 (250mm×4.6mm, 5µm) column kept at 30°C. The mobile phase consisted of a gradient mixture of mobile phase A (water-0.1% formic acid pH 4.5, 80:20, v/v) and B (ACN-water-0.1% formic acid pH 4.5, 65:15:20, v/v/v) pumped at a flow rate of 1.0mL/min. UV detection was performed at 290nm. The method was validated for its robustness, sensitivity, precision, accuracy and linearity based on ICH guidelines. The robustness study was performed by means of an experimental design and multivariate analysis. Satisfactory results were obtained from the validation studies. The use of volatile mobile phases allowed for the identification of three main impurities present above the identification threshold using mass spectrometry (MS). The developed LC method has been applied for the assay and impurity determination of GF drug substances and tablets. The method could be very useful for the quality control of GF and its impurities in bulk and formulated dosage forms.

Development and validation of a reversed phase liquid chromatographic method for analysis of oxytetracycline and related impurities.

A simple, robust and fast high-performance liquid chromatographic method is described for the analysis of oxytetracycline and its related impurities. The principal peak and impurities are all baseline separated in 20 min using an Inertsil C8 (150 mm × 4.6 mm, 5 µm) column kept at 50 °C. The mobile phase consists of a gradient mixture of mobile phases A (0.05% trifluoroacetic acid in water) and B (acetonitrile-methanol-tetrahydrofuran, 80:15:5, v/v/v) pumped at a flow rate of 1.3 ml/min. UV detection was performed at 254 nm. The developed method was validated for its robustness, sensitivity, precision and linearity in the range from limit of quantification (LOQ) to 120%. The limits of detection (LOD) and LOQ were found to be 0.08 µg/ml and 0.32 µg/ml, respectively. This method allows the separation of oxytetracycline from all known and 5 unknown impurities, which is better than previously reported in the literature. Moreover, the simple mobile phase composition devoid of non-volatile buffers made the method suitable to interface with mass spectrometry for further characterization of unknown impurities. The developed method has been applied for determination of related substances in oxytetracycline bulk samples available from four manufacturers. The validation results demonstrate that the method is reliable for quantification of oxytetracycline and its impurities.

Development and validation of a reversed-phase liquid chromatographic method for analysis of demeclocycline and related impurities.

A simple, robust, and rapid reversedphase high-performance liquid chromatographic method for the analysis of demeclocycline and its impurities is described. Chromatographic separations were achieved on a Symmetry Shield RP8 (75 mm × 4.6 mm, 3.5 µm) column kept at 40°C. The mobile phase was a gradient mixture of acetonitrile, 0.06 M sodium edetate (pH 7.5), 0.06 M tetrapropylammonium hydrogen sulphate (pH 7.5) and water, A (2:35:35:28 v/v/v/v) and B (30:35:35:0 v/v/v/v) pumped at a flow rate of 1 mL/min. UV detection was performed at 280 nm. The developed method was validated according to the ICH guidelines for specificity, limit of detection, limit of quantification, linearity, precision, and robustness. An experimental design was applied for robustness study. Results show that the peak shape, chromatographic resolution between the impurities, and the total analysis time are satisfactory and better than previous methods. The method has been applied for the analysis of commercial demeclocycline bulk samples available on the market.